WorldWideScience

Sample records for cd46 splicing variants

  1. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel......RNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....

  2. A TIMP-1 splice variant transcript

    DEFF Research Database (Denmark)

    Øbro, Nina Friesgård; Lademann, Ulrik Axel; Birkenkamp-Demtroder, Karin

    2008-01-01

    A splice variant of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA lacking exon 2 (TIMP-1-v2) has been identified in human cancer cells and in colorectal and breast cancer tumors. The purpose of this study was (1) to study the level of full length TIMP-1 and TIMP-1-v2 transcripts in color...... of TIMP-1 pre-mRNA to TIMP-1-v2 mRNA might be involved in regulating TIMP-1 expression.......A splice variant of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA lacking exon 2 (TIMP-1-v2) has been identified in human cancer cells and in colorectal and breast cancer tumors. The purpose of this study was (1) to study the level of full length TIMP-1 and TIMP-1-v2 transcripts...... in colorectal tumors; (2) to investigate if TIMP-1-v2 is translated to protein. Full length TIMP-1 and TIMP-1-v2 mRNA levels were compared between colorectal tumors and normal mucosa by Q-PCR. Both full length TIMP-1 and TIMP-1-v2 transcripts were upregulated in tumor tissue. However, the level of TIMP-1-v2...

  3. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-14-1-0480 TITLE: Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy PRINCIPAL INVESTIGATOR...Splice Variants and Resistance to Taxane Chemotherapy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0480 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...During this reporting period, we have obtained approval for a no-cost extension of this award and change of research focus in the final year. This

  4. Two new splice variants in porcine PPARGC1A

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    Peelman Luc J

    2008-12-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. Findings This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa, of which the first 291 aa would be the same compared to the complete protein (796 aa. Conclusion Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.

  5. Splice variants of porcine PPHLN1 encoding periphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Momeni, Jamal; Farajzadeh, Leila

    2017-01-01

    of the periphilin-1 protein. Thus, variants Sp1 and Sp1 are the result of alternative splicing. The porcine PPHLN1 gene was mapped to chromosome 5. The porcine PPHLN1 gene was found to be differentially expressed in various porcine organs and tissues. The sequence of the porcine PPHLN1 cDNA, encoding the periphilin......The periphilin-1 protein is encoded by the PPHLN1 gene. Periphilin-1 is found in the cornified cell envelope during the terminal differentiation of keratinocyte at the outer layer of epidermis. In the current study we report on the cloning and characterization of the porcine PPHLN1 cDNA and two...... splice variants hereof. RT-PCR cloning using oligonucleotide primers derived from in silico sequences resulted in three PPHLN1 transcripts: a full-length mRNA and two transcript variant resulting in shorter proteins. The longest encoded periphilin-1, consisting of 373 amino acids, displays a high...

  6. Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

    DEFF Research Database (Denmark)

    Ahlborn, Lise B; Dandanell, Mette; Steffensen, Ane Y

    2015-01-01

    needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined...... by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c...... to have no or an uncertain effect on the protein level, whereas one variant (c.5072C>T/p.Thr1691Ile) were shown to have a strong effect on the protein level as well. In conclusion, our study emphasizes that in silico splicing prediction and mini-gene splicing analysis are important for the classification...

  7. Alternative splice variants of the human PD-1 gene

    DEFF Research Database (Denmark)

    Nielsen, Christian; Ohm-Laursen, Line; Barington, Torben

    2005-01-01

    PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1......Deltaex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Deltaex3 is therefore likely to encode a soluble form of PD-1. PD-1Deltaex2,3 lacks exon 2 and 3. These three variants have unaffected open...

  8. Splice variants of androgen receptor and prostate cancer

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    Orazio Caffo

    2016-05-01

    Full Text Available Over the last ten years, two new-generation hormonal drugs and two chemotherapeutic agents have been approved for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, some patients have primary resistance to them and the others eventually develop secondary resistance. It has recently been suggested that the presence of androgen receptor splice variants plays a leading role in the primary and secondary resistance to the new hormonal drugs, whereas their presence seem to have only a partial effect on the activity of the chemotherapeutic agents. The aim of this paper is to review the published data concerning the role of androgen receptor splice variants in prostate cancer biology, and their potential use as biomarkers when making therapeutic decisions.

  9. ISVASE: identification of sequence variant associated with splicing event using RNA-seq data.

    Science.gov (United States)

    Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Yu, Jun; Hu, Songnian

    2017-06-28

    Exon recognition and splicing precisely and efficiently by spliceosome is the key to generate mature mRNAs. About one third or a half of disease-related mutations affect RNA splicing. Software PVAAS has been developed to identify variants associated with aberrant splicing by directly using RNA-seq data. However, it bases on the assumption that annotated splicing site is normal splicing, which is not true in fact. We develop the ISVASE, a tool for specifically identifying sequence variants associated with splicing events (SVASE) by using RNA-seq data. Comparing with PVAAS, our tool has several advantages, such as multi-pass stringent rule-dependent filters and statistical filters, only using split-reads, independent sequence variant identification in each part of splicing (junction), sequence variant detection for both of known and novel splicing event, additional exon-exon junction shift event detection if known splicing events provided, splicing signal evaluation, known DNA mutation and/or RNA editing data supported, higher precision and consistency, and short running time. Using a realistic RNA-seq dataset, we performed a case study to illustrate the functionality and effectiveness of our method. Moreover, the output of SVASEs can be used for downstream analysis such as splicing regulatory element study and sequence variant functional analysis. ISVASE is useful for researchers interested in sequence variants (DNA mutation and/or RNA editing) associated with splicing events. The package is freely available at https://sourceforge.net/projects/isvase/ .

  10. Cephalopod eye evolution was modulated by the acquisition of Pax-6 splicing variants

    National Research Council Canada - National Science Library

    Yoshida, Masa-aki; Yura, Kei; Ogura, Atsushi

    2014-01-01

    Previous studies have reported that the developmental processes of vertebrate eyes are controlled by four Pax-6 splicing variants, each modulating different downstream genes, whereas those of insect...

  11. Cephalopod eye evolution was modulated by the acquisition of Pax-6 splicing variants.

    Science.gov (United States)

    Yoshida, Masa-aki; Yura, Kei; Ogura, Atsushi

    2014-03-05

    Previous studies have reported that the developmental processes of vertebrate eyes are controlled by four Pax-6 splicing variants, each modulating different downstream genes, whereas those of insect eyes are controlled by duplicated Pax-6 genes. Cephalopods belong to the Protostomes but possess a camera-type eye similar to those in vertebrates. We examined Pax-6 variations in the squid and found five types of Pax-6 splicing variants but no duplication of the Pax-6 gene. In the five splicing variants, the splicing patterns were produced by the combination of two additional exons to the ortholog and one jettisoned exon containing most of the Homeobox domain (HD). These five variants show spatio-temporal patterns of gene expression during development in the squid. Our study suggests that cephalopods acquired Pax-6 splicing variants independent of those in vertebrates and that these variants were similarly utilized in the development of the squid eye.

  12. A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma.

    Science.gov (United States)

    Luo, Zai-Li; Cheng, Shu-Qun; Shi, Jie; Zhang, Hui-Lu; Zhang, Cun-Zhen; Chen, Hai-Yang; Qiu, Bi-Jun; Tang, Liang; Hu, Cong-Li; Wang, Hong-Yang; Li, Zhong

    2015-10-07

    Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 ((Δ2-4)Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that (Δ2-4)Merlin interferes with the capacity of wild-type Merlin to bind β-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, (Δ2-4)Merlin overexpression increases the expression levels of β-catenin and stemness-related genes, induces the epithelium-mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the (Δ2-4)Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis.

  13. Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants

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    Furuichi Teiichi

    2007-04-01

    Full Text Available Abstract Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2 is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3 is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD. CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f, and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and

  14. The role of CD44 splice variants in human metastatic cancer

    NARCIS (Netherlands)

    Sleeman, J.; Moll, J.; Sherman, L.; Dall, P.; Pals, S. T.; Ponta, H.; Herrlich, P.

    1995-01-01

    The large family of CD44 splice variants are likely to serve multiple functions in the embryo and in the adult organism. This is reflected in their complex patterns of expression. In molecular terms these functions are largely unknown. Certain splice variants (CD44v) can promote the metastatic

  15. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

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    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  16. Expression of androgen receptor splice variants in clinical breast cancers.

    Science.gov (United States)

    Hickey, Theresa E; Irvine, Connie M; Dvinge, Heidi; Tarulli, Gerard A; Hanson, Adrienne R; Ryan, Natalie K; Pickering, Marie A; Birrell, Stephen N; Hu, Dong Gui; Mackenzie, Peter I; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V; Dehm, Scott M; Plymate, Stephen R; Bradley, Robert K; Tilley, Wayne D; Selth, Luke A

    2015-12-29

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.

  17. Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma.

    Science.gov (United States)

    Fu, Yao; Zhao, Xingli; Li, Zhaohui; Wei, Jun; Tian, Yu

    2016-08-01

    The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma.

  18. Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1.

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    Ling Pan

    Full Text Available The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3' or 5' splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1, providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.

  19. Clinical Significance of HER-2 Splice Variants in Breast Cancer Progression and Drug Resistance

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    Claire Jackson

    2013-01-01

    Full Text Available Overexpression of human epidermal growth factor receptor (HER-2 occurs in 20–30% of breast cancers and confers survival and proliferative advantages on the tumour cells making HER-2 an ideal therapeutic target for drugs like Herceptin. Continued delineation of tumour biology has identified splice variants of HER-2, with contrasting roles in tumour cell biology. For example, the splice variant 16HER-2 (results from exon 16 skipping increases transformation of cancer cells and is associated with treatment resistance; conversely, Herstatin (results from intron 8 retention and p100 (results from intron 15 retention inhibit tumour cell proliferation. This review focuses on the potential clinical implications of the expression and coexistence of HER-2 splice variants in cancer cells in relation to breast cancer progression and drug resistance. “Individualised” strategies currently guide breast cancer management; in accordance, HER-2 splice variants may prove valuable as future prognostic and predictive factors, as well as potential therapeutic targets.

  20. A Newly Identified Natural Splice Variant ASN Enhances Hepatitis B Virus Amplification.

    Science.gov (United States)

    Zhang, Xiumin; Zhu, Sibo; Zhu, Wei; Li, Aijun; Zhu, Naishuo

    2016-01-01

    Chronic hepatitis B virus (HBV) infection causes approximately one-third of all the cases of liver cirrhosis and more than three-quarters of hepatocellular carcinoma (HCC) worldwide. There are eight different genotypes (A-H) of HBV, among which B and C are the major types of HBV in China. There is a positive correlation between viral load and level of viral splicing variants and the high risk of HCC. The aim of this study was to investigate the splicing variants of HBV circulating in HCC patients. Twenty-four carcinoma and adjacent liver tissues collected from HCC patients were studied. Using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, we identified a new type of natural splice variant with nucleotides 2448-489 and 910-2120 deleted, and we named it ASN. We also found that a higher viral load and splicing variant level existed in liver carcinoma tissues compared to paracarcinoma tissues. In the investigation of our splicing variant, we found its enhancing effect on HBV replication in vitro. Although splicing variants are not essential for the replication of HBV, they may have an important influence.

  1. Alternative splicing modulated by genetic variants demonstrates accelerated evolution regulated by highly conserved proteins.

    Science.gov (United States)

    Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Lin, Xianzhi; Chan, Tak-Ming; Wang, Rena; Xiao, Xinshu

    2016-04-01

    Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting that cis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon. © 2016 Hsiao et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing

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    Zhilong Chen

    2018-02-01

    Full Text Available Titin (TTN is a major disease-causing gene in cardiac muscle. Titin (TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20. Three isoforms of TTN protein are produced by mutually exclusive exons 45 (Novex 1, 46 (Novex 2, and 48 (Novex 3. Alternatively splicing in Novex isoforms across species and whether Novex isoforms are associated with heart disease remains completely unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is more highly conserved across all species than exons 46 and 48. Importantly, a conserved region between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase chain reaction (RT-PCR and DNA sequencing confirmed a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2 primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46 retention is mainly expressed in the human and frog heart, another variant with co-expression of exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns 45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart, we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3 could be associated with dilated cardiomyopathy (DCM and/or arrhythmogenic right ventricular cardiomyopathy (ARVC. Taken together, our study reveals that splicing diversity of Novex exons across species and Novex variants might play a role in cardiomyopathy.

  3. Functional characterization of BRCA1 gene variants by mini-gene splicing assay

    DEFF Research Database (Denmark)

    Steffensen, Ane Y; Dandanell, Mette; Jønson, Lars

    2014-01-01

    are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants...... of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213-1G>A, c.670+1delG, c.4185+1G>A, and c.5075-1G......>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302-15C>G, c.547+14delG, c.4676-20A>G, c.4987-21G>T, and c.5278-14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays...

  4. Alternative Splicing Generates Different 5′ UTRs in OCT4B Variants

    OpenAIRE

    Poursani, Ensieh M.; Mehravar, Majid; Shahryari, Alireza; Mowla, Seyed Javad; Mohammad Soltani, Bahram

    2017-01-01

    Background: The human OCT4 gene, responsible for pluripotency and self-renewal of Embryonic Stem (ES) and Embryonic Carcinoma (EC) cells, can generate several transcripts (OCT4A, OCT4B-variant 2, OCT4B-variant 3, OCT4B-variant 5, OCT4B1, OCT4 B2 and OCT4B3) by alternative splicing and alternative promoters. OCT4A that is responsible for ES and EC cell stemness properties is transcribed from a promoter upstream of Exon1a in those cells. The OCT4B group variants (OCT4B-variant2, OCT4B-variant3,...

  5. RBM20 and RBM24 cooperatively promote the expression of short enh splice variants.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2016-07-01

    PDZ-LIM protein ENH1 is a scaffold protein for protein kinases and transcriptional regulators. While ENH1 promotes the hypertrophic growth of cardiomyocytes, its short splice variant (ENH3) prevents the hypertrophic growth. The mechanism underlying the alternative splicing of enh mRNA between ENH short and long isoforms has remained unknown. Here, we found that two splicing factors, RNA-binding motif 20 (RBM20) and RNA-binding motif 24 (RBM24) together promoted the expression of short enh splice variants and bound the 5' intronic region of exon 11 containing an in-phase stop codon. In addition, expression of both RBMs is repressed by hypertrophic stimulations. Collectively, our results suggest that, in healthy conditions, RBM20 and RBM24 cooperate to promote the expression of short ENH isoforms. © 2016 Federation of European Biochemical Societies.

  6. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

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    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  7. Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

    Science.gov (United States)

    Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

    2010-01-01

    This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

  8. Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

    Science.gov (United States)

    Sun, Shihua; Sprenger, Cynthia C.T.; Vessella, Robert L.; Haugk, Kathleen; Soriano, Kathryn; Mostaghel, Elahe A.; Page, Stephanie T.; Coleman, Ilsa M.; Nguyen, Holly M.; Sun, Huiying; Nelson, Peter S.; Plymate, Stephen R.

    2010-01-01

    Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand. PMID:20644256

  9. A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements.

    Science.gov (United States)

    Churbanov, Alexander; Vorechovský, Igor; Hicks, Chindo

    2010-01-12

    Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II) for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS) sensor used in our tool allows inference on non-canonical exons. Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD). SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

  10. A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2010-01-01

    Full Text Available Abstract Background Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS sensor used in our tool allows inference on non-canonical exons. Results Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD. SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. Conclusions We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

  11. A novel ERCC6 splicing variant associated with a mild Cockayne syndrome phenotype.

    Science.gov (United States)

    Swartz, Jonathan M; Akinci, Aysehan; Andrew, Shayne F; Siğirci, Ahmet; Hirschhorn, Joel N; Rosenfeld, Ron G; Dauber, Andrew; Hwa, Vivian

    2014-01-01

    Cockayne syndrome is an autosomal recessive, heterogeneous syndrome with classical features, including short stature, microcephaly, developmental delay, neuropathy, and photosensitivity. New genomic approaches offer improved molecular diagnostic potential. Whole-exome sequencing was employed to study a consanguineous extended family with severe short stature and variable presentations of peripheral neuropathy, lipoatrophy, photosensitivity, webbed neck, and hirsutism. We identified a novel homozygous ERCC6 variant at the donor splice site of intron 9 (c.1992 + 3A>G), which was predicted to only slightly perturb splicing efficiencies. Assessment of primary fibroblast-derived mRNAs, however, revealed a dominant splicing species that utilized an unsuspected putative donor splice site within exon 9, resulting in predicted early protein termination (p.Arg637Serfs*34). We describe a new splicing ERCC6 defect causal of Cockayne syndrome. The application of exome sequence analysis was integral to diagnosis, given the complexity of phenotypic presentation in the affected family members. The novel splicing defect, furthermore, illustrates how a seemingly minor change in the relative strength of a splice site can have significant biological consequences. 2014 S. Karger AG, Basel

  12. Characterization and alternative splicing of the complex I 19-kD subunit in Dunaliella salina: expression and mutual correlation of splice variants under diverse stresses.

    Science.gov (United States)

    Cao, Yu; Jin, Nan; Xu, Hui; Liu, Yi; Zhu, Wei Hua; Li, Xin Ran; Qiao, Dai Rong; Cao, Yi

    2010-01-01

    Complex I is the first enzyme in the mitochondrial respiratory chain. It extracts energy from NADH, which is produced by the oxidation of sugars and fats, and traps the energy by virtue of a potential difference or voltage across the mitochondrial inner membrane. Herein, the genomic sequence and four splice variants encoding the complex I 19-kD subunit were isolated from Dunaliella salina. There were four transcripts coding for the complex I 19-kD subunit due to alternative splicing in algae, and the four transcripts were translated to two protein isoforms with varying C-terminals. We report the splicing pattern in the 3'-region of the D. salina 19-kD subunit, in which three of the exons (5, 6, and 7) could be alternatively spliced. Moreover, we found that four alternatively spliced variants were subject to coordinated transcription in response to different stresses by real-time quantitative PCR.

  13. Expression of CD44 splice variants in human primary brain tumors

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; Morsink, F.; Keehnen, R. M.; Leenstra, S.; Bosch, D. A.; Pals, S. T.

    1995-01-01

    Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of

  14. Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization1

    OpenAIRE

    Cowles, Chad L.; Wu, Yi-Ying; Barnett, Scott D.; Lee, Michael T; Burkin, Heather R.; Buxton, Iain L. O.

    2015-01-01

    TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (

  15. Characterization and functional analysis of four HYH splicing variants in Arabidopsis hypocotyl elongation.

    Science.gov (United States)

    Li, Chen; Zheng, Lanlan; Zhang, Jingxuan; Lv, Yanxia; Liu, Jianping; Wang, Xuanbin; Palfalvi, Gergo; Wang, Guodong; Zhang, Yonghong

    2017-07-01

    Arabidopsis thaliana LONG HYPOCOTYL5 (HY5) is a positive regulator of the light signaling pathway. The hy5 mutant has an elongated hypocotyl in all light conditions, whereas the hy5 homolog (hyh) mutant has a very weak phenotype, but only in blue light. However, overexpression of HYH rescues the elongated hypocotyl phenotype in the hy5 null mutant. Here, we report the identification of four HYH splicing variants in Arabidopsis. Alternative splicing in the 5' region of the HYH gene occurred such that the proteins encoded by all four HYH variants retained their bZIP domain. In hypocotyl tissue, transcript levels of HYH.2, HYH.3, and HYH.4 were higher than those of HYH.1. Like HY5, all HYH variants were induced by light. Functional analysis of the four HYH variants, based on their abilities to complement the hy5 mutant, indicated that they have similar roles in hypocotyl development, and may function redundantly with HY5. Our results indicate that the bZIP domain in HYH is critical for the function of four variants in the compensation of hy5 mutant in hypocotyl development. Additionally, while HY5/HYH is found in plant species ranging from green algae to flowering plants, the potential alternative splicing events are distinct in different species, with certain HYH variants found with greater frequency in some species than others. Copyright © 2017. Published by Elsevier B.V.

  16. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members.

    Science.gov (United States)

    Thomassen, Mads; Blanco, Ana; Montagna, Marco; Hansen, Thomas V O; Pedersen, Inge S; Gutiérrez-Enríquez, Sara; Menéndez, Mireia; Fachal, Laura; Santamariña, Marta; Steffensen, Ane Y; Jønson, Lars; Agata, Simona; Whiley, Phillip; Tognazzo, Silvia; Tornero, Eva; Jensen, Uffe B; Balmaña, Judith; Kruse, Torben A; Goldgar, David E; Lázaro, Conxi; Diez, Orland; Spurdle, Amanda B; Vega, Ana

    2012-04-01

    Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of

  17. A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5 Protein that Is Selectively Expressed in Retina.

    Directory of Open Access Journals (Sweden)

    Susan N Bolch

    Full Text Available Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5 protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina.Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins.PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium

  18. Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization.

    Science.gov (United States)

    Cowles, Chad L; Wu, Yi-Ying; Barnett, Scott D; Lee, Michael T; Burkin, Heather R; Buxton, Iain L O

    2015-11-01

    TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (TREK-1 variants could interfere with TREK-1 function or expression. To investigate a potential role for these variants, immunofluorescence, cell surface assays, Western blots, and patch clamp were employed to study TREK-1 and TREK-1 variants expressed in HEK293T cells. The results of this study demonstrate that coexpression of TREK-1 with TREK-1 variants alters TREK-1 expression and suppresses channel function. Each variant affected TREK-1 in a disparate manner. In HEK293T cells coexpressing TREK-1 and each variant, TREK-1 membrane expression was diminished with compartmentalization inside the cell. When expressed alone, individual variants displayed channel properties that were significantly decreased compared to full-length TREK-1. In coexpression studies using patch clamp, basal TREK-1 currents were reduced by ∼64% (4.3 vs. 12.0 pA/pF) on average at 0 mV when coexpressed with each variant. TREK-1 currents that were activated by intracellular acidosis were reduced an average of ∼77% (21.4 vs. 94.5 pA/pF) at 0 mV when cells were transfected with TREK-1 and any one of the splice variants. These data correlate the presence of TREK-1 variants to reduced TREK-1 activity, suggesting a pathological role for TREK-1 variants in preterm labor. © 2015 by the Society for the Study of Reproduction, Inc.

  19. Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization1

    Science.gov (United States)

    Cowles, Chad L.; Wu, Yi-Ying; Barnett, Scott D.; Lee, Michael T.; Burkin, Heather R.; Buxton, Iain L.O.

    2015-01-01

    TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (TREK-1 variants could interfere with TREK-1 function or expression. To investigate a potential role for these variants, immunofluorescence, cell surface assays, Western blots, and patch clamp were employed to study TREK-1 and TREK-1 variants expressed in HEK293T cells. The results of this study demonstrate that coexpression of TREK-1 with TREK-1 variants alters TREK-1 expression and suppresses channel function. Each variant affected TREK-1 in a disparate manner. In HEK293T cells coexpressing TREK-1 and each variant, TREK-1 membrane expression was diminished with compartmentalization inside the cell. When expressed alone, individual variants displayed channel properties that were significantly decreased compared to full-length TREK-1. In coexpression studies using patch clamp, basal TREK-1 currents were reduced by ∼64% (4.3 vs. 12.0 pA/pF) on average at 0 mV when coexpressed with each variant. TREK-1 currents that were activated by intracellular acidosis were reduced an average of ∼77% (21.4 vs. 94.5 pA/pF) at 0 mV when cells were transfected with TREK-1 and any one of the splice variants. These data correlate the presence of TREK-1 variants to reduced TREK-1 activity, suggesting a pathological role for TREK-1 variants in preterm labor. PMID:26400398

  20. Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

    Directory of Open Access Journals (Sweden)

    Tanja Dodenhof

    Full Text Available Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.

  1. Analysis of Maxi-K alpha subunit splice variants in human myometrium

    Directory of Open Access Journals (Sweden)

    Morrison John J

    2004-09-01

    Full Text Available Abstract Background Large-conductance, calcium-activated potassium (Maxi-K channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP, and at Caesarean section, at elective (pregnant not-in-labour, PNL and intrapartum (pregnant in-labour, PL procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at

  2. Registered report: androgen receptor splice variants determine taxane sensitivity in prostate cancer

    Directory of Open Access Journals (Sweden)

    Xiaochuan Shan

    2015-09-01

    Full Text Available The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from “Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer” by Thadani-Mulero and colleagues (2014 published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR, and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

  3. Registered report: androgen receptor splice variants determine taxane sensitivity in prostate cancer.

    Science.gov (United States)

    Shan, Xiaochuan; Danet-Desnoyers, Gwenn; Fung, Juan José; Kosaka, Alan H; Tan, Fraser; Perfito, Nicole; Lomax, Joelle; Iorns, Elizabeth

    2015-01-01

    The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

  4. Transcriptional activity of human brain estrogen receptor-α splice variants: evidence for cell type-specific regulation

    NARCIS (Netherlands)

    Ishunina, T. A.; Sluiter, A. A.; Swaab, D. F.; Verwer, R. W. H.

    2013-01-01

    Estrogen receptor α (ERα) isoforms with complex types of alternative splicing are naturally present in the human brain and may affect canonical receptor signaling. In the present study we investigated transcriptional activity of common ERα splice variants from this group with different molecular

  5. Evaluation of a 5-tier scheme proposed for classification of sequence variants using bioinformatic and splicing assay data

    DEFF Research Database (Denmark)

    Walker, Logan C; Whiley, Phillip J; Houdayer, Claude

    2013-01-01

    Splicing assays are commonly undertaken in the clinical setting to assess the clinical relevance of sequence variants in disease predisposition genes. A 5-tier classification system incorporating both bioinformatic and splicing assay information was previously proposed as a method to provide...

  6. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Oncogenicity and Selective Inhibition of ERG Splicing Variants in Prostate Cancer

    Science.gov (United States)

    2012-03-01

    other oncogenes [32]. Indeed, ERG- expressing cells, but not controls, stained positive for senescence biomarker ( beta )- Galactosidase (Figure 9C). None...prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products , a group of related native ERG isoforms is also highly over-expressed in...variants of the normal ERG gene product have been described, arising from a combination of alternative splicing, polyadenilation and transcriptional

  8. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

    2016-08-05

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  9. Cancer-associated splicing variant of tumor suppressor AIMP2/p38: pathological implication in tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Jin Woo Choi

    2011-03-01

    Full Text Available Although ARS-interacting multifunctional protein 2 (AIMP2, also named as MSC p38 was first found as a component for a macromolecular tRNA synthetase complex, it was recently discovered to dissociate from the complex and work as a potent tumor suppressor. Upon DNA damage, AIMP2 promotes apoptosis through the protective interaction with p53. However, it was not demonstrated whether AIMP2 was indeed pathologically linked to human cancer. In this work, we found that a splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2 is highly expressed by alternative splicing in human lung cancer cells and patient's tissues. AIMP2-DX2 compromised pro-apoptotic activity of normal AIMP2 through the competitive binding to p53. The cells with higher level of AIMP2-DX2 showed higher propensity to form anchorage-independent colonies and increased resistance to cell death. Mice constitutively expressing this variant showed increased susceptibility to carcinogen-induced lung tumorigenesis. The expression ratio of AIMP2-DX2 to normal AIMP2 was increased according to lung cancer stage and showed a positive correlation with the survival of patients. Thus, this work identified an oncogenic splicing variant of a tumor suppressor, AIMP2/p38, and suggests its potential for anti-cancer target.

  10. Clinical Relevance of Androgen Receptor Splice Variants in Castration-Resistant Prostate Cancer.

    Science.gov (United States)

    Maughan, Benjamin L; Antonarakis, Emmanuel S

    2015-12-01

    Metastatic castration-resistant prostate cancer (mCRPC) currently benefits from a wealth of treatment options, yet still remains lethal in the vast majority of patients. It is becoming increasingly understood that this disease entity continues to evolve over time, acquiring additional and diverse resistance mechanisms with each subsequent therapy used. This dynamic relationship between treatment pressure and disease resistance can be challenging for the managing clinician. The recent discovery of alternate splice variants of the androgen receptor (AR) is one potential mechanism of escape in mCRPC, and recognizing this resistance mechanism might be important for optimal treatment selection for our patients. AR-V7 appears to be the most relevant AR splice variant, and early clinical data suggest that it is a negative prognostic marker in mCRPC. Emerging evidence also suggests that detection of AR-V7 may be associated with resistance to novel hormonal therapy (abiraterone and enzalutamide) but may be compatible with sensitivity to taxane chemotherapy (docetaxel and cabazitaxel). Adding to this complexity is the observation that AR-V7 is a dynamic marker whose status may change across time and depending on selective pressures induced by different therapies. Finally, it is possible that AR-V7 may represent a therapeutic target in mCRPC if drugs can be designed that degrade or inhibit AR splice variants or block their transcriptional activity. Several such agents (including galeterone, EPI-506, and bromodomain/BET inhibitors) are now in clinical development.

  11. An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

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    Kevin C Miranda

    2010-03-01

    Full Text Available The vacuolar-type H(+-ATPase (V-ATPase is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

  12. Elevated Oestrogen Receptor Splice Variant ERαΔ5 Expression in Tumour-adjacent Hormone-responsive Tissue

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    Pierre L. Martin-Hirsch

    2010-11-01

    Full Text Available Susceptibility to prostate or endometrial cancer is linked with obesity, a state of oestrogen excess. Oestrogen receptor (ER splice variants may be responsible for the tissue-level of ER activity. Such micro-environmental regulation may modulate cancer initiation and/or progression mechanisms. Real-time reverse transcriptase (RT polymerase chain reaction (PCR was used to quantitatively assess the levels of four ER splice variants (ERαΔ3, ERαΔ5, ERβ2 and ERβ5, plus the full-length parent isoforms ERα and ERβ1, in high-risk [tumour-adjacent prostate (n = 10 or endometrial cancer (n = 9] vs. low-risk [benign prostate (n = 12 or endometrium (n = 9], as well as a comparison of UK (n = 12 vs. Indian (n = 15 benign prostate. All three tissue groups expressed the ER splice variants at similar levels, apart from ERαΔ5. This splice variant was markedly raised in all of the tumour-adjacent prostate samples compared to benign tissues. Immunofluorescence analysis for ERβ2 in prostate tissue demonstrated that such splice variants are present in comparable, if not greater, amounts as the parent full-length isoform. This small pilot study demonstrates the ubiquitous nature of ER splice variants in these tissue sites and suggests that ERαΔ5 may be involved in progression of prostate adenocarcinoma.

  13. Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

    Science.gov (United States)

    Grinnell, Steven G; Ansonoff, Michael; Marrone, Gina F; Lu, Zhigang; Narayan, Ankita; Xu, Jin; Rossi, Grace; Majumdar, Susruta; Pan, Ying-Xian; Bassoni, Daniel L; Pintar, John; Pasternak, Gavril W

    2016-10-01

    Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors. © 2016 Wiley Periodicals, Inc.

  14. Study of USH1 splicing variants through minigenes and transcript analysis from nasal epithelial cells.

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    María José Aparisi

    Full Text Available Usher syndrome type I (USH1 is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient's tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient's tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A, three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG and one PCDH15 (c.3717+2dupTT variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects.

  15. Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5 and altered Axin-1 expression during tumour progression

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    Reich Jens G

    2006-06-01

    Full Text Available Abstract Background Splicing processes might play a major role in carcinogenesis and tumour progression. The Wnt pathway is of crucial relevance for cancer progression. Therefore we focussed on the Wnt/β-catenin signalling pathway in order to validate the expression of sequences predicted as alternatively spliced by bioinformatic methods. Splice variants of its key molecules were selected, which may be critical components for the understanding of colorectal tumour progression and may have the potential to act as biological markers. For some of the Wnt pathway genes the existence of splice variants was either proposed (e.g. β-Catenin and CTNNB1 or described only in non-colon tissues (e.g. GSK3β or hitherto not published (e.g. LRP5. Results Both splice variants – normal and alternative form – of all selected Wnt pathway components were found to be expressed in cell lines as well as in samples derived from tumour, normal and healthy tissues. All splice positions corresponded totally with the bioinformatical prediction as shown by sequencing. Two hitherto not described alternative splice forms (CTNNB1 and LRP5 were detected. Although the underlying EST data used for the bioinformatic analysis suggested a tumour-specific expression neither a qualitative nor a significant quantitative difference between the expression in tumour and healthy tissues was detected. Axin-1 expression was reduced in later stages and in samples from carcinomas forming distant metastases. Conclusion We were first to describe that splice forms of crucial genes of the Wnt-pathway are expressed in human colorectal tissue. Newly described splicefoms were found for β-Catenin, LRP5, GSK3β, Axin-1 and CtBP1. However, the predicted cancer specificity suggested by the origin of the underlying ESTs was neither qualitatively nor significant quantitatively confirmed. That let us to conclude that EST sequence data can give adequate hints for the existence of alternative splicing

  16. The hTERTα Splice Variant is a Dominant Negative Inhibitor of Telomerase Activity

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    Lorel M. Colgin

    2000-09-01

    Full Text Available The telomerase catalytic subunit (hTERT is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a “deletion” variant (hTERTα that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTα inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity.

  17. A splice-site variant in FLVCR1 produces retinitis pigmentosa without posterior column ataxia.

    Science.gov (United States)

    Yusuf, Imran H; Shanks, Morag E; Clouston, Penny; MacLaren, Robert E

    2017-12-01

    FLVCR1 (feline leukemia virus subgroup c receptor 1) is a transmembrane protein involved in the trafficking of intracellular heme. Homozygous variants in FLVCR1 have been described in association with a clinical syndrome of posterior column ataxia with retinitis pigmentosa (PCARP). Here, we describe a patient with non-syndromic retinitis pigmentosa homozygous for a splice-site variant in FLVCR1 (c.1092 + 5G>A) without evidence of posterior column ataxia or cerebellar degeneration. We suggest an association between intronic splice-site variants in FLVCR1 and the absence of posterior column degeneration and suggest a hypothesis to explain this observation. Should this association be proven, it would provide valuable prognostic information for patients. Retinal degeneration appears to be the sole clinical manifestation of this FLVCR1 variant; gene therapy approaches using an adeno-associated viral vector with sub-retinal delivery may therefore represent a therapeutic approach to halting retinal degeneration in this patient group.

  18. Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants.

    Science.gov (United States)

    Pagenstecher, Constanze; Wehner, Maria; Friedl, Waltraut; Rahner, Nils; Aretz, Stefan; Friedrichs, Nicolaus; Sengteller, Marlies; Henn, Wolfram; Buettner, Reinhard; Propping, Peter; Mangold, Elisabeth

    2006-03-01

    Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G > C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G > C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G > A and MLH1,c.677G > A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A > G, MLH1,c.588+5G > A, MLH1,c.790+4A > G and MLH1,c.1984A > C. In contrast, five missense mutations (MSH2,c.4G > A, MSH2,c.2123T > A, MLH1,c.464T > G, MLH1,c.875T > C and MLH1,c.2210A > T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level.

  19. Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants.

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    Michel Fausther

    Full Text Available Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and

  20. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Sun, Shiqin [College of Pharmacy, Harbin Medical University-Daqing, Daqing, Heilongjiang 163319 (China); Chen, Xiangmei, E-mail: xm_chen6176@bjmu.edu.cn [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Lu, Fengmin [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-04-25

    Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

  1. Splice variants of enigma homolog, differentially expressed during heart development, promote or prevent hypertrophy.

    Science.gov (United States)

    Yamazaki, Tomoko; Wälchli, Sébastien; Fujita, Toshitsugu; Ryser, Stephan; Hoshijima, Masahiko; Schlegel, Werner; Kuroda, Shun'ichi; Maturana, Andrés D

    2010-06-01

    Proteins with a PDZ (for PSD-95, DLG, ZO-1) and one to three LIM (for Lin11, Isl-1, Mec-3) domains are scaffolding sarcomeric and cytoskeletal elements that form structured muscle fibres and provide for the link to intracellular signalling by selectively associating protein kinases, ion channels, and transcription factors with the mechanical stress-strain sensors. Enigma homolog (ENH) is a PDZ-LIM protein with four splice variants: ENH1 with an N-terminal PDZ domain and three C-terminal LIM domains and ENH2, ENH3, and ENH4 without LIM domains. We addressed the functional role of ENH alternative splicing. We studied the expression of the four ENH isoforms in the heart during development and in a mouse model of heart hypertrophy. All four isoforms are expressed in the heart but the pattern of expression is clearly different between embryonic, neonatal, and adult stages. ENH1 appears as the embryonic isoform, whereas ENH2, ENH3, and ENH4 are predominant in adult heart. Moreover, alternative splicing of ENH was changed following induction of heart hypertrophy, producing an ENH isoform pattern similar to that of neonatal heart. Next, we tested a possible causal role of ENH1 and ENH4 in the development of cardiac hypertrophy. When overexpressed in rat neonatal cardiomyocytes, ENH1 promoted the expression of hypertrophy markers and increased cell volume, whereas, on the contrary, ENH4 overexpression prevented these changes. Antagonistic splice variants of ENH may play a central role in the adaptive changes of the link between mechanical stress-sensing and signalling occurring during embryonic development and/or heart hypertrophy.

  2. Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

    Directory of Open Access Journals (Sweden)

    Gomez-Roman Javier

    2010-06-01

    Full Text Available Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

  3. Dominant Negative Effects of a Non-conducting TREK1 Splice Variant Expressed in Brain*

    OpenAIRE

    Veale, Emma L; Rees, Kathryn A.; Mathie, Alistair; Trapp, Stefan

    2010-01-01

    Two-pore domain potassium (K2P) channels modulate neuronal excitability throughout the entire CNS. The stretch-activated channel TREK1 (K2P2.1) is expressed widely in brain and has been linked to depression, neuroprotection, pain perception, and epilepsy. Little, however, is known about the regulation of TREK1 expression on the transcriptional and translational level or about its trafficking to the plasma membrane. Here we have used PCR techniques to identify a splice variant of TREK1 express...

  4. Expression of a Splice Variant of CYP26B1 in Betel Quid-Related Oral Cancer

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    Ping-Ho Chen

    2014-01-01

    Full Text Available Betel quid (BQ is a psychostimulant, an addictive substance, and a group 1 carcinogen that exhibits the potential to induce adverse health effects. Approximately, 600 million users chew a variety of BQ. Areca nut (AN is a necessary ingredient in BQ products. Arecoline is the primary alkaloid in the AN and can be metabolized through the cytochrome P450 (CYP superfamily by inducing reactive oxygen species (ROS production. Full-length CYP26B1 is related to the development of oral pharyngeal cancers. We investigated whether a splice variant of CYP26B1 is associated with the occurrence of ROS related oral and pharyngeal cancer. Cytotoxicity assays were used to measure the effects of arecoline on cell viability in a dose-dependent manner. In vitro and in vivo studies were conducted to evaluate the expression of the CYP26B1 splice variant. The CYP26B1 splice variant exhibited lower expression than did full-length CYP26B1 in the human gingival fibroblast-1 and Ca9-22 cell models. Increased expression of the CYP26B1 splice variant was observed in human oral cancer tissue compared with adjacent normal tissue, and increased expression was observed in patients at a late tumor stage. Our results suggested that the CYP26B1 splice variant is associated with the occurrence of BQ-related oral cancer.

  5. A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

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    Maria Valeria Esposito

    2016-12-01

    Full Text Available About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.

  6. Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine.

    Science.gov (United States)

    Verzillo, Vittorio; Madia, Priyanka A; Liu, Nai-Jiang; Chakrabarti, Sumita; Gintzler, Alan R

    2014-09-01

    The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction. © 2014 International Society for Neurochemistry.

  7. The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability.

    Science.gov (United States)

    Yang, Yun-Chiu; Chen, Pei-Ni; Wang, Siou-Yu; Liao, Chen-Yi; Lin, Yu-Ying; Sun, Shih-Rhong; Chiu, Chun-Ling; Hsieh, Yih-Shou; Shieh, Jia-Ching; Chang, Jinghua Tsai

    2015-07-01

    Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.

  8. New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes.

    Science.gov (United States)

    Okumoto, Kanji; Ono, Tatsuaki; Toyama, Ryusuke; Shimomura, Ayako; Nagata, Aiko; Fujiki, Yukio

    2017-12-08

    Microtubule-dependent long-distance movement of peroxisomes occurs in mammalian cells. However, its molecular mechanisms remain undefined. In this study, we identified three distinct splicing variants of human mitochondrial Rho GTPase-1 (Miro1), each containing amino acid sequence insertions 1 (named Miro1-var2), 2 (Miro1-var3), and both 1 and 2 (Miro1-var4), respectively, at upstream of the transmembrane domain. Miro1-var4 and Miro1-var2 are localized to peroxisomes in a manner dependent on the insertion 1 that is recognized by the cytosolic receptor Pex19p. Exogenous expression of Miro1-var4 induces accumulation of peroxisomes at the cell periphery and augments long-range movement of peroxisomes along microtubules. Depletion of all Miro1 variants by knocking down MIRO1 suppresses the long-distance movement of peroxisomes. Such abrogated movement is restored by reexpression of peroxisomal Miro1 variants. Collectively, our findings identify for the first time peroxisome-localized Miro1 variants as adapter proteins that link peroxisomes to the microtubule-dependent transport complexes including TRAK2 in the intracellular translocation of peroxisomes in mammalian cells. © 2018 Okumoto et al.

  9. Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor

    DEFF Research Database (Denmark)

    Krishnaveni, Manda S; Hansen, Jakob Lerche; Seeger, Werner

    2006-01-01

    , but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand...

  10. Dominant negative effects of a non-conducting TREK1 splice variant expressed in brain.

    Science.gov (United States)

    Veale, Emma L; Rees, Kathryn A; Mathie, Alistair; Trapp, Stefan

    2010-09-17

    Two-pore domain potassium (K(2P)) channels modulate neuronal excitability throughout the entire CNS. The stretch-activated channel TREK1 (K(2P)2.1) is expressed widely in brain and has been linked to depression, neuroprotection, pain perception, and epilepsy. Little, however, is known about the regulation of TREK1 expression on the transcriptional and translational level or about its trafficking to the plasma membrane. Here we have used PCR techniques to identify a splice variant of TREK1 expressed in the brain, which encodes a heavily truncated TREK1 protein retaining a single transmembrane domain. Functional expression of this splice variant TREK1ΔEx4 in tsA201 cells in the presence or absence of wild type TREK1 revealed that TREK1ΔEx4 has no channel activity itself but reduced TREK1 whole cell current amplitude. Confocal analysis of the expression of fluorescently tagged TREK1 variants revealed that TREK1ΔEx4 is translated, but it is retained in the intracellular compartment. Additionally, TREK1ΔEx4 reduced the level of TREK1 expression in the plasma membrane. Long and short forms of TREK1 derived from alternative translation initiation are differentially affected by TREK1ΔEx4, with the short form (lacking the first 41 amino acids at its N terminus) unaffected. This differential regulatory role of TREK1ΔEx4 will alter the functional profile of TREK1 current in neurons where they are expressed. These results indicate that the N-terminal domain and first transmembrane domain of TREK1 are likely to be important for channel dimerization and trafficking to the plasma membrane.

  11. Insights into the structural perturbations of spliced variants of CD44: a modeling and simulation approach.

    Science.gov (United States)

    Patel, Shanaya; Shaikh, Faraz; Devaraji, Vinod; Radadiya, Ashish; Shah, Kanisha; Shah, Anamik; Rawal, Rakesh

    2017-02-01

    Transient interactions between cancer stem cells and components of the tumor microenvironment initiate various signaling pathways crucial for carcinogenesis. Predominant hyaluronan (HA) receptor, CD44 is structurally and functionally one of the most variable cell surface receptors having the potential to generate a diverse repertory of CD44 isoforms by alternative splicing of variant exons and post-translational modifications. A structurally distinctive variant of CD44, CD44v10, has an inevitable role in malignant progression, invasion, and metastasis. This can be attributed to the binding of HA with CD44v10, which demonstrates a completely different behavioral pattern as compared to the other spliced variants of CD44 molecule. Absence of a comprehensively predicted crystal structure of human CD44s and CD44v10 is an impediment in understanding the resultant structural alterations caused by the binding of HA. Thus, in this study, we aim to predict the CD44s and CD44v10 structures to their closest native confirmation and study the HA binding-induced structural perturbations using homology modeling, molecular docking, and MD simulation approach. The results depicted that modeled 3D structures of CD44s and CD44v10 isoforms were found to be stable throughout MD simulations; however, a substantial decrease was observed in the binding affinity of HA with CD44v10 (-5.355 kcal/mol) as compared to CD44s. Furthermore, loss and gain of several H-bonds and hydrophobic interactions in CD44v10-HA complex during the simulation process not only elucidated the reason for decreased binding affinity for HA but also prompted toward the plausible role of HA-induced structural perturbations in occurrence and progression of carcinogenesis.

  12. Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

    Science.gov (United States)

    Schroeter, Annett; Walzik, Stefan; Blechschmidt, Steve; Haufe, Volker; Benndorf, Klaus; Zimmer, Thomas

    2010-07-01

    Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na(v)1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na(v)1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na(v)1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na(v)1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed. Copyright 2010 Elsevier Ltd. All rights reserved.

  13. OCA2 splice site variant in German Spitz dogs with oculocutaneous albinism

    Science.gov (United States)

    Caduff, Madleina; Bauer, Anina; Jagannathan, Vidhya

    2017-01-01

    We investigated a German Spitz family where the mating of a black male to a white female had yielded three puppies with an unexpected light brown coat color, lightly pigmented lips and noses, and blue eyes. Combined linkage and homozygosity analysis based on a fully penetrant monogenic autosomal recessive mode of inheritance identified a critical interval of 15 Mb on chromosome 3. We obtained whole genome sequence data from one affected dog, three wolves, and 188 control dogs. Filtering for private variants revealed a single variant with predicted high impact in the critical interval in LOC100855460 (XM_005618224.1:c.377+2T>G LT844587.1:c.-45+2T>G). The variant perfectly co-segregated with the phenotype in the family. We genotyped 181 control dogs with normal pigmentation from diverse breeds including 22 unrelated German Spitz dogs, which were all homozygous wildtype. Comparative sequence analyses revealed that LOC100855460 actually represents the 5’-end of the canine OCA2 gene. The CanFam 3.1 reference genome assembly is incorrect and separates the first two exons from the remaining exons of the OCA2 gene. We amplified a canine OCA2 cDNA fragment by RT-PCR and determined the correct full-length mRNA sequence (LT844587.1). Variants in the OCA2 gene cause oculocutaneous albinism type 2 (OCA2) in humans, pink-eyed dilution in mice, and similar phenotypes in corn snakes, medaka and Mexican cave tetra fish. We therefore conclude that the observed oculocutaneous albinism in German Spitz is most likely caused by the identified variant in the 5’-splice site of the first intron of the canine OCA2 gene. PMID:28973042

  14. OCA2 splice site variant in German Spitz dogs with oculocutaneous albinism.

    Directory of Open Access Journals (Sweden)

    Madleina Caduff

    Full Text Available We investigated a German Spitz family where the mating of a black male to a white female had yielded three puppies with an unexpected light brown coat color, lightly pigmented lips and noses, and blue eyes. Combined linkage and homozygosity analysis based on a fully penetrant monogenic autosomal recessive mode of inheritance identified a critical interval of 15 Mb on chromosome 3. We obtained whole genome sequence data from one affected dog, three wolves, and 188 control dogs. Filtering for private variants revealed a single variant with predicted high impact in the critical interval in LOC100855460 (XM_005618224.1:c.377+2T>G LT844587.1:c.-45+2T>G. The variant perfectly co-segregated with the phenotype in the family. We genotyped 181 control dogs with normal pigmentation from diverse breeds including 22 unrelated German Spitz dogs, which were all homozygous wildtype. Comparative sequence analyses revealed that LOC100855460 actually represents the 5'-end of the canine OCA2 gene. The CanFam 3.1 reference genome assembly is incorrect and separates the first two exons from the remaining exons of the OCA2 gene. We amplified a canine OCA2 cDNA fragment by RT-PCR and determined the correct full-length mRNA sequence (LT844587.1. Variants in the OCA2 gene cause oculocutaneous albinism type 2 (OCA2 in humans, pink-eyed dilution in mice, and similar phenotypes in corn snakes, medaka and Mexican cave tetra fish. We therefore conclude that the observed oculocutaneous albinism in German Spitz is most likely caused by the identified variant in the 5'-splice site of the first intron of the canine OCA2 gene.

  15. OCA2 splice site variant in German Spitz dogs with oculocutaneous albinism.

    Science.gov (United States)

    Caduff, Madleina; Bauer, Anina; Jagannathan, Vidhya; Leeb, Tosso

    2017-01-01

    We investigated a German Spitz family where the mating of a black male to a white female had yielded three puppies with an unexpected light brown coat color, lightly pigmented lips and noses, and blue eyes. Combined linkage and homozygosity analysis based on a fully penetrant monogenic autosomal recessive mode of inheritance identified a critical interval of 15 Mb on chromosome 3. We obtained whole genome sequence data from one affected dog, three wolves, and 188 control dogs. Filtering for private variants revealed a single variant with predicted high impact in the critical interval in LOC100855460 (XM_005618224.1:c.377+2T>G LT844587.1:c.-45+2T>G). The variant perfectly co-segregated with the phenotype in the family. We genotyped 181 control dogs with normal pigmentation from diverse breeds including 22 unrelated German Spitz dogs, which were all homozygous wildtype. Comparative sequence analyses revealed that LOC100855460 actually represents the 5'-end of the canine OCA2 gene. The CanFam 3.1 reference genome assembly is incorrect and separates the first two exons from the remaining exons of the OCA2 gene. We amplified a canine OCA2 cDNA fragment by RT-PCR and determined the correct full-length mRNA sequence (LT844587.1). Variants in the OCA2 gene cause oculocutaneous albinism type 2 (OCA2) in humans, pink-eyed dilution in mice, and similar phenotypes in corn snakes, medaka and Mexican cave tetra fish. We therefore conclude that the observed oculocutaneous albinism in German Spitz is most likely caused by the identified variant in the 5'-splice site of the first intron of the canine OCA2 gene.

  16. Expression of activation-induced cytidine deaminase splicing variants in patients with ankylosing spondylitis.

    Science.gov (United States)

    Kim, Ji-Young; Yoon, Hee-Kyung; Song, Seung Taek; Park, Seok-Rae; Shim, Seung-Cheol

    2017-12-01

    To investigate the expression patterns of activation-induced cytidine deaminase (AID) variants in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and examine their clinical implications, we isolated PBMCs from healthy controls (HC, n = 33) and patients with AS (n = 62), and measured mRNA expression of AID variants and translesion synthesis (TLS) polymerases using quantitative real-time polymerase chain reaction. The proportion of patients with AS in whom AID splicing variant (sv) 2 was expressed was significantly higher than that of HC (p = .031). 80.7% of AS patients were treated with tumour necrosis factor inhibitor (TNFi). Significantly higher proportion of the TNFi-treated group expressed sv2 compared to the TNF-naïve group (p = .037). And we compared the level of AID variants expression between the TNFi-treated group and the TNF-naïve group. The expression levels of AID full-length (FL) and sv1 were significantly lower in the TNFi-treated group than the TNF-naïve group (FL: p = .002, sv1: p = .045). In addition, we investigated mRNA expression levels of translesion synthesis (TLS) polymerases in PBMCs from patients with AS and HC. The expression level of TLS pol ι was significantly lower in patients with AS than in HC (p = .007). In conclusion, AS patients expressed significantly higher levels of sv2 than HC. TNFi treatment restored the gene expression of the AID variants (FL, sv1, and sv2) in patients with AS. A clear understanding of the underlying cellular and molecular mechanisms will help to identify the pathogenesis of AS better and to develop novel therapeutic targets.

  17. In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features.

    Science.gov (United States)

    Ibáñez-Costa, Alejandro; Gahete, Manuel D; Rivero-Cortés, Esther; Rincón-Fernández, David; Nelson, Richard; Beltrán, Manuel; de la Riva, Andrés; Japón, Miguel A; Venegas-Moreno, Eva; Gálvez, Ma Ángeles; García-Arnés, Juan A; Soto-Moreno, Alfonso; Morgan, Jennifer; Tsomaia, Natia; Culler, Michael D; Dieguez, Carlos; Castaño, Justo P; Luque, Raúl M

    2015-03-04

    Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas compared with normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24-72 h) increased GH and ACTH secretion, Ca(2+) and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors.

  18. In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features

    Science.gov (United States)

    Ibáñez-Costa, Alejandro; Gahete, Manuel D.; Rivero-Cortés, Esther; Rincón-Fernández, David; Nelson, Richard; Beltrán, Manuel; de la Riva, Andrés; Japón, Miguel A.; Venegas-Moreno, Eva; Gálvez, Ma Ángeles; García-Arnés, Juan A.; Soto-Moreno, Alfonso; Morgan, Jennifer; Tsomaia, Natia; Culler, Michael D.; Dieguez, Carlos; Castaño, Justo P.; Luque, Raúl M.

    2015-01-01

    Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas comparedwith normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24–72 h) increased GH and ACTH secretion, Ca2+ and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors. PMID:25737012

  19. Differential expression of splicing variants of the human caldesmon gene (CALD1) in glioma neovascularization versus normal brain microvasculature

    NARCIS (Netherlands)

    P.P. Zheng (Pingpin); A.M. Sieuwerts (Anieta); T.M. Luider (Theo); M.M. van der Weiden (Marcel); J.M. Kros (Johan); P.A.E. Sillevis Smitt (Peter)

    2004-01-01

    textabstractCaldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal

  20. Survivin 2α: a novel Survivin splice variant expressed in human malignancies

    Directory of Open Access Journals (Sweden)

    Honsey Laura E

    2005-03-01

    Full Text Available Abstract Background Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. Results In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2α. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2α is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2α attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2α suggests a physical interaction with survivin. Conclusion We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.

  1. Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

    DEFF Research Database (Denmark)

    Williamson, M; Hauser, F; Grimmelikhuijzen, C J

    2000-01-01

    of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme......, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined...... the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in mammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead...

  2. ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T.

    Science.gov (United States)

    Menotta, Michele; Biagiotti, Sara; Spapperi, Chiara; Orazi, Sara; Rossi, Luigia; Chessa, Luciana; Leuzzi, Vincenzo; D'Agnano, Daniela; Soresina, Annarosa; Micheli, Roberto; Magnani, Mauro

    2017-07-05

    Ataxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1. In the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new "ATMdexa1 variants" originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients' blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment. For the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration.

  3. Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

    Science.gov (United States)

    Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

    2008-06-01

    Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

  4. Whole-genome sequencing of two probands with hereditary spastic paraplegia reveals novel splice-donor region variant and known pathogenic variant in SPG11.

    Science.gov (United States)

    Yu, Allen Chi-Shing; Chan, Anne Yin-Yan; Au, Wing Chi; Shen, Yun; Chan, Ting Fung; Chan, Ho-Yin Edwin

    2016-11-01

    Hereditary spastic paraplegias (HSPs) are a group of heterogeneous neurodegenerative disorders, which are often presented with overlapping phenotypes such as progressive paraparesis and spasticity. To assist the diagnosis of HSP subtypes, next-generation sequencing is often used to provide supporting evidence. In this study, we report the case of two probands from the same family with HSP symptoms, including bilateral lower limb weakness, unsteady gait, cognitive decline, dysarthria, and slurring of speech since the age of 14. Subsequent whole-genome sequencing revealed that the patients are compound heterozygous for variants in the SPG11 gene, including the paternally inherited c.6856C>T (p.Arg2286*) variant and the novel maternally inherited c.2316+5G>A splice-donor region variant. Variants in SPG11 are the common cause of autosomal recessive spastic paraplegia type 11. According to the ClinVar database, there are already 101 reported pathogenic variants in SPG11 that are associated with HSPs. To our knowledge, this is the first report of SPG11 variants in our local population. The novel splice variant identified in this study enriches the catalog of SPG11 variants, potentially leading to better genetic diagnosis of HSPs.

  5. Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia

    Science.gov (United States)

    Stasiak, Grazyna; Zaleska, Joanna; Kielbus, Michal; Czapinski, Jakub; Schunn, Matthias; Correa, Stephany C.; Glodkowska-Mrowka, Eliza; Sundaram, Reddy Chakkarappan; Jankowska-Lecka, Olga; Schlenk, Richard F.; Döhner, Hartmut; Döhner, Konstanze; Stepulak, Andrzej; Bullinger, Lars; Giannopoulos, Krzysztof

    2017-01-01

    Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients. PMID:29221119

  6. Targeting a Single Alternative Polyadenylation Site Coordinately Blocks Expression of Androgen Receptor mRNA Splice Variants in Prostate Cancer.

    Science.gov (United States)

    Van Etten, Jamie L; Nyquist, Michael; Li, Yingming; Yang, Rendong; Ho, Yeung; Johnson, Rachel; Ondigi, Olivia; Voytas, Daniel F; Henzler, Christine; Dehm, Scott M

    2017-10-01

    Prostate cancer is the second leading cause of male cancer deaths due to disease progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) splice variants including AR-V7 function as constitutively active transcription factors in CRPC cells, thereby promoting resistance to AR-targeted therapies. To date, there are no AR variant-specific treatments for CRPC. Here we report that the splicing of AR variants AR-V7 as well as AR-V1 and AR-V9 is regulated coordinately by a single polyadenylation signal in AR intron 3. Blocking this signal with morpholino technology or silencing of the polyadenylation factor CPSF1 caused a splice switch that inhibited expression of AR variants and blocked androgen-independent growth of CRPC cells. Our findings support the development of new therapies targeting the polyadenylation signal in AR intron 3 as a strategy to prevent expression of a broad array of AR variants in CRPC. Cancer Res; 77(19); 5228-35. ©2017 AACR. ©2017 American Association for Cancer Research.

  7. Cisplatin-induced haemolytic uraemic syndrome associated with a novel intronic mutation of CD46 treated with eculizumab.

    Science.gov (United States)

    Gilbert, Rodney D; Stanley, Louise K; Fowler, Darren J; Angus, Elizabeth M; Hardy, Steven A; Goodship, Timothy H

    2013-08-01

    A 2-year-old patient with a neuroblastoma developed haemolytic uraemic syndrome (HUS) following treatment with cisplatin and carboplatin. Following treatment with eculizumab, there was a substantial improvement in renal function with the recovery of the platelet count and the cessation of haemolysis. Subsequent investigations showed a novel, heterozygous CD46 splice site mutation with reduced peripheral blood neutrophil CD46 expression. Withdrawal of eculizumab was followed by the recurrence of disease activity, which resolved with re-introduction of therapy. Abnormal regulation of complement may be associated with other cases of cisplatin-induced HUS and treatment with eculizumab may be appropriate for other affected individuals.

  8. Existence of splicing variants in homologues of Theileria lestoquardi clone-5 gene's transcripts in Theileria annulata and Theileria parva.

    Science.gov (United States)

    Bakheit, Mohammed A; Ahmed, Jabbar S; Seitzer, Ulrike

    2008-12-01

    Clone 5 has been described as an immunogenic protein and was used to establish an ELISA for malignant theileriosis. Molecular characterization of the gene product revealed alternative splicing at the single intron resulting in two mRNA transcripts, translating into a long and a short protein form. Homologues of clone 5 exist in Theileria annulata and T. parva according to the available annotated GenBank sequences, showing however only the long protein forms in these parasites (GenBank accession numbers CAI73679, EAN33624). The present study aimed to determine whether two splice variants of homologues of clone 5 occur in T. annulata and T. parva.

  9. Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agents

    Science.gov (United States)

    Zhang, Guanyi; Liu, Xichun; Li, Jianzhuo; Ledet, Elisa; Alvarez, Xavier; Qi, Yanfeng; Fu, Xueqi; Sartor, Oliver; Dong, Yan; Zhang, Haitao

    2015-01-01

    Docetaxel-based chemotherapy is established as a first-line treatment and standard of care for patients with metastatic castration-resistant prostate cancer. However, half of the patients do not respond to treatment and those do respond eventually become refractory. A better understanding of the resistance mechanisms to taxane chemotherapy is both urgent and clinical significant, as taxanes (docetaxel and cabazitaxel) are being used in various clinical settings. Sustained signaling through the androgen receptor (AR) has been established as a hallmark of CRPC. Recently, splicing variants of AR (AR-Vs) that lack the ligand-binding domain (LBD) have been identified. These variants are constitutively active and drive prostate cancer growth in a castration-resistant manner. In taxane-resistant cell lines, we found the expression of a major variant, AR-V7, was upregulated. Furthermore, ectopic expression of two clinically relevant AR-Vs (AR-V7 and ARV567es), but not the full-length AR (AR-FL), reduced the sensitivities to taxanes in LNCaP cells. Treatment with taxanes inhibited the transcriptional activity of AR-FL, but not those of AR-Vs. This could be explained, at least in part, due to the inability of taxanes to block the nuclear translocation of AR-Vs. Through a series of deletion constructs, the microtubule-binding activity was mapped to the LBD of AR. Finally, taxane-induced cytoplasm sequestration of AR-FL was alleviated when AR-Vs were present. These findings provide evidence that constitutively active AR-Vs maintain the AR signaling axis by evading the inhibitory effects of microtubule-targeting agents, suggesting that these AR-Vs play a role in resistance to taxane chemotherapy. PMID:26160840

  10. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

    Full Text Available Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S. Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  11. Levels of estrogen receptor B splice variant (ERBΔ5 mRNA correlates with progesterone receptor in breast carcinomas

    Directory of Open Access Journals (Sweden)

    Mandušić Vesna

    2010-01-01

    Full Text Available It is well known that breast tumors which are estrogen positive ER(+ are more likely to respond to hormone therapy. However, a certain percentage of ER(+/PR(+ tumors do not respond to this therapy. Identification of the second estrogen receptor, named estrogen receptor beta (ERβ, as well as the existence of numerous isoforms/splice variants of both ERα and ERβ, suggests that a complex regulation of estrogen action exists. In this study, we analyzed the expression ratio of ERβ1 isoform and ERβΔ5 splice variant mRNAs, and its correlation with ER/PR status by quantitative RT-PCR and clinical and histopathological parameters. We found that the relative proportion of ERβΔ5 in the total ERβ1 transcript 'pool' inversely correlates with the PR level (p = -0,359, p< 0,003, Spearman. It may be that the ERβΔ5 variant modulates the ERα activity of downstream targets. In addition, we suggest that the determination of the expression profiles of ERα and ERβ isoforms and splice variants in the defined groups of patients are necessary for elucidating their involvement in endocrine resistance.

  12. Characterization of the 3' end of the mouse SERCA 3 gene and tissue distribution of mRNA spliced variants.

    Science.gov (United States)

    Ozog, A; Pouzet, B; Bobe, R; Lompré, A M

    1998-05-15

    The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) type 1 and 2 genes are alternatively spliced at their 3' end. We hypothesized that similar mechanism may occur for SERCA 3. Two spliced variants were identified by RNase protection analysis. We then isolated and sequenced the 3' end portion of the mouse SERCA 3 gene, and confirmed the presence of an alternative mRNA transcript by sequencing a cDNA fragment obtained by RT-PCR. Tissue distribution of the alternatively spliced mRNAs was studied by RT-PCR: SERCA 3b was the only isoform expressed in endothelial cells from aorta and heart and also was the major isoform in lung and kidney whereas SERCA 3a and 3b were coexpressed in trachea, intestine, thymus, spleen, and fetal liver.

  13. The oncogenic role of the spliced somatostatin receptor sst5TMD4 variant in prostate cancer.

    Science.gov (United States)

    Hormaechea-Agulla, Daniel; Jiménez-Vacas, Juan M; Gómez-Gómez, Enrique; L-López, Fernando; Carrasco-Valiente, Julia; Valero-Rosa, José; Moreno, María M; Sánchez-Sánchez, Rafael; Ortega-Salas, Rosa; Gracia-Navarro, Francisco; Culler, Michael D; Ibáñez-Costa, Alejandro; Gahete, Manuel D; Requena, María J; Castaño, Justo P; Luque, Raúl M

    2017-11-01

    sst5TMD4, a splice variant of the sst5 gene, is overexpressed and associated with aggressiveness in various endocrine-related tumors, but its presence, functional role, and mechanisms of actions in prostate cancer (PCa)-the most common cancer type in males-is completely unexplored. In this study, formalin-fixed, paraffin-embedded prostate pieces from patients with localized PCa, which included tumoral and nontumoral adjacent regions ( n = 45), fresh biopsies from patients with high-risk PCa ( n = 52), and healthy fresh prostates from cystoprostatectomies ( n = 14) were examined. In addition, PCa cell lines and xenograft models were used to determine the presence and functional role of sst5TMD4. Results demonstrated that sst5TMD4 is overexpressed (mRNA/protein) in PCa samples, and this is especially drastic in metastatic and/or high Gleason score tumor samples. Remarkably, sst5TMD4 expression was associated with an altered frequency of 2 single-nucleotide polymorphisms: rs197055 and rs12599155. In addition, PCa cell lines and xenograft models were used to demonstrate that sst5TMD4 overexpression increases cell proliferation and migration in PCa cells and induces larger tumors in nude mice, whereas its silencing decreased proliferation and migration. Remarkably, sst5TMD4 overexpression activated multiple intracellular pathways (ERK/JNK, MYC/MAX, WNT, retinoblastoma), altered oncogenes and tumor suppressor gene expression, and disrupted the normal response to somatostatin analogs in PCa cells. Altogether, we demonstrate that sst5TMD4 is overexpressed in PCa, especially in those patients with a worse prognosis, and plays an important pathophysiologic role in PCa, which suggesting its potential as a biomarker and/or therapeutic target.-Hormaechea-Agulla, D., Jiménez-Vacas, J. M., Gómez-Gómez, E., L.-López, F., Carrasco-Valiente, J., Valero-Rosa, J., Moreno, M. M., Sánchez-Sánchez, R., Ortega-Salas, R., Gracia-Navarro, F., Culler, M. D., Ibáñez-Costa, A., Gahete

  14. Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database.

    Science.gov (United States)

    Betsalel, Ofir T; Rosenberg, Efraim H; Almeida, Ligia S; Kleefstra, Tjitske; Schwartz, Charles E; Valayannopoulos, Vassili; Abdul-Rahman, Omar; Poplawski, Nicola; Vilarinho, Laura; Wolf, Philipp; den Dunnen, Johan T; Jakobs, Cornelis; Salomons, Gajja S

    2011-01-01

    The X-linked creatine transporter defect is caused by mutations in the SLC6A8 gene. Until now, 66 synonymous and intronic variants in SLC6A8 were detected in our laboratory. To gain more insight in the effect of the detected variants, we applied five free web-based splice-site analysis tools to 25 published variants that were stratified as (non-)disease causing. All were correctly predicted to have no effect (n=18) or to cause erroneous splicing (n=7), with the exception of a pathogenic de novo 24 bp intronic deletion. Second, 41 unclassified variants, including 28 novel, were subjected to analysis by these tools. At least four splice-site analysis tools predicted that three of the variants would affect splicing as the mutations disrupted the canonical splice site. Urinary creatine/creatinine and brain MRS confirmed creatine transporter deficiency in five patients (four families), including one female. Another variant was predicted to moderately affect splicing by all five tools. However, transient transfection of a minigene containing the variant in a partial SLC6A8 segment showed no splicing errors, and thus was finally classified as non-disease causing. This study shows that splice tools are useful for the characterization of the majority of variants, but also illustrates that the actual effect can be misclassified in rare occasions. Therefore, further laboratory studies should be considered before final conclusions on the disease-causing nature are drawn. To provide an accessible database, the 109 currently known SLC6A8 variants, including 35 novel ones, are included in a newly developed LOVD DNA variation database.

  15. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

    NARCIS (Netherlands)

    E. van der Wal (Erik); A.J. Bergsma (Atze); J. Pijnenburg (Joon); A.T. van der Ploeg (Ans); W.W.M.P. Pijnappel (Pim)

    2017-01-01

    textabstractThe most common variant causing Pompe disease is c.-32-13T>G (IVS1) in the acid α-glucosidase (GAA) gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult

  16. MAPT isoforms: differential transcriptional profiles related to 3R and 4R splice variants.

    Science.gov (United States)

    Chen, Shufen; Townsend, Kirk; Goldberg, Terry E; Davies, Peter; Conejero-Goldberg, Concepcion

    2010-01-01

    Tau aggregation in neurofibrillary tangles is a pathological hallmark in tauopathies including Alzheimer's disease (AD). The predominant aggregation of certain MAPT (tau gene) isoforms, either the 4-repeat (4R tau) or the 3-repeat (3R tau) isoform has been widely described in tauopathies. Alterations of the 4R tau to 3R tau ratio may be a key for tau-related neurodegeneration. To study the biological consequences in expression between tau splicing isoforms 4R and 3R, we analyzed the main neurobiological effects of inclusion of the repeat region coded by exon 10 in MAPT. We compared the transcriptional profiles of the 4R tau isoforms to 3R tau isoforms using whole-genome gene expression profiling microarrays using human neuroblastoma SH-SY5Y cell lines overexpressing either human 4R tau or 3R tau isoforms. We identified 68 transcripts that differed significantly (at p 4R and 3R isoforms as conditioned on a second variant, the so-called 2N inclusion. We extended these findings in a 2 × 2 ANOVA to examine interaction effects of these variants. Transcripts involved in embryonic development were downregulated when exon 10 was present, while transcripts related to outgrowth of neurites were generally upregulated. An important pathway implicated in AD also differed between the 3R and 4R cell lines, Wnt signaling. These studies demonstrate expression differences between MAPT isoforms 4R tau and 3R tau due to the inclusion/exclusion of the repeat region coded for by exon 10. Our data add to complex findings on the role of 3R/4R in normal and abnormal neuronal function and highlight several molecular mechanisms that might drive neurodegeneration, or perhaps, set the stage for it.

  17. Biological impact of the TSH-beta splice variant in health and disease

    Directory of Open Access Journals (Sweden)

    John R. Klein

    2014-04-01

    Full Text Available Thyroid stimulating hormone (TSH, a glycoprotein hormone composed of alpha and beta chains, is produced by thryrotrope cells of the anterior pituitary. Within the conventional endocrine loop, pituitary-derived TSH binds to receptors in the thyroid, resulting in the release of the thyroid hormones thyroxine (T4 and triiodothyronine (T3. T4 and T3 in turn regulate nearly every aspect of mammalian physiology, including basal metabolism, growth and development, and mood and cognition. Although TSH-beta has been known for years to be produced by cells of the immune system, the significance of that has remained largely unclear. Recently, a splice variant of TSH-beta (TSH-beta-v, which consists of a truncated but biologically functional portion of the native form of TSH-beta, was shown to be produced by bone marrow cells and peripheral blood leukocytes, particularly cells of the myeloid/monocyte lineage. In contrast, full-length native TSH-beta is minimally produced by cells of the immune system. The present article will describe the discovery of the TSH-beta-v and will discuss its potential role in immunity and autoimmunity, inflammation, and bone remodeling.

  18. Molecular characterization of a Theileria lestoquardi gene encoding for immunogenic protein splice variants.

    Science.gov (United States)

    Bakheit, M A; Scholzen, T; Ahmed, J S; Seitzer, U

    2006-12-01

    A Theileria lestoquardi schizont cDNA library was screened using sera collected from sheep recovering from a natural malignant theileriosis infection. An immunogenic clone (clone-5) was isolated and its full sequence was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR) technique. PCR experiments and sequencing demonstrated the presence of two transcript forms of the gene, resulting from splicing variation at the single intron found in the gene. Both gene products, clone-5 long and clone-5 short variants with calculated molecular weights of 99.9 and 72.7 kDa, respectively, were expressed in a T. lestoquardi-infected cell line. BLAST searches suggested the presence of homologues of the gene in both the Theileria parva and Theileria annulata genomes, with identities of 53 and 62% on the DNA level, respectively. The intron was preserved in size, sequence, and location within the gene in these parasites. Analysis of the subcellular localization of the clone-5 proteins showed a predominant parasite membrane association in T. lestoquardi-infected cells. Both recombinantly produced forms were found to be reactive with sera from infected animals. Bioinformatic analyses were employed to address the possible function of the gene products in the biology of T. lestoquardi.

  19. Novel kinin B₁ receptor splice variant and 5'UTR regulatory elements are responsible for cell specific B₁ receptor expression.

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    Faang Y Cheah

    Full Text Available The kinin B₁ receptor (B₁R is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood. We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV of human B₁R gene with a shorter 5'untranslated region. This novel SV is ≈35% less stable than the wild-type (WT transcript in lung adenocarcinoma cells (H2126, but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE -1020 to -766 bp in H2126; positive response element (PRE -766 to -410 bp in 16HBE; -410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells were found. These findings reveal complex regulation of B₁R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.

  20. Exploration of Potential Roles of a New LOXL2 Splicing Variant Using Network Knowledge in Esophageal Squamous Cell Carcinoma

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    Bing-Li Wu

    2014-01-01

    Full Text Available LOXL2 (lysyl oxidase-like 2, an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC. A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs, following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.

  1. Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinshang, E-mail: sanmaosound@163.com; Zhao, Heng, E-mail: hengzhao2000@gmail.com; Chen, Yeyu, E-mail: cyyleaf@126.com; Luo, Huiying, E-mail: luohuiying@caas.cn; Yao, Bin, E-mail: binyao@caas.cn

    2014-10-24

    Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.

  2. Clinical Isolates of Measles Virus Use CD46 as a Cellular Receptor†

    Science.gov (United States)

    Manchester, Marianne; Eto, Danelle S.; Valsamakis, Alexandra; Liton, Paloma B.; Fernandez-Muñoz, Rafael; Rota, Paul A.; Bellini, William J.; Forthal, Donald N.; Oldstone, Michael B. A.

    2000-01-01

    Laboratory strains of measles viruses (MV), such as Edmonston and Halle, use the complement regulatory protein CD46 as a cell surface receptor. The receptor usage of clinical isolates of MV, however, remains unclear. Receptor usage by primary patient isolates of MV was compared to isolates that had been passaged on a variety of tissue culture cell lines. All of the isolates could infect cells in a CD46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes versus fibroblasts). The results indicate that patient isolates that have not been adapted to tissue culture cell lines use CD46 as a receptor. In addition, passaging primary MV patient isolates in B95-8 cells selected variants that had alternate receptor usage compared to the original isolate. Thus, changes in receptor usage by MV are dependent upon the cell type used for isolation. Furthermore, our results confirm the relevance of the CD46 receptor to natural measles infection. PMID:10756008

  3. Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations

    DEFF Research Database (Denmark)

    Nielsen, Karsten Bork; Sørensen, Suzette; Cartegni, Luca

    2007-01-01

    The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro...... assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing...... a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3' splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE...

  4. Characterization of subcellular localization and stability of a splice variant of G alphai2

    Directory of Open Access Journals (Sweden)

    Wedegaertner Philip B

    2002-05-01

    Full Text Available Abstract Background Alternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sαi2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas αi2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal. Results This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sαi2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to αi2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and αi2(1–331, in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes. Finally, sαi2 displays a propensity to localize to potential aggresome-like structures. Conclusions Thus, instead of the novel C-terminus of sαi2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2.

  5. Genetic analysis of membrane cofactor protein (CD46) of the complement system in women with and without preeclamptic pregnancies.

    Science.gov (United States)

    Lokki, A Inkeri; Aalto-Viljakainen, Tia; Meri, Seppo; Laivuori, Hannele

    2015-01-01

    Preeclampsia is a common disorder of pregnancy characterized by endothelial dysfunction. It may be life-threatening for the mother and fetus in severe cases. Dysregulation of the complement system has been suggested to predispose women to preeclampsia. Complement is part of the innate and adaptive immune systems and potentially capable of causing inflammation and tissue damage. Membrane cofactor protein MCP (CD46) is among the potent complement regulators that have recently been linked to a severe form of preeclampsia with or without an underlying autoimmune phenotype. Mutations in CD46 predispose to thrombotic microangiopathy with endothelial cell dysfunction. The exome of CD46 were sequenced in 95 Finnish women with severe preeclampsia. Genetic variations discovered in the full exome were compared to those observed in 95 control women who did not develop preeclampsia. Because A304V (rs35366573) was associated with preeclampsia in one previous study, we sequenced the transmembrane region including the A304V variant and part of the cytoplasmic tail in 95 additional controls. We did not discover any association between A304V or other CD46 SNPs and preeclampsia. This study describes a carefully characterized cohort of severely preeclamptic Finnish women and found no potentially predisposing variants in CD46. However, it is possible that other genetic components of the complement system may affect the pathogenesis of severe preeclampsia and related diseases.

  6. Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Jansen-Olesen, Inger; Olesen, Jes

    2011-01-01

    The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned f...

  7. Calcitriol Modulates the CD46 Pathway in T Cells

    Science.gov (United States)

    Kickler, Karoline; Ni Choileain, Siobhan; Williams, Anna; Richards, Anna; Astier, Anne L.

    2012-01-01

    The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)2D3 or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS. PMID:23144765

  8. The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness.

    Science.gov (United States)

    Hormaechea-Agulla, Daniel; Gahete, Manuel D; Jiménez-Vacas, Juan M; Gómez-Gómez, Enrique; Ibáñez-Costa, Alejandro; L-López, Fernando; Rivero-Cortés, Esther; Sarmento-Cabral, André; Valero-Rosa, José; Carrasco-Valiente, Julia; Sánchez-Sánchez, Rafael; Ortega-Salas, Rosa; Moreno, María M; Tsomaia, Natia; Swanson, Steve M; Culler, Michael D; Requena, María J; Castaño, Justo P; Luque, Raúl M

    2017-08-29

    The Ghrelin-system is a complex, pleiotropic family composed of several peptides, including native-ghrelin and its In1-ghrelin splicing variant, and receptors (GHSR 1a/b), which are dysregulated in various endocrine-related tumors, where they associate to pathophysiological features, but the presence, functional role, and mechanisms of actions of In1-ghrelin splicing variant in prostate-cancer (PCa), is completely unexplored. Herein, we aimed to determine the presence of key ghrelin-system components (native-ghrelin, In1-ghrelin, GHSR1a/1b) and their potential pathophysiological role in prostate cancer (PCa). In1-ghrelin and native-ghrelin expression was evaluated by qPCR in prostate tissues from patients with high PCa-risk (n = 52; fresh-tumoral biopsies), and healthy-prostates (n = 12; from cystoprostatectomies) and correlated with clinical parameters using Spearman-test. In addition, In1-ghrelin and native-ghrelin was measured in plasma from an additional cohort of PCa-patients with different risk levels (n = 30) and control-healthy patients (n = 20). In vivo functional (proliferation/migration) and mechanistic (gene expression/signaling-pathways) assays were performed in PCa-cell lines in response to In1-ghrelin and native-ghrelin treatment, overexpression and/or silencing. Finally, tumor progression was monitored in nude-mice injected with PCa-cells overexpressing In1-ghrelin, native-ghrelin and empty vector (control). In1-ghrelin, but not native-ghrelin, was overexpressed in high-risk PCa-samples compared to normal-prostate (NP), and this expression correlated with that of PSA. Conversely, GHSR1a/1b expression was virtually absent. Remarkably, plasmatic In1-ghrelin, but not native-ghrelin, levels were also higher in PCa-patients compared to healthy-controls. Furthermore, In1-ghrelin treatment/overexpression, and to a much lesser extent native-ghrelin, increased aggressiveness features (cell-proliferation, migration and PSA secretion) of NP and PCa

  9. Cloning and Characterization of Spliced Variants of the Porcine G Protein Coupled Receptor 120

    Directory of Open Access Journals (Sweden)

    Tongxing Song

    2015-01-01

    Full Text Available The polyunsaturated fatty acids (PUFAs receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120 has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform was detected in spleen, while the transcript 2 (coding 310-aa isoform was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891 and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2 phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.

  10. The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

    Science.gov (United States)

    Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

    2004-09-01

    Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

  11. Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

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    Ganesh Ambigapathy

    Full Text Available Brain-derived neurotrophic factor (BDNF has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

  12. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  13. Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database

    NARCIS (Netherlands)

    Betsalel, O.T.; Rosenberg, E.H.; Almeida, L.S.; Kleefstra, T.; Schwartz, C.E.; Valayannopoulos, V.; Abdul-Rahman, O.; Poplawski, N.; Vilarinho, L.; Wolf, P.; Dunnen, J.T. den; Jakobs, C.; Salomons, G.S.

    2011-01-01

    The X-linked creatine transporter defect is caused by mutations in the SLC6A8 gene. Until now, 66 synonymous and intronic variants in SLC6A8 were detected in our laboratory. To gain more insight in the effect of the detected variants, we applied five free web-based splice-site analysis tools to 25

  14. DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

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    V. D. Cvetkova

    2016-01-01

    Full Text Available The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM are poorely understood.The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8, and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM. Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow

  15. Internal ribosome entry site-mediated translational regulation of ATF4 splice variant in mammalian unfolded protein response.

    Science.gov (United States)

    Chan, Ching-Ping; Kok, Kin-Hang; Tang, Hei-Man Vincent; Wong, Chi-Ming; Jin, Dong-Yan

    2013-10-01

    Activating transcription factor 4 (ATF4) is a master regulator of genes involved in unfolded protein response (UPR) and its translation is regulated through reinitiation at upstream open reading frames. Here, we demonstrate internal ribosome entry site (IRES)-mediated translation of an alternatively spliced variant of human ATF4. This variant that contains four upstream open reading frames in the 5' leader region was expressed in leukocytes and other tissues. mRNA and protein expression of this variant was activated in the UPR. Its translation was neither inhibited by steric hindrance nor affected by eIF4G1 inactivation, indicating a cap-independent and IRES-dependent mechanism not mediated by ribosome scanning-reinitiation. The IRES activity mapped to a highly structured region that partially overlaps with the third and fourth open reading frames was unlikely attributed to cryptic promoter or splicing, but was activated by PERK-induced eIF2α phosphorylation. Taken together, our findings reveal a new mechanism for translational regulation of ATF4 in mammalian UPR. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Quantification of hTERT Splice Variants in Melanoma by SYBR Green Real-time Polymerase Chain Reaction Indicates a Negative Regulatory Role for the β Deletion Variant

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    Lisa F. Lincz

    2008-10-01

    Full Text Available Telomerase activity is primarily determined by transcriptional regulation of the catalytic subunit, human telomerase reverse transcriptase (hTERT. Several mRNA splice variants for hTERT have been identified, but it is not clear if telomerase activity is determined by the absolute or relative levels of full-length (functional and variant hTERT transcripts. We have developed an SYBR green-based reverse transcription-quantitative polymerase chain reaction assay for the enumeration of the four common hTERT mRNA variants and correlated these with telomerase activity and telomere length in 24 human melanoma cell lines. All except five of the lines expressed four hTERT transcripts, with an overall significant level of co-occurrence between absolute mRNA levels of full-length α+/β+ hTERT and the three splice variants α-/β+, α+/β-, and α-/β-. On average, α+/β+ made up the majority (48.1% of transcripts, followed by α+/β- (44.6%, α-/β- (4.4%, and α-/β+ (2.9%. Telomerase activity ranged from 1 to 247 relative telomerase activity and correlated most strongly with the absolute amount of α+/β+ (R = 0.791, P = .000004 and the relative amount of α+/β- (R = -0.465, P = .022. This study shows that telomerase activity in melanoma cells is best determined by the absolute expression of full-length hTERT mRNA and indicates a role for the hTERT β deletion variant in the negative regulation of enzyme activity.

  17. MDS shows a higher expression of hTERT and alternative splice variants in unactivated T-cells.

    Science.gov (United States)

    Dong, Wen; Wu, Lei; Sun, Houfang; Ren, Xiubao; Epling-Burnette, Pearlie K; Yang, Lili

    2016-11-01

    Telomere instability and telomerase reactivation are believed to play an important role in the development of myelodysplastic syndromes (MDS). Abnormal enzymatic activity of human telomerase reverse transcriptase (hTERT), and its alternative splice variants have been reported to account for deregulated telomerase function in many cancers. In this study, we aim to compare the differences in expression of hTERT and hTERT splice variants, as well as telomere length and telomerase activity in unstimulated T-cells between MDS subgroups and healthy controls. Telomere length in MDS cases was significantly shorter than controls (n = 20, pMDS using World Health Organization classification (WHO subgroups versus control: RARS, p= 0.009; RCMD, p=0.0002; RAEB1/2, p=0.004, respectively) and the International Prognostic Scoring System (IPSS subgroups: Low+Int-1, pMDS patients (n=20) had significantly higher telomerase activity (p=0.002), higher total hTERT mRNA levels (p=0.001) and hTERT α+β- splice variant expression (pMDS (r=0.58, p=0.007). This data is in sharp contrast to data published previously by our group showing a reduction in telomerase and hTERT mRNA in MDS T-cells after activation. In conclusion, this study provides additional insight into hTERT transcript patterns and activity in peripheral T-cells of MDS patients. Additional studies are necessary to better understand the role of this pathway in MDS development and progression.

  18. The Survivin −31 Snp in Human Colorectal Cancer Correlates with Survivin Splice Variant Expression and Improved Overall Survival

    Directory of Open Access Journals (Sweden)

    Anna G. Antonacopoulou

    2010-01-01

    Full Text Available Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated.

  19. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

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    Erik van der Wal

    2017-06-01

    Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

  20. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease.

    Science.gov (United States)

    van der Wal, Erik; Bergsma, Atze J; Pijnenburg, Joon M; van der Ploeg, Ans T; Pijnappel, W W M Pim

    2017-06-16

    The most common variant causing Pompe disease is c.-32-13T>G (IVS1) in the acid α-glucosidase (GAA) gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs) to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA)-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. A splice site variant in the bovine RNF11 gene compromises growth and regulation of the inflammatory response.

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    Arnaud Sartelet

    Full Text Available We report association mapping of a locus on bovine chromosome 3 that underlies a Mendelian form of stunted growth in Belgian Blue Cattle (BBC. By resequencing positional candidates, we identify the causative c124-2A>G splice variant in intron 1 of the RNF11 gene, for which all affected animals are homozygous. We make the remarkable observation that 26% of healthy Belgian Blue animals carry the corresponding variant. We demonstrate in a prospective study design that approximately one third of homozygous mutants die prematurely with major inflammatory lesions, hence explaining the rarity of growth-stunted animals despite the high frequency of carriers. We provide preliminary evidence that heterozygous advantage for an as of yet unidentified phenotype may have caused a selective sweep accounting for the high frequency of the RNF11 c124-2A>G mutation in Belgian Blue Cattle.

  2. Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

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    Rou-Mu Hu

    Full Text Available SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5, has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

  3. Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis.

    Science.gov (United States)

    Luo, Hua; Zhou, Yang; Li, Yingxia; Li, Qifa

    2013-07-01

    Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation. Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation.

  4. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    Science.gov (United States)

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

  5. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    Directory of Open Access Journals (Sweden)

    Cecilia Herraiz

    Full Text Available The melanocortin 1 receptor gene (MC1R expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH. Human MC1R has an inefficient poly(A site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3. Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR might promote an isoform switch from canonical MC1R (MC1R-001 to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

  6. Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex.

    Science.gov (United States)

    Coussen, Françoise; Perrais, David; Jaskolski, Frédéric; Sachidhanandam, Shankar; Normand, Elisabeth; Bockaert, Joel; Marin, Philippe; Mulle, Christophe

    2005-08-18

    Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

  7. A truncated splice variant of human lysyl oxidase-like 2 promotes migration and invasion in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Zou, Hai-Ying; Lv, Guo-Qing; Dai, Li-Hua; Zhan, Xiu-Hui; Jiao, Ji-Wei; Liao, Lian-Di; Zhou, Tai-Mei; Li, Chun-Quan; Wu, Bing-Li; Xu, Li-Yan; Li, En-Min

    2016-06-01

    Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family, which plays an important role in extracellular matrix protein biosynthesis and tumor progression. In the present study, we identified a novel splice variant, LOXL2Δ72, which encodes a peptide having the same N- and C-termini as wild-type LOXL2 (LOXL2WT), but lacks 72 nucleotides encoding 24 amino acids. LOXL2Δ72 had dramatically reduced enzymatic activity, and was no longer secreted. However, LOXL2Δ72 promoted greater cell migration and invasion than LOXL2WT. Furthermore, a dual luciferase reporter assay indicated that LOXL2Δ72 activates distinct signal transduction pathways compared to LOXL2WT, consistent with cDNA microarray data showing different expression levels of cell migration- and invasion-related genes induced following over-expression of each LOXL2 isoform. In particular, LOXL2Δ72 distinctly promoted esophageal squamous cell carcinoma (ESCC) cell migration via up-regulating the C-C motif chemokine ligand 28 (CCL28). Our results suggest that the new LOXL2 splice variant contributes to tumor progression by novel molecular mechanisms different from LOXL2WT. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Effects of eccentric cycling exercise on IGF-I splice variant expression in the muscles of young and elderly people

    DEFF Research Database (Denmark)

    Hameed, M.; Toft, A.D.; Harridge, S.D.

    2008-01-01

    . No difference was observed between the baseline levels of the two splice variants between the two subject groups. Eccentric cycling exercise resulted in a significant increase in the mean MGF mRNA in both young and old subjects but did not alter IGF-IEa mRNA levels in either age group. As reported previously...... growth factor (MGF) were studied in response to 1 h of eccentric cycling exercise in young and old individuals. Subjects (nine young, aged 20-27 years and eight elderly, aged 67-75 years) completed an eccentric exercise protocol that consisted of 60 min of reverse pedal cycling. Workloads were chosen......Recovery from micro damage resulting from intensive exercise has been shown to take longer in older muscles. To investigate the factors that may contribute to muscle repair, we have studied the expression of two splice variants of the insulin-like growth factor-I (IGF-I) gene. IGF-IEa and mechano...

  9. Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

    Science.gov (United States)

    Willemse, Hermia; Theodoratos, Angelo; Smith, Paul N; Dulhunty, Angela F

    2016-02-01

    The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

  10. The Identification of Splice Variants as Molecular Markers in Parkinson’s Disease

    Science.gov (United States)

    2008-09-01

    Purpose: Alternative splicing is responsible for producing several products from a single transcript and can cause pathogenic changes in RNA in...or submitted for publication some of these findings and in addition, are carrying out gene expression studies to localize the aberrant spice

  11. Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Sahebi, Reza; Malakootian, Mahshid; Balalaee, Baharak; Shahryari, Alireza; Khoshnia, Masoud; Abbaszadegan, Mohammad Reza; Moradi, Abdolvahab; Javad Mowla, Seyed

    2016-10-01

    Similar characteristics of molecular pathways between cellular reprogramming events and tumorigenesis have been accentuated in recent years. Reprogramming-related transcription factors, also known as Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC), are also well-known oncogenes promoting cancer initiation, progression, and cellular transformation into cancer stem cells. Long non-coding RNAs (lncRNAs) are a major class of RNA molecules with emerging roles in stem cell pluripotency, cellular reprogramming, cellular transformation, and tumorigenesis. The long intergenic non-coding RNA ROR (lincRNA-ROR, linc-ROR) acts as a regulator of cellular reprograming through sponging miR-145 that normally negatively regulates the expression of the stemness factors NANOG, OCT4, and SOX2. Here, we employed a real-time PCR approach to determine the expression patterns of linc-ROR and its two novel spliced variants (variants 2 and 4) in esophageal squamous cell carcinoma (ESCC). The quantitative real-time RT-PCR results revealed a significant up-regulation of linc-ROR (P=0.0098) and its variants 2 (P=0.0250) and 4 (P=0.0002) in tumor samples of ESCC, compared to their matched non-tumor tissues obtained from the margin of same tumors. Our data also demonstrated a significant up-regulation of variant 4 in high-grade tumor samples, in comparison to the low-grade ones (P=0.04). Moreover, the ROC curve analysis demonstrated that the variant 4 of ROR has a potential to discriminate between tumor and non-tumor samples (AUC=0.66, PROR and its variants 2 and 4 in ESCC tissue samples.

  12. Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A*S⃞

    Science.gov (United States)

    Carrithers, Michael D.; Chatterjee, Gouri; Carrithers, Lisette M.; Offoha, Roosevelt; Iheagwara, Uzoma; Rahner, Christoph; Graham, Morven; Waxman, Stephen G.

    2009-01-01

    Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells. PMID:19136557

  13. Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.

    Science.gov (United States)

    Carrithers, Michael D; Chatterjee, Gouri; Carrithers, Lisette M; Offoha, Roosevelt; Iheagwara, Uzoma; Rahner, Christoph; Graham, Morven; Waxman, Stephen G

    2009-03-20

    Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

  14. Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

    Science.gov (United States)

    Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert M; Chuturgoon, Anil A

    2016-10-01

    Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. A novel actin mRNA splice variant regulates ACTG1 expression.

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    Meghan C Drummond

    Full Text Available Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1 transcript that includes a previously unidentified exon (3a. Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD. To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST.

  16. Identification and characterization of alternative splice variants of the mouse Trek2/Kcnk10 gene

    OpenAIRE

    Mirkovic, Kelsey; Wickman, Kevin

    2011-01-01

    Two-pore domain K+ (K2P) channels underlie leak or background potassium conductances in many cells. The Trek subfamily of K2P channels, which includes Trek1/Kcnk2 and Trek2/Kcnk10 and has been implicated in depression, nociception, and cognition, exhibits complex regulation and can modulate cell excitability in response to a wide array of stimuli. While alternative translation initiation and alternative splicing contribute to the structural and functional diversity of Trek1, the impact of pos...

  17. Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy.

    Science.gov (United States)

    Ames, E G; Lawson, M J; Mackey, A J; Holmes, J W

    2013-09-01

    Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are re-expressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (Phypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3

    OpenAIRE

    Patel, Hiren; Bhartiya, Deepa; Parte, Seema; Gunjal, Pranesh; Yedurkar, Snehal; Bhatt, Mithun

    2013-01-01

    Background We have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants ?progenitors? termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FS...

  19. Porcine ubiquitin-like 5 (UBL5) gene: genomic organization, polymorphisms, mRNA cloning, splicing variants and association study.

    Science.gov (United States)

    Masopust, Martin; Weisz, Filip; Bartenschlager, Heinz; Knoll, Aleš; Vykoukalová, Zuzana; Geldermann, Hermann; Cepica, Stanislav

    2014-01-01

    Ubiquitin-like 5 (UBL5), which is supposed to be involved in regulation of feed intake, energy metabolism, obesity and type 2 diabetes, is located at position 62.1 cM on the pig chromosome 2 region harbouring quantitative trait loci for carcass and meat quality. The 4,354 bp genomic sequence (FR798948) of the porcine gene encompassing the promoter and entire gene was cloned by polymerase chain reaction. Comparative sequencing revealed 13 polymorphisms in noncoding regions. Synthesis of full-length cDNA sequences using rapid amplification of 5' and 3' ends showed three splice variants. Variants 1 and 2 differ in transcription length for the untranslated part of exon 1 with deduced protein of 73 amino acid (aa) residues and 100 % identities between human, mouse and other species. Variant 3, with 4 bp deletion at the 3' end of exon 2, encodes a truncated protein with 28 aa residues. In a Wild boar×Meishan F2 population (n = 334) with 47 recorded traits, loci FR798948:g.2788G>A and FR798948:g.2141T>C were associated at nominal P A at adjusted P C showed suggestive association with growth (adjusted P = 0.0690). As association mapping conducted in the outbred MLW population is more precise than in the three generation F2 population the UBL5 gene tends to be associated with growth rather than with fat accretion.

  20. Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera.

    Science.gov (United States)

    Démares, Fabien; Drouard, Florian; Massou, Isabelle; Crattelet, Cindy; Lœuillet, Aurore; Bettiol, Célia; Raymond, Valérie; Armengaud, Catherine

    2014-09-01

    Glutamate-gated chloride channels (GluCl) belong to the cys-loop ligand-gated ion channel superfamily and their expression had been described in several invertebrate nervous systems. In the honeybee, a unique gene amel_glucl encodes two alternatively spliced subunits, Amel_GluCl A and Amel_GluCl B. The expression and differential localization of those variants in the honeybee brain had been previously reported. Here we characterized the involvement of each variant in olfactory learning and memory processes, using specific small-interfering RNA (siRNA) targeting each variant. Firstly, the efficacy of the two siRNAs to decrease their targets' expression was tested, both at mRNA and protein levels. The two proteins showed a decrease of their respective expression 24h after injection. Secondly, each siRNA was injected into the brain to test whether or not it affected olfactory memory by using a classical paradigm of conditioning the proboscis extension reflex (PER). Amel_GluCl A was found to be involved only in retrieval of 1-nonanol, whereas Amel_GluCl B was involved in the PER response to 2-hexanol used as a conditioned stimulus or as new odorant. Here for the first time, a differential behavioral involvement of two highly similar GluCl subunits has been characterized in an invertebrate species. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Pharmacological Profile of Brain-derived Neurotrophic Factor (BDNF) Splice Variant Translation Using a Novel Drug Screening Assay

    Science.gov (United States)

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L. M.; Vicario, Annalisa; Tongiorgi, Enrico

    2014-01-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5′- and 3′UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5′- and 3′UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5′UTR or a 3′UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5′UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3′UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent “a quantitative code” for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. PMID:25074925

  2. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  3. A splice variant of the myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading

    Science.gov (United States)

    Reho, John J.; Kenchegowda, Doreswamy; Asico, Laureano D.

    2016-01-01

    The cGMP activated kinase cGK1α is targeted to its substrates via leucine zipper (LZ)-mediated heterodimerization and thereby mediates vascular smooth muscle (VSM) relaxation. One target is myosin phosphatase (MP), which when activated by cGK1α results in VSM relaxation even in the presence of activating calcium. Variants of MP regulatory subunit Mypt1 are generated by alternative splicing of the 31 nt exon 24 (E24), which, by changing the reading frame, codes for isoforms that contain or lack the COOH-terminal LZ motif (E24+/LZ−; E24−/LZ+). Expression of these isoforms is vessel specific and developmentally regulated, modulates in disease, and is proposed to confer sensitivity to nitric oxide (NO)/cGMP-mediated vasorelaxation. To test this, mice underwent Tamoxifen-inducible and smooth muscle-specific knockout of E24 (E24 cKO) after weaning. Deletion of a single allele of E24 (shift to Mypt1 LZ+) enhanced vasorelaxation of first-order mesenteric arteries (MA1) to diethylamine-NONOate (DEA/NO) and to cGMP in permeabilized and calcium-clamped arteries and lowered blood pressure. There was no further effect of deletion of both E24 alleles, indicating high sensitivity to shift of Mypt1 isoforms. However, a unique property of MA1s from homozygous E24 cKOs was significantly reduced force generation to α-adrenergic activation. Furthermore 2 wk of high-salt (4% NaCl) diet increased MA1 force generation to phenylephrine in control mice, a response that was markedly suppressed in the E24 cKO homozygotes. Thus Mypt1 E24 splice variants tune arterial reactivity and could be worthy targets for lowering vascular resistance in disease states. PMID:27084390

  4. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Directory of Open Access Journals (Sweden)

    Adrián Ochoa-Leyva

    Full Text Available After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα2, (βα4, (βα5, (βα6, and (βα7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to

  5. A functional splice variant of the human Golgi CMP-sialic acid transporter.

    Science.gov (United States)

    Salinas-Marín, Roberta; Mollicone, Rosella; Martínez-Duncker, Iván

    2016-12-01

    The human Golgi Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Lex antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

  6. Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants.

    Science.gov (United States)

    Zhu, Rong; Chen, Zhong-yuan; Wang, Jun; Yuan, Jiang-di; Liao, Xiang-yong; Gui, Jian-Fang; Zhang, Qi-Ya

    2014-02-01

    A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis.

    Science.gov (United States)

    Börner, Kenneth; Becker, Peter B

    2016-09-01

    SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange. © 2016. Published by The Company of Biologists Ltd.

  8. High frequency of fusion transcripts involving TCF7L2 in colorectal cancer: novel fusion partner and splice variants.

    Directory of Open Access Journals (Sweden)

    Torfinn Nome

    Full Text Available VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC, found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes [1]. To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines, and identified the gene RP11-57H14.3 (ENSG00000225292 as a novel fusion partner for TCF7L2. The fusion was discovered from both genome and transcriptome data in the HCT116 cell line. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 CRC cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs, revealing that the TCF7L2 fusion transcripts are neither specific to cancer nor to the colon and rectum. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3, and demonstrated detectable levels in a large fraction of CRC samples, as well as in normal colonic mucosa and other tissue types. We suggest that the fusion transcripts observed in a high frequency of samples are transcription induced chimeras that are expressed at low levels in most samples. The similar fusion transcripts induced by genomic rearrangements observed in individual cancer cell lines may yet have oncogenic potential as suggested in the original study by Bass et al.

  9. High frequency of fusion transcripts involving TCF7L2 in colorectal cancer: novel fusion partner and splice variants.

    Science.gov (United States)

    Nome, Torfinn; Hoff, Andreas M; Bakken, Anne Cathrine; Rognum, Torleiv O; Nesbakken, Arild; Skotheim, Rolf I

    2014-01-01

    VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC), found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes [1]. To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines, and identified the gene RP11-57H14.3 (ENSG00000225292) as a novel fusion partner for TCF7L2. The fusion was discovered from both genome and transcriptome data in the HCT116 cell line. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 CRC cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs, revealing that the TCF7L2 fusion transcripts are neither specific to cancer nor to the colon and rectum. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3, and demonstrated detectable levels in a large fraction of CRC samples, as well as in normal colonic mucosa and other tissue types. We suggest that the fusion transcripts observed in a high frequency of samples are transcription induced chimeras that are expressed at low levels in most samples. The similar fusion transcripts induced by genomic rearrangements observed in individual cancer cell lines may yet have oncogenic potential as suggested in the original study by Bass et al.

  10. Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

    Directory of Open Access Journals (Sweden)

    Surget S

    2013-12-01

    Full Text Available Sylvanie Surget,1,2 Marie P Khoury,1,2 Jean-Christophe Bourdon1,21Dundee Cancer Centre, 2Jacqui Wood Cancer Centre, Ninewells Hospital, University of Dundee, Dundee, UKAbstract: Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets.Keywords: p53, isoforms, p63, p73, alternative splicing, cancer

  11. T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

    Science.gov (United States)

    Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

    2016-09-01

    TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

  12. A splicing variant of TERT identified by GWAS interacts with menopausal estrogen therapy in risk of ovarian cancer.

    Science.gov (United States)

    Lee, Alice W; Bomkamp, Ashley; Bandera, Elisa V; Jensen, Allan; Ramus, Susan J; Goodman, Marc T; Rossing, Mary Anne; Modugno, Francesmary; Moysich, Kirsten B; Chang-Claude, Jenny; Rudolph, Anja; Gentry-Maharaj, Aleksandra; Terry, Kathryn L; Gayther, Simon A; Cramer, Daniel W; Doherty, Jennifer A; Schildkraut, Joellen M; Kjaer, Susanne K; Ness, Roberta B; Menon, Usha; Berchuck, Andrew; Mukherjee, Bhramar; Roman, Lynda; Pharoah, Paul D; Chenevix-Trench, Georgia; Olson, Sara; Hogdall, Estrid; Wu, Anna H; Pike, Malcolm C; Stram, Daniel O; Pearce, Celeste Leigh

    2016-12-15

    Menopausal estrogen-alone therapy (ET) is a well-established risk factor for serous and endometrioid ovarian cancer. Genetics also plays a role in ovarian cancer, which is partly attributable to 18 confirmed ovarian cancer susceptibility loci identified by genome-wide association studies. The interplay among these loci, ET use and ovarian cancer risk has yet to be evaluated. We analyzed data from 1,414 serous cases, 337 endometrioid cases and 4,051 controls across 10 case-control studies participating in the Ovarian Cancer Association Consortium (OCAC). Conditional logistic regression was used to determine the association between the confirmed susceptibility variants and risk of serous and endometrioid ovarian cancer among ET users and non-users separately and to test for statistical interaction. A splicing variant in TERT, rs10069690, showed a statistically significant interaction with ET use for risk of serous ovarian cancer (pint  = 0.013). ET users carrying the T allele had a 51% increased risk of disease (OR = 1.51, 95% CI 1.19-1.91), which was stronger for long-term ET users of 10+ years (OR = 1.85, 95% CI 1.28-2.66, pint  = 0.034). Non-users showed essentially no association (OR = 1.08, 95% CI 0.96-1.21). Two additional genomic regions harboring rs7207826 (C allele) and rs56318008 (T allele) also had significant interactions with ET use for the endometrioid histotype (pint  = 0.021 and pint  = 0.037, respectively). Hence, three confirmed susceptibility variants were identified whose associations with ovarian cancer risk are modified by ET exposure; follow-up is warranted given that these interactions are not adjusted for multiple comparisons. These findings, if validated, may elucidate the mechanism of action of these loci. © 2016 UICC.

  13. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members

    DEFF Research Database (Denmark)

    Thomassen, Mads; Blanco, Ana; Montagna, Marco

    2012-01-01

    Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortiu...

  14. Study of the Expression of Survivin & Its Splice Variants; ΔEx3, 2b and 3b as Diagnostic Molecular Markers in Breast Cancer

    Directory of Open Access Journals (Sweden)

    E Babaei

    2009-07-01

    Full Text Available Introduction: Survivin is a new member of the Inhibitor Apotosis Protein family (IAP which plays an important role in the regulation of both cell cycle and apoptosis. Its distinct expression in tumor cells as compared to normal adult cells introduces Survivin as the fourth transcriptom demonstrated in tumors. Breast cancer is the most common malignancy among women and scientist`s efforts to classify it has lead to various molecular subtypes and controversial results. Because of the high prevalence of these tumors and lack of suitable molecular markers for diagnosis and prognosis, there are ongoing efforts to find molecular markers which can distinguish nontumoral from tumor tissues. In this study we evaluate the potential usefulness of Survivin and its splice variants ΔEx3, 2b and 3b as molecular markers in breast cancer. Methods: We studied 18 tumor and 17 non tumor adjacent tissues. Transcription levels were measured by Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR and normalized by ß2m as an internal control. Results: 1Survivin and its splice variants; Δex3, 2b and 3b showed differentially higher expression levels in tumors than adjacent normal tissues. 2 The expression levels of Survivin, Survivin-ΔEx3 and Survivin-3b were significantly correlated with the type of tumors. 3 Survivin-2b was expressed in a few samples. 4 Survivin-3b was detected only in tumor samples. Also, our results showed that ΔEx3 variant can be introduced as a dominant expressed variant in breast cancer. Conclusion: Our data indicated that the expression of Survivin, Survivin ∆Ex3 and especially, Survivin-3b were correlated with cancerous nature of tumors and Survivin-∆Ex3 was the most common expressed variant in breast carcinomas. These results besides confirming the potential usefulness of Survivin and its splice variants as molecular markers in breast cancer, demonstrated the role of the gene and its splice variants, especially 3b

  15. An Intronic MBTPS2 Variant Results in a Splicing Defect in Horses with Brindle Coat Texture

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    Leonardo Murgiano

    2016-09-01

    Full Text Available We investigated a family of horses exhibiting irregular vertical stripes in their hair coat texture along the neck, back, hindquarters, and upper legs. This phenotype is termed “brindle” by horse breeders. We propose the term “brindle 1 (BR1” for this specific form of brindle. In some BR1 horses, the stripes were also differentially pigmented. Pedigree analyses were suggestive of a monogenic X-chromosomal semidominant mode of inheritance. Haplotype analyses identified a 5 Mb candidate region on chromosome X. Whole genome sequencing of four BR1 and 60 nonbrindle horses identified 61 private variants in the critical interval, none of them located in an exon of an annotated gene. However, one of the private variants was close to an exon/intron boundary in intron 10 of the MBTPS2 gene encoding the membrane bound transcription factor peptidase, site 2 (c.1437+4T>C. Different coding variants in this gene lead to three related genodermatoses in human patients. We therefore analyzed MBTPS2 transcripts in skin, and identified an aberrant transcript in a BR1 horse, which lacked the entire exon 10 and parts of exon 11. The MBTPS2:c1437+4T>C variant showed perfect cosegregation with the brindle phenotype in the investigated family, and was absent from 457 control horses of diverse breeds. Altogether, our genetic data, and previous knowledge on MBTPS2 function in the skin, suggest that the identified MBTPS2 intronic variant leads to partial exon skipping, and causes the BR1 phenotype in horses.

  16. Anti-inflammatory properties and expression in selected organs of canine interleukin-1β splice variant 1.

    Science.gov (United States)

    Kiczak, L; Wałecka-Zacharska, E; Bania, J; Sambor, I; Stefaniak, T; Dzięgiel, P; Zacharski, M; Tomaszek, A; Rybińska, I; Pasławska, U

    2015-10-15

    The IL-1β gene can be also be spliced with the intron 4 retention; the result is a IL-1β splice variant 1 (IL-1βsv1), which was significantly up-regulated in failing myocardium of dogs suffering from chronic degenerative valvular disease (CDVD). Expression of IL-1βsv1 was assessed, at both RNA and protein levels, in organs affected by heart failure, namely, kidneys, liver, and lungs from 35 dogs suffering chronic degenerative valvular disease (CDVD) and in 20 disease free control dogs. IL-1βsv1 RNA was detected in the dogs from both groups. In the CDVD group, the highest RNA and protein IL-1βsv1 levels were observed in lungs, followed, in that order, by the liver and kidneys. IL-1βsv1 protein was found in the cytoplasm of hepatocytes and IL-1βsv1-overexpressing DH82 cells. In lungs, IL-1βsv1 was localized in the cytoplasm and in the nuclei of bronchiolar epithelial and smooth-muscle cells. Cytoplasmic and nuclear IL-1βsv1 expression was observed in macrophages, and a strong nuclear signal was detected in epithelial cells of the alveolar sacs. Following lipopolysaccharide (LPS) stimulation, overexpression of IL-1βsv1 in DH82 cells decreased the pro-inflammatory response. Our results indicate that IL-1βsv1 is constitutively expressed in both normal tissues and in tissues from cases of heart failure. The presence of IL-1βsv1 in tissues exposed to invading agents and its anti-inflammatory activity in DH82 cells may point to its immunomodulatory role in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. A teleost CD46 is involved in the regulation of complement activation and pathogen infection.

    Science.gov (United States)

    Li, Mo-Fei; Sui, Zhi-Hai; Sun, Li

    2017-11-03

    In mammals, CD46 is involved in the inactivation of complement by factor I (FI). In teleost, study on the function of CD46 is very limited. In this study, we examined the immunological property of a CD46 molecule (CsCD46) from tongue sole, a teleost species with important economic value. We found that recombinant CsCD46 (rCsCD46) interacted with FI and inhibited complement activation in an FI-dependent manner. rCsCD46 also interacted with bacterial pathogens via a different mechanism to that responsible for the FI interaction, involving different rCsCD46 sites. Cellular study showed that CsCD46 was expressed on peripheral blood leukocytes (PBL) and protected the cells against the killing effect of complement. When the CsCD46 on PBL was blocked by antibody before incubation of the cells with bacterial pathogens, cellular infection was significantly reduced. Consistently, when tongue sole were infected with bacterial pathogens in the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria.

  18. Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

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    McCann Richard O

    2006-12-01

    Full Text Available Abstract Background Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene. Results We show here that, based on their distribution in cDNA libraries, Talin-a and Talin-b are differentially expressed during C. intestinalis development. The I/LWEQ modules of the two proteins also have different affinities for F-actin. Consistent with the hypothesis that Talin-a and Talin-b have different roles in cell adhesion, the distinct I/LWEQ modules of Talin-a and Talin-b possess different subcellular targeting determinants. The I/LWEQ module of Talin-a is targeted to focal adhesions, where it most likely serves as the link between integrin and the actin cytoskeleton. The Talin-b I/LWEQ module is not targeted to focal adhesions, but instead preferentially labels F-actin stress fibers. These different properties of C. intestinalis the Talin-a and Talin-b I/LWEQ modules mimic the differences between mammalian

  19. Minnelide Inhibits Androgen Dependent, Castration Resistant Prostate Cancer Growth by Decreasing Expression of Androgen Receptor Full Length and Splice Variants.

    Science.gov (United States)

    Isharwal, Sumit; Modi, Shrey; Arora, Nivedita; Uhlrich, Charles; Giri, Bhuwan; Barlass, Usman; Soubra, Ayman; Chugh, Rohit; Dehm, Scott M; Dudeja, Vikas; Saluja, Ashok; Banerjee, Sulagna; Konety, Badrinath

    2017-05-01

    With almost 30,000 deaths per year, prostate cancer is the second-leading cause of cancer-related death in men. Androgen Deprivation Therapy (ADT) has been the corner stone of prostate cancer treatment for decades. However, despite an initial response of prostate cancer to ADT, this eventually fails and the tumors recur, resulting in Castration Resistant Prostate Cancer (CRPC). Triptolide, a diterpene triepoxide, has been tested for its anti-tumor properties in a number of cancers for over a decade. Owing to its poor solubility in aqueous medium, its clinical application had been limited. To circumvent this problem, we have synthesized a water-soluble pro-drug of triptolide, Minnelide, that is currently being evaluated in a Phase 1 clinical trial against gastrointestinal tumors. In the current study, we assessed the therapeutic potential of Minnelide and its active compound triptolide against androgen dependent prostate cancer both in vitro as well as in vivo. Cell viability was measured by a MTT based assay after treating prostate cancer cells with multiple doses of triptolide. Apoptotic cell death was measured using a caspase 3/7 activity. Androgen Receptor (AR) promoter-binding activity was evaluated by using luciferase reporter assay. For evaluating the effect in vivo, 22Rv1 cells were implanted subcutaneously in animals, following which, treatment was started with 0.21 mg/kg Minnelide. Our study showed that treatment with triptolide induced apoptotic cell death in CRPC cells. Triptolide treatment inhibited AR transcriptional activity and decreased the expression of AR and its splice variants both at the mRNA and the protein level. Our studies show that triptolide inhibits nuclear translocation of Sp1, resulting in its decreased transcriptional activity leading to downregulation of AR and its splice variants in prostate cancer cells. In vivo, Minnelide (0.21 mg/kg) regressed subcutaneous tumors derived from CRPC 22RV1 at our study endpoint. Our animal

  20. Regulation of Translational Efficiency by Disparate 5′-UTRs of PPARγ Splice Variants

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    Shawn McClelland

    2009-01-01

    Full Text Available The PPAR-γ gene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the 5′-untranslated region (UTR. The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the 5′-UTR in regulating the efficiency with which the message is translated to protein. A coupled in vitro transcription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. An in vivo reporter gene assay using each 5′-UTR upstream of the firefly luciferase gene showed that the 5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the 5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each 5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the 5′-UTR, and the length of the 5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

  1. Expression of insulin receptor spliced variants and their functional correlates in muscle from patients with non-insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Hansen, Torben; Bjørbaek, C; Vestergaard, H

    1993-01-01

    Due to alternative splicing of exon 11 of the receptor gene, the human insulin receptor exists in two forms, that have distinct tissue-specific expression and are functionally different. Needle biopsies obtained from vastus lateralis muscle from 20 patients with noninsulin-dependent diabetes...... mellitus (NIDDM) and 20 normal control subjects were analyzed for the relative expression of insulin receptor mRNA variants in a novel assay using fluorescence-labeled primers and subsequent analysis on an automated DNA sequencer. In subgroups of patients and control subjects, insulin binding and tyrosine...... kinase activity were examined in wheat germ agglutinin-purified insulin receptors isolated from muscle biopsies. Moreover, insulin-stimulated glucose disposal was studied by means of the euglycemic hyperinsulinemic clamp technique. No difference in the relative expression of spliced variants...

  2. A novel splice variant of folate receptor 4 predominantly expressed in regulatory T cells

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    Tian Yi

    2012-06-01

    Full Text Available Abstract Background Regulatory T cells (Tregs are required for proper maintenance of immunological self-tolerance and immune homeostasis. Folate receptor 4 (FR4 is expressed at high levels in transforming growth factor-beta (TGF-β-induced Tregs and natural Tregs. Moreover, antibody-mediated targeting of FR4 is sufficient to mediate Treg depletion. Results In this study, we describe a novel FR4 transcript variant, FR4D3, in which exon 3 is deleted. The mRNA of FR4D3 encodes a FR4 variant truncated by 189 bp. FR4D3 was found to be predominantly expressed in CD4+CD25+ Treg cells. Overexpression of FR4D3 in CD4+CD25+ Treg cells in vitro stimulated proliferation, which may modulate the ability of these cells to bind and incorporate folic acid. Conclusions Our results suggested that high levels of FR4D3 may be critical to support the substantial proliferative capacity of Treg cells.

  3. Localization and Regulation of the N Terminal Splice Variant of PGC-1α in Adult Skeletal Muscle Fibers

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    Tiansheng Shen

    2012-01-01

    Full Text Available The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1–270 was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.

  4. Complex functions of Mef2 splice variants in the differentiation of endoderm and of a neuronal cell type in a sea anemone.

    Science.gov (United States)

    Genikhovich, Grigory; Technau, Ulrich

    2011-11-01

    In triploblastic animals, mesoderm gives rise to many tissues and organs, including muscle. By contrast, the representatives of the diploblastic phylum Cnidaria (corals, sea anemones, jellyfish and hydroids) lack mesoderm but possess muscle. In vertebrates and insects, the transcription factor Mef2 plays a pivotal role in muscle differentiation; however, it is also an important regulator of neuron differentiation and survival. In the sea anemone Nematostella vectensis, an organism that lacks mesoderm but has muscles and neurons, Mef2 (Nvmef2) has been reported in single ectodermal cells of likely neural origin. To our surprise, we found that Nvmef2 is alternatively spliced, forming differentially expressed variants. Using morpholino-mediated knockdown and mRNA injection, we demonstrate that specific splice variants of Nvmef2 are required for the proliferation and differentiation of endodermal cells and for the development of ectodermal nematocytes, a neuronal cell type. Moreover, we identified a small conserved motif in the transactivation domain that is crucially involved in the endodermal function of Nvmef2. The identification of a crucial and conserved motif in the transactivation domain predicts a similarly important role in vertebrate Mef2 function. This is the first functional study of a determinant of several mesodermal derivatives in a diploblastic animal. Our data suggest that the involvement of alternative splice variants of Mef2 in endomesoderm and neuron differentiation predates the cnidarian-bilaterian split.

  5. Full-Length L1CAM and Not Its Δ2Δ27 Splice Variant Promotes Metastasis through Induction of Gelatinase Expression

    Science.gov (United States)

    Gerg, Michael; Schäfer, Michael K.; Pfeifer, Marco; Hazin, John; Schelter, Florian; Weidle, Ulrich H.; Ramser, Juliane; Volkmann, Juliane; Meindl, Alfons; Schmitt, Manfred; Schrötzlmair, Florian; Altevogt, Peter; Krüger, Achim

    2011-01-01

    Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression. PMID:21541352

  6. Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression.

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    Stephanie Hauser

    Full Text Available Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM, which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM and a splice variant lacking exons 2 and 27 (SV-L1CAM. It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.

  7. Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3.

    Science.gov (United States)

    Patel, Hiren; Bhartiya, Deepa; Parte, Seema; Gunjal, Pranesh; Yedurkar, Snehal; Bhatt, Mithun

    2013-01-01

    We have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants 'progenitors' termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FSH-R3 have biological activity. Present study was undertaken to identify FSHR isoforms mediating FSH action on ovarian stem cells, using sheep OSE cells culture as the study model. Cultures of sheep OSE cells (a mix of epithelial cells, VSELs, OGSCs and few contaminating red blood cells) were established with and without FSH 5IU/ml treatment. Effect of FSH treatment on self-renewal of VSELs and their differentiation into OGSCs was studied after 15 hrs by qRT-PCR using markers specific for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4). FSH receptors and its specific transcripts (R1 and R3) were studied after 3 and 15 hrs of FSH treatment by immunolocalization, in situ hybridization and qRT-PCR. FSHR and OCT-4 were also immuno-localized on sheep ovarian sections, in vitro matured follicles and early embryos. FSH treatment resulted in increased stem cells self-renewal and clonal expansion evident by the appearance of stem cell clusters. FSH receptors were expressed on ovarian stem cells whereas the epithelial cells were distinctly negative. An increase in R3 mRNA transcripts was noted after 3 hrs of FSH treatment and was reduced to basal levels by 15 hrs, whereas R1 transcript expression remained unaffected. Both FSHR and OCT-4 were immuno-localized in nuclei of stem cells, showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles. FSH modulates ovarian stem cells via FSH-R3 to undergo potential self-renewal, clonal

  8. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

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    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  9. Cloning and tissue distribution of novel splice variants of the ovine ghrelin gene.

    Science.gov (United States)

    Menzies, Moira; Seim, Inge; Josh, Peter; Nagaraj, Shivashankar H; Lees, Michael; Walpole, Carina; Chopin, Lisa K; Colgrave, Michelle; Ingham, Aaron

    2014-09-06

    The ghrelin axis is involved in the regulation of metabolism, energy balance, and the immune, cardiovascular and reproductive systems. The manipulation of this axis has potential for improving economically valuable traits in production animals, and polymorphisms in the ghrelin (GHRL) and ghrelin receptor (GHSR) genes have been associated with growth and carcass traits. Here we investigate the structure and expression of the ghrelin gene (GHRL) in sheep, Ovis aries. We identify two ghrelin mRNA isoforms, which we have designated Δex2 preproghrelin and Δex2,3 preproghrelin. Expression of Δex2,3 preproghrelin is likely to be restricted to ruminants, and would encode truncated ghrelin and a novel C-terminal peptide. Both Δex2 preproghrelin and canonical preproghrelin mRNA isoforms were expressed in a range of tissues. Expression of the Δex2,3 preproghrelin isoform, however, was restricted to white blood cells (WBC; where the wild-type preproghrelin isoform is not co-expressed), and gastrointestinal tissues. Expression of Δex2 preproghrelin and Δex2,3 preproghrelin mRNA was elevated in white blood cells in response to parasitic worm (helminth) infection in genetically susceptible sheep, but not in resistant sheep. The restricted expression of the novel preproghrelin variants and their distinct WBC expression pattern during parasite infection may indicate a novel link between the ghrelin axis and metabolic and immune function in ruminants.

  10. Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL: molecular cloning, expression distribution, splice variants and DNA methylation profile

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    Wang ShaoHua

    2007-03-01

    Full Text Available Abstract Background The genetic basis of telomere length heterogeneity among mammalian species is still not well understood. Recently, a gene named regulator of telomere length elongation helicase (RTEL was identified and predicted to be an essential participant in species-specific telomere length regulation in two murine species. To obtain broader insights into its structure and biological functions and to ascertain whether RTEL is also a candidate gene in the regulation of telomere length diversity in other mammalian species, data from other mammals may be helpful. Results Here we report the cDNA cloning, genomic structure, chromosomal location, alternative splicing pattern, expression distribution and DNA methylation profile of the bovine homolog of RTEL. The longest transcript of bovine RTEL is 4440 nt, encompassing 24.8 kb of genomic sequence that was mapped to chromosome 13q2.2. It encodes a conserved helicase-like protein containing seven characterized helicase motifs in the first 750 aa and a PIP box in the C-terminus. Four splice variants were identified within the transcripts in both the coding and 5'-untranslated regions; Western blot revealed that the most abundant splice variant SV-1 was translated to a truncated isoform of RTEL. The different 5'UTRs imply alternative transcription start sites in the promoter; Bovine RTEL was transcribed at the blastocyst stage, and expression levels were highest in adult testis, liver and ovary. DNA methylation analysis of tissues that differed significantly in expression level indicated that relatively low DNA methylation is associated with higher expression. Conclusion In this study, we have identified and characterized a bovine RTEL homolog and obtained basic information about it, including gene structure, expression distribution, splice variants and profile of DNA methylation around two putative transcription start sites. These data may be helpful for further comparative and functional analysis

  11. Splice-mediated Variants of Proteins (SpliVaP – data and characterization of changes in signatures among protein isoforms due to alternative splicing

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    Thanaraj Thangavel

    2008-10-01

    Full Text Available Abstract Background It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. Results (i We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. Conclusion (i Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b When swapping of signatures occurs

  12. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells

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    Satoru Kanto

    2016-04-01

    second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein. Discussion. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a single Runx2 gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

  13. CD44 Splice Variant v8-10 as a Marker of Serous Ovarian Cancer Prognosis.

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    Amanda Sosulski

    Full Text Available CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10 has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20% compared to those with the lowest expression (bottom 10 and 20% (p = 0.0181, p = 0.0262 respectively. Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05. Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites

  14. CD44 Splice Variant v8-10 as a Marker of Serous Ovarian Cancer Prognosis.

    Science.gov (United States)

    Sosulski, Amanda; Horn, Heiko; Zhang, Lihua; Coletti, Caroline; Vathipadiekal, Vinod; Castro, Cesar M; Birrer, Michael J; Nagano, Osamu; Saya, Hideyuki; Lage, Kasper; Donahoe, Patricia K; Pépin, David

    2016-01-01

    CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be

  15. Splice variants and regulatory networks associated with host resistance to the intestinal worm Cooperia oncophora in cattle.

    Science.gov (United States)

    Li, Robert W; Wu, Sitao; Li, Cong-Jun; Li, Weizhong; Schroeder, Steven G

    2015-07-30

    To elucidate the molecular mechanism of host resistance, we characterized the jejunal transcriptome of Angus cattle selected for parasite resistance for over 20 years in response to infection caused by the intestinal worm Cooperia oncophora. The transcript abundance of 56 genes, such as that of mucin 12 (MUC12) and intestinal alkaline phosphatase (ALPI), was significantly higher in resistant cattle. Novel splicing variants, exon skipping events, and gene fusion events, were also detected. An algorithm for the reconstruction of accurate cellular networks (ARACNE) was used to infer de novo regulatory molecular networks in the interactome between the parasite and host. Under a combined cutoff of an error tolerance (ϵ = 0.10) and a stringent P-value threshold of mutual information (1.0 × 10(-5)), a total of 229,100 direct interactions controlled by 20,288 hub genes were identified. Among these hub genes, 7651 genes had ≥ 100 direct neighbors while the top 9778 hub genes controlled more than 50% of total direct interactions. Three lysozyme genes (LYZ1, LYZ2, and LYZ3), which are co-located in bovine chromosome 5 in tandem and are strongly upregulated in resistant cattle, shared a common regulatory network of 55 genes. These ancient antimicrobials were likely involved in regulating host-parasite interactions by affecting host gut microbiome. Notably, ALPI, known as a gut mucosal defense factor, controlled a molecular network consisting 410 genes, including 14 transcription factors (TF) and 10 genes that were significantly regulated in resistant cattle. Several large regulatory networks were controlled by TF, such as STAT6, SREBF1, and ELF4. Gene ontology (GO) processes significantly enriched in the regulatory network controlled by STAT6 included lipid metabolism. Our findings provide insights into the immune regulation of host-parasite interactions and the molecular mechanisms of host resistance in cattle. Published by Elsevier B.V.

  16. Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.

    Science.gov (United States)

    Malone, Andrew F; Funk, Steven D; Alhamad, Tarek; Miner, Jeffrey H

    2017-06-01

    Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.

  17. Alternative Splicing in CKD.

    Science.gov (United States)

    Stevens, Megan; Oltean, Sebastian

    2016-06-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. Copyright © 2016 by the American Society of Nephrology.

  18. Expression of Tetrahymena snRNA gene variants including a U1 gene with mutations in the 5' splice site recognition sequence

    DEFF Research Database (Denmark)

    Eugen-Olsen, J; Hagemeister, J J; Hellung-Larsen, P

    1997-01-01

    The expression of U1, U2 and U5 snRNA gene variants has been studied under different physiological states of Tetrahymena. Variants of all three snRNA genes are expressed. Among the snRNAs detected is U1-3, a variant with 66 mutations compared to the normal U1 snRNA. Three of these mutations affec...... the 5' splice site recognition sequence. The U1-3 snRNA is present in a few hundred copies per cell. The expression of Tetrahymena snRNA genes is independent of the physiological state of the cell.......The expression of U1, U2 and U5 snRNA gene variants has been studied under different physiological states of Tetrahymena. Variants of all three snRNA genes are expressed. Among the snRNAs detected is U1-3, a variant with 66 mutations compared to the normal U1 snRNA. Three of these mutations affect...

  19. Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

    Science.gov (United States)

    Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

    2012-01-01

    Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

  20. IGF1 mRNA splicing variants in Liaoning cashmere goat: identification, characterization, and transcriptional patterns in skin and visceral organs.

    Science.gov (United States)

    Bai, Wen L; Yin, Rong H; Yin, Rong L; Wang, Jiao J; Jiang, Wu Q; Luo, Guang B; Zhao, Zhi H

    2013-01-01

    Insulin-like growth factor I (IGF1) is a member of the insulin superfamily. It performs important roles in the proliferation and differentiation of skin cell and control of hair cycles and is thought to be a potential candidate gene for goat cashmere traits. In this work, we isolated and characterized three kinds of IGF1 mRNA splicing variants from the liver of Liaoning Cashmere goat, and the expression characterization of the IGF1 mRNA splicing variants were investigated in skin and other tissues of Liaoning cashmere goat. The sequencing results indicated that the classes 1w, 1, and 2 of IGF1 cDNAs in Liaoning cashmere goat, each included an open reading frame encoding the IGF1 precursor protein. The deduced amino acid sequences of the three IGF1 precursor proteins differed only in their NH2-terminal leader peptides. Through removal of the signal peptide and extension peptide, the three IGF1 mRNA splicing variants (classes 1w, 1, and 2) resulted in the same mature IGF1 protein in Liaoning cashmere goat. In skin tissue of Liaoning cashmere goat, class 1 and class 2 were detected in all stages of hair follicle cycling, and they had the highest transcription level at anagen, and then early anagen; whereas at telogen both classes 1 and 2 had the lowest expression in mRNA level, but the class 1 appears to be relatively more abundant than class 2 in skin tissue of Liaoning cashmere goat. However, the class 1w transcript was not detected in the skin tissues. Three classes of IGF1 mRNA were transcribed in a variety of tissues, including heart, brain, spleen, lung, kidney, liver, and skeletal muscle, but class 1 IGF1 mRNA was more abundant than classes 1w and 2 in the investigated tissues.

  1. Contrasting changes in DRD1 and DRD2 splice variant expression in schizophrenia and affective disorders, and associations with SNPs in postmortem brain

    DEFF Research Database (Denmark)

    Kaalund, S S; Newburn, E N; Ye, Tuo

    2014-01-01

    of working memory that relies on normal dorsolateral prefrontal cortex (DLPFC) function. To better understand the association of dopamine receptors with SCZ, we studied the expression of three DRD2 splice variants and the DRD1 transcript in DLPFC, hippocampus and caudate nucleus in a large cohort of subjects......Dopamine 2 receptor (DRD2) is of major interest to the pathophysiology of schizophrenia (SCZ) both as a target for antipsychotic drug action as well as a SCZ-associated risk gene. The dopamine 1 receptor (DRD1) is thought to mediate some of the cognitive deficits in SCZ, including impairment...

  2. CD46 accelerates macrophage-mediated host susceptibility to meningococcal sepsis in a murine model.

    Science.gov (United States)

    Wang, Xiao; Zhang, Ding; Sjölinder, Mikael; Wan, Yi; Sjölinder, Hong

    2017-01-01

    CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement-mediated attack. Human transgenic CD46 homozygous mice (CD46+/+ ) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46+/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro-inflammatory mediators such as IL-6, TNF, IL-12, and IL-1β In the presence of M-CSF or GM-CSF, CD46 signaling enhances monocyte-macrophage differentiation. Additionally, CD46+/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46+/+ peritoneal macrophages aggravated septic responses in wild-type mice, but the depletion of macrophages partially alleviated septic reactions in CD46+/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.

    Directory of Open Access Journals (Sweden)

    Tatiana A Soboleva

    2017-02-01

    Full Text Available The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1, from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II. Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS, but also at the beginning of the gene body (but being excluded from the +1 nucleosome compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

  4. A novel TMEM16A splice variant lacking the dimerization domain contributes to calcium-activated chloride secretion in human sweat gland epithelial cells.

    Science.gov (United States)

    Ertongur-Fauth, Torsten; Hochheimer, Andreas; Buescher, Joerg Martin; Rapprich, Stefan; Krohn, Michael

    2014-11-01

    Sweating is an important physiological process to regulate body temperature in humans, and various disorders are associated with dysregulated sweat formation. Primary sweat secretion in human eccrine sweat glands involves Ca(2+) -activated Cl(-) channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia; however, their molecular identity in sweat glands remained elusive. Here, we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(acΔe3) lacking the TMEM16A-dimerization domain. Chloride flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca(2+) -dependent Cl(-) secretion in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(acΔe3) is forming a functional CaCC, with basal and Ca(2+) -activated Cl(-) permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl(-) secretion providing opportunities to develop sweat gland-specific therapeutics for treatment of sweating disorders. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Androgen Receptor Splice Variant 7 and Efficacy of Taxane Chemotherapy in Patients With Metastatic Castration-Resistant Prostate Cancer

    Science.gov (United States)

    Antonarakis, Emmanuel S.; Lu, Changxue; Luber, Brandon; Wang, Hao; Chen, Yan; Nakazawa, Mary; Nadal, Rosa; Paller, Channing J.; Denmeade, Samuel R.; Carducci, Michael A.; Eisenberger, Mario A.; Luo, Jun

    2015-01-01

    IMPORTANCE We previously showed that detection of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from men with castration-resistant prostate cancer (CRPC) was associated with primary resistance to enzalutamide and abiraterone therapy, but the relevance of AR-V7 status in the context of chemotherapy is unknown. OBJECTIVE To investigate whether AR-V7–positive patients would retain sensitivity to taxane chemotherapy and whether AR-V7 status would have a differential impact on taxane-treated men compared with enzalutamide- or abiraterone-treated men. DESIGN, SETTING, AND PARTICIPANTS We examined CTCs for AR-V7 mRNA using a reverse-transcription polymerase chain reaction assay. From January 2013 to July 2014, we prospectively enrolled patients with metastatic CRPC initiating taxane chemotherapy (docetaxel or cabazitaxel) at a single academic institution (Johns Hopkins). Our prespecified statistical plan required a sample size of 36 taxane-treated men. MAIN OUTCOMES AND MEASURES We evaluated associations between AR-V7 status and prostate-specific antigen (PSA) response rates. PSA progression-free survival (PSA PFS), and clinical and/or radiographic progression-free survival (PFS). After incorporating updated data from our prior study of 62 patients treated with enzalutamide or abiraterone, we also investigated the interaction between AR-V7 status (positive or negative) and treatment type (taxane vs enzalutamide or abiraterone). RESULTS Of 37 taxane-treated patients enrolled. 17 (46%) had detectable AR-V7 in CTCs. Prostate-specific antigen responses were achieved in both AR-V7–positive and AR-V7–negative men (41% vs 65%; P = .19) Similarly, PSA PFS (hazard ratio [HR], 1.7, 95% CI, 0.6-5.0; P = .32) and PFS (HR, 2.7, 95% CI, 0.8-8.8; P = .11) were comparable in AR-V7–positive and AR-V7–negative patients. A significant interaction was observed between AR-V7 status and treatment type (P < .001). Clinical outcomes were superior with

  6. Human GluR6c, a functional splicing variants of GluR6, is mainly expressed in non-nervous cells.

    Science.gov (United States)

    Barbon, Alessandro; Gervasoni, Annalisa; LaVia, Luca; Orlandi, Cesare; Jaskolski, Frédéric; Perrais, David; Barlati, Sergio

    2008-03-21

    Kainate-type glutamate receptors (KARs) are receptor channels with a variety of distinct physiological functions in synaptic transmission, depending on their sub-cellular location in functional neuronal compartments. The kainate receptor subunit GluR6 presents different splice variants involving the C-terminal domain, namely GluR6a, GluR6b and GluR6c. In this study, we report the analysis of the three human splicing isoforms and in particular of the uncharacterized hGluR6c. When expressed in COS-7 cells, hGluR6a receptor subunit was highly present on the surface of the plasma membrane, whereas hGluR6b and hGluRc were poorly transported to the membrane. Electrophysiological studies of homomeric receptors showed that hGluR6c subunit can generate functional receptors with characteristics similar to the GluR6b variant. mRNA expression analysis demonstrated that hGluR6c variant is mainly expressed in non-neuronal cells and barely expressed in neuronal ones. Interestingly, undifferentiated NT2 cells expressing only the hGluR6c isoform, during neuronal differentiation induced by retinoic acid, increased the expression level of the neuronal form hGluR6a with a parallel decreased of hGluR6c. Overall, our data indicate that hGluR6c might have unique properties in non-nervous cells and in the first stages of CNS development.

  7. Molecular cloning, genomic organization, chromosome mapping, tissues expression pattern and identification of a novel splicing variant of porcine CIDEb gene

    Energy Technology Data Exchange (ETDEWEB)

    Li, YanHua, E-mail: liyanhua.1982@aliyun.com [Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, China International Science and Technology Cooperation base of Child development and Critical Disorders, Children’s Hospital of Chongqing Medical University, Chongqing 400014 (China); Li, AiHua [Chongqing Cancer Institute & Hospital & Cancer Center, Chongqing 404100 (China); Yang, Z.Q. [Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China)

    2016-09-09

    Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open reading frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were

  8. The vinculin-DeltaIn20/21 mouse: characteristics of a constitutive, actin-binding deficient splice variant of vinculin.

    Directory of Open Access Journals (Sweden)

    Susanna Marg

    Full Text Available BACKGROUND: The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate vinculin-DeltaEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-DeltaEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-DeltaIn20/21 allele and shows defective head-to-tail interaction. Homozygous VCL-DeltaIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-DeltaEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-DeltaEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-DeltaEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. CONCLUSIONS/SIGNIFICANCE: Vinculin-DeltaEx20 is an active variant supporting adhesion site stabilization without an enhanced

  9. Persistent detection of alternatively spliced BCR‐ABL variant results in a failure to achieve deep molecular response

    OpenAIRE

    Yuda, Junichiro; Miyamoto, Toshihiro; Odawara, Jun; Ohkawa, Yasuyuki; Semba, Yuichiro; Hayashi, Masayasu; Miyamura, Koichi; Tanimoto, Mitsune; Yamamoto, Kazuhito; Taniwaki, Masafumi; Akashi, Koichi

    2017-01-01

    Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI‐resistant BCR‐ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR‐ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR‐ABL and its mutants, including alternatively spliced BCR‐ABL with an insertion of 35 intronic nucleotides ( BCR‐ ABL I ns35bp ) between ABL exons 8 and 9, which intro...

  10. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

    Science.gov (United States)

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

    2014-10-03

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects.

    Science.gov (United States)

    Majumdar, Susruta; Grinnell, Steven; Le Rouzic, Valerie; Burgman, Maxim; Polikar, Lisa; Ansonoff, Michael; Pintar, John; Pan, Ying-Xian; Pasternak, Gavril W

    2011-12-06

    Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.

  12. In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters.

    Science.gov (United States)

    Luque, Raul M; Sampedro-Nuñez, Miguel; Gahete, Manuel D; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D; Castaño, Justo P; Marazuela, Mónica

    2015-08-14

    Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value.

  13. In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters

    Science.gov (United States)

    Gahete, Manuel D.; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D.; Castaño, Justo P.; Marazuela, Mónica

    2015-01-01

    Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value. PMID:26124083

  14. HE4 Transcription- and Splice Variants-Specific Expression in Endometrial Cancer and Correlation with Patient Survival

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    Shi-Wen Jiang

    2013-11-01

    Full Text Available We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.

  15. A highly susceptible CD46 transgenic mouse model of subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis.

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    Yoshida, Haruno; Takahashi, Tetsufumi; Nakamura, Masahiko; Øverby, Anders; Takahashi, Takashi; Ubukata, Kimiko; Matsui, Hidenori

    2016-04-01

    The Streptococcus dysgalactiae subspecies equisimilis (SDSE) possesses clinical similarities to group A streptococcus (GAS) and has recently been recognized as a causative pathogen of life-threatening streptococcal infections. Human membrane cofactor protein (CD46), a complement regulatory protein ubiquitously expressed on every cell type except for erythrocytes, has been implicated as a receptor for human-specific pathogens including GAS. In the present report, SDSE strain GGS_124 was isolated from a patient suffering from streptococcal toxic shock syndrome. When CD46-expressing transgenic (Tg) and non-Tg mice were infected subcutaneously into a hind footpad with 1 × 10(7) colony-forming units of GGS_124, both CD46 Tg and non-Tg mice showed similar levels of colonization in the popliteal lymph nodes at day 3 after infection. However, the following differences were found between CD46 Tg and non-Tg mice after infection. First, there was a statistically significant difference in mortality rates between CD46 Tg (33%) and non-Tg (0%) mice within 35 days after infection. Second, all surviving CD46 Tg mice developed ankle arthritis at day 35 after infection, whereas non-Tg mice did not develop ankle arthritis on the infected hind paws. Finally, CD46 Tg mice developed a pus-filled abscess accompanied by renal failure at day 6 or later after infection. These observations suggest that CD46, the host cell-surface pathogen receptor, functioned to attract GGS_124 into deep tissues, so that the subcutaneous infection with GGS_124 induced invasive streptococcal diseases in CD46 Tg mice. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  16. A novel exon 15-deleted, splicing variant of Slit2 shows potential for growth inhibition in addition to invasion inhibition in lung cancer.

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    Lin, Yu-Ying; Yang, Chun-Hung; Sheu, Gwo-Tarng; Huang, Chi-Ying F; Wu, Yu-Chung; Chuang, Shu-Ming; Fann, Ming-Ji; Chang, Han; Lee, Huei; Chang, Jinghua Tsai

    2011-08-01

    The axon guidance cue molecule Slit2 has been shown to suppress cancer cell invasion. However, the role of Slit2 in growth inhibition is still controversial. The authors identified a novel exon 15 (AKEQYFIP)-deleted slit2, located at the end of the second leucine-rich repeat (LRR2). Because LRR2 interacts with Robo1 receptor to inhibit invasion, they hypothesized that exon 15 plays an important role in modulating Slit2 function. Slit2 expression was assessed via microarray analysis in 27 lung adenocarcinomas. Exon 15-deleted slit2 (slit2-ΔE15) and exon 15-containing slit2 (slit2-WT) were cloned and expressed in the CL1-5 lung cancer cell line. The effect of exon 15 on Slit2-mediated cell growth was evaluated by a xenografted model and in vitro cell growth assays. The effect of exon 15 on Slit2-mediated invasion was analyzed with a modified Boyden chamber in vitro. Tumor growth from CL1-5/Slit2-WT cells was comparable to that from CL1-5 cells bearing empty vector. However, tumor size from CL1-5/Slit2-ΔE15 cells was much smaller than that from Slit2-WT cells or vector control cells in the xenografted model. In vitro analyses demonstrated that Slit2-WT inhibits invasion of CL1-5 cells. In addition to inhibiting invasion, Slit2-ΔE15 greatly suppresses cell growth. The data demonstrated that exon 15 modulates Slit2 function in growth inhibition of lung cancer cells. Because slit2-ΔE15 splice variant is present in low invasive cancer cells and nontumor lung tissues, loss of this splice variant is an important event in tumor progression and invasion. Copyright © 2011 American Cancer Society.

  17. A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant.

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    Alexandre Bolze

    Full Text Available We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3. Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the 'rescue' role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic.

  18. Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells.

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    Shafi, Ayesha A; Putluri, Vasanta; Arnold, James M; Tsouko, Efrosini; Maity, Suman; Roberts, Justin M; Coarfa, Cristian; Frigo, Daniel E; Putluri, Nagireddy; Sreekumar, Arun; Weigel, Nancy L

    2015-10-13

    Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.

  19. Identification and functional evaluation of two STAT3 variants in grass carp: Implication for the existence of specific alternative splicing of STAT3 gene in teleost.

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    Du, Linyong; Zhou, Hong; Qin, Lei; Wei, He; Zhang, Anying; Yang, Kun; Wang, Xinyan

    2017-11-01

    A STAT family member, STAT3, becomes activated as a DNA binding protein in response to cytokines and growth factors. In teleost, STAT3 cDNA has been cloned and identified in a few species, but only a single STAT3 transcript is revealed in these studies. In the present study, two variants of STAT3 gene generated by alternative splicing were isolated from grass carp and nominated as STAT3α1 and STAT3α2 based on the homology with their mammalian orthologs. In particular, the homologs of STAT3α1/2 were also found in various fish species, including zebrafish, takifugu, tilapia, medaka and goldfish. Intriguingly, sequence alignment and genomic structure analysis revealed that fish STAT3α1/2 are generated through similar alternative splicing events, implying the potential physiological significance of generating STAT3 variants in fish. Grass carp STAT3α1/2 (gcSTAT3α1/2) were ubiquitously expressed although the transcript levels of STAT3α2 were markedly higher than STAT3α1 in all examined tissues. In vivo and in vitro studies showed that the expression patterns of these two variants were similar under the stimulation of immune stimuli. To reveal the role of gcSTAT3α1/2 in fish immunity, their phosphorylation and involvement in IL-17A/F1 mRNA expression were demonstrated in grass carp peripheral blood lymphocytes upon LPS or PHA challenge, providing evidence for the functional conservation of STAT3 signaling in fish. These findings also raise a question of whether both gcSTAT3α1/2 participate in transcriptional regulation in fish. Actually, our results showed that both of them had the ability to translocate into the nucleus upon activation, and to amplify IL-10 signaling, indicating the existence of STAT3 isoforms with functional redundancy in teleost. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The truncated splice variant of peroxisome proliferator-activated receptor alpha, PPARα-tr, autonomously regulates proliferative and pro-inflammatory genes.

    Science.gov (United States)

    Thomas, Maria; Bayha, Christine; Klein, Kathrin; Müller, Simon; Weiss, Thomas S; Schwab, Matthias; Zanger, Ulrich M

    2015-06-30

    The peroxisome proliferator-activated receptor alpha (PPARα) controls lipid/energy homeostasis and inflammatory responses. The truncated splice variant PPARα-tr was suggested to exert a dominant negative function despite being unable to bind consensus PPARα DNA response elements. The distribution and variability factor of each PPARα variant were assessed in the well-characterized cohort of human liver samples (N = 150) on the mRNA and protein levels. Specific siRNA-mediated downregulation of each transcript as well as specific overexpression with subsequent qRT-PCR analysis of downstream genes was used for investigation of specific functional roles of PPARα-wt and PPARα-tr forms in primary human hepatocytes. Bioinformatic analyses of genome-wide liver expression profiling data suggested a possible role of PPARα-tr in downregulating proliferative and pro-inflammatory genes. Specific gene silencing of both forms in primary human hepatocytes showed that induction of metabolic PPARα-target genes by agonist WY14,643 was prevented by PPARα-wt knock-down but neither prevented nor augmented by PPARα-tr knock-down. WY14,643 treatment did not induce proliferative genes including MYC, CDK1, and PCNA, and knock-down of PPARα-wt had no effect, while PPARα-tr knock-down caused up to 3-fold induction of these genes. Similarly, induction of pro-inflammatory genes IL1B, PTGS2, and CCL2 by IL-6 was augmented by knock-down of PPARα-tr but not of PPARα-wt. In contrast to human proliferative genes, orthologous mouse genes were readily inducible by WY14,643 in PPARα-tr non-expressing AML12 mouse hepatocytes. Induction was augmented by overexpression of PPARα-wt and attenuated by overexpression of PPARα-tr. Pro-inflammatory genes including IL-1β, CCL2 and TNFα were induced by WY14,643 in mouse and human cells and both PPARα forms attenuated induction. As potential mechanism of PPARα-tr inhibitory action we suggest crosstalk with WNT/β-catenin pathway. Finally

  1. Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma

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    Reza Sahebi

    2016-10-01

    Results: The quantitative real-time RT-PCR results revealed a significant up-regulation of linc-ROR (P=0.0098 and its variants 2 (P=0.0250 and 4 (P=0.0002 in tumor samples of ESCC, compared to their matched non-tumor tissues obtained from the margin of same tumors. Our data also demonstrated a significant up-regulation of variant 4 in high-grade tumor samples, in comparison to the low-grade ones (P=0.04. Moreover, the ROC curve analysis demonstrated that the variant 4 of ROR has a potential to discriminate between tumor and non-tumor samples (AUC=0.66, P

  2. Differential expression, distinct localization and opposite effect on Golgi structure and cell differentiation by a novel splice variant of human PRMT5.

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    Sohail, Muhammad; Zhang, Manli; Litchfield, David; Wang, Lisheng; Kung, Sam; Xie, Jiuyong

    2015-10-01

    Alternative splicing contributes greatly to the proteomic diversity of metazoans. Protein arginine methyltransferase 5 (PRMT5) methylates arginines of Golgi components and other factors exerting diverse effects on cell growth/differentiation, but the underlying molecular basis for its subcellular distribution and diverse roles has not been fully understood. Here we show the detailed properties of an evolutionarily emerged splice variant of human PRMT5 (PRMT5S) that is distinct from the original isoform (PRMT5L). The isoforms are differentially expressed among mammalian cells and tissues. The PRMT5S is distributed all over the cell but PRMT5L mainly colocalizes with Giantin, a Golgi marker. PRMT5 knockdown led to an enlarged Giantin pattern, which was prevented by the expression of either isoform. Rescuing PRMT5S also increased the percentage of cells with an interphase Giantin pattern compacted at one end of the nucleus, consistent with its cell cycle-arresting effect, while rescuing PRMT5L increased that of the mitotic Giantin patterns of dynamically fragmented structures. Moreover, the isoforms are differentially expressed during neuronal or dendritic cell differentiation, and their ectopic expression showed an opposite effect on dendritic cell differentiation. Furthermore, besides their differential regulation of gene expression, both isoforms also similarly regulate over a thousand genes particularly those involved in apoptosis and differentiation. Taking these properties together, we propose that their differential expression and subcellular localization contribute to spatial and temporal regulation of arginine methylation and gene expression to exert different effects. The novel PRMT5S likely contributes to the observed diverse effects of PRMT5 in cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Glucocorticoid receptor alpha and beta variant expression is associated with ASF/SF2 splicing factor upregulation in HT-29 colon cancer and MCF-7 breast carcinoma cells.

    Science.gov (United States)

    Piotrowska, Hanna; Jagodzinski, Pawel P

    2009-04-01

    Transcriptional activity of NF-kappaB is inhibited by the liganded glucocorticoid receptor (GR), which exists mainly in two splice variants as functional GRalpha and nonfunctional GRbeta. We investigated the effect of 5-aza-2'-deoxycytidine (5-dAzaC), trichostatin A (TSA), and sodium butyrate (NaBu) on GRalpha,GRbeta and ASF/SF2 splicing factor expression in HT-29 colon and MCF-7 breast carcinoma cells. HT-29 and MCF-7 cells were cultured in the absence or in the presence of 5-dAzaC, TSA, and NaBu, followed by RNA and protein isolation. The transcript and protein levels of GRalpha, GRbeta ASF/SF2 were determined by reverse transcription, real-time quantitative PCR and Western blot analysis. We found that 5-dAzaC, TSA, and NaBu lead to an increase in GRalpha and ASF/SF2 transcript levels and a decrease in GRbeta transcript levels in HT-29 and MCF-7 cells. The 5-dAzaC, TSA, and NaBu resulted in increased GRalpha and ASF/SF2 protein levels and GRbeta protein downregulation in HT-29 cells. The most increased GRalpha protein expression in MCF-7 cells was observed with NaBu. However, all of these compounds inhibited GRbeta protein expression in MCF-7 cells. The MCF-7 cells treated with NaBu demonstrated a remarkable increase in ASF/SF2 protein expression. Because NF-kappaB is considered to be a factor in the augmentation of malignant properties of cells, treatment of tumors with 5-dAzaC, TSA, and NaBu may provide a novel approach to the enhancement of therapeutic effects of glucocorticoids in epithelial carcinomas.

  4. Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes.

    Science.gov (United States)

    Mutti, Navdeep S; Wang, Ying; Kaftanoglu, Osman; Amdam, Gro V

    2011-01-01

    Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life. Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees. Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.

  5. Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant

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    Slat Emily A

    2007-07-01

    Full Text Available Abstract Background Voltage-gated Na+ channel β1 (Scn1b subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that β1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel α subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of β1 subunits in vivo we cloned two β1-like subunit cDNAs from D. rerio. Results Two β1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian β1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian β1 subunits. In

  6. A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins.

    Science.gov (United States)

    Stutika, Catrin; Gogol-Döring, Andreas; Botschen, Laura; Mietzsch, Mario; Weger, Stefan; Feldkamp, Mirjam; Chen, Wei; Heilbronn, Regine

    2015-11-11

    Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study

  7. A splice variant of the two-pore domain potassium channel TREK-1 with only one pore domain reduces the surface expression of full-length TREK-1 channels.

    Science.gov (United States)

    Rinné, Susanne; Renigunta, Vijay; Schlichthörl, Günter; Zuzarte, Marylou; Bittner, Stefan; Meuth, Sven G; Decher, Niels; Daut, Jürgen; Preisig-Müller, Regina

    2014-08-01

    We have identified a novel splice variant of the human and rat two-pore domain potassium (K2P) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents.

  8. Detection and Quantization of the Expression of Two mu-Opioid Receptor Splice Variants mRNA (hMOR-1A and hMOR-1O in Peripheral Blood Lymphocytes of Long-Term Abstinent Former Opioid Addicts

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    N Vousooghi, Pharm

    2012-05-01

    Full Text Available

    Background and Objectives

    The mu-Opioid receptor (MOR exerts a critical role on effects of opiodis. The objective of this study is to find a peripheral bio-marker in addiction studies through quantization of the expression of two MOR splice variants mRNA (hMOR-1A and hMOR-1O in peripheral blood lymphocytes (PBLs of long-term abstinent former opioids addicts.

    Methods

    In this case-control study, case and control people were male and divided in two groups: people who gave up addiction to opioids (case and healthy individuals without history of addiction (control. The mRNA expression in PBLs of participants was detected and measured by real-time Polymerase Chain Reaction (PCR using SYBR Green Dye.

    Results

    The hMOR-1A mRNA expression in PBLs of abstinent group was significantly reduced and reached to 0.33 of the control group (p<0.001. Similar results were obtained for the other splice variant with the mRNA expression of hMOR-1O in PBLs of abstinent group reaching to 0.38 of that of the control group (p < 0.001.

    Conclusion

    mRNA expression deficiency of two mu-opioid receptor splice variants, hMOR-1A and nMOR-1O, seams to be a risk factor making individuals vulnerable to drug addiction. Based on this analysis measuring the amount of mRNA expression of these two splice variants in PBLs can serve as a peripheral bio-marker for detecting people at risk.

  9. Missense splice variant (g.20746A>G, p.Ile183Val of interferon gamma receptor 1 (IFNGR1 coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions

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    Agnieszka Bińczak-Kuleta

    2016-08-01

    Full Text Available Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1 susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100 was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val. No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205, possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003% was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity.

  10. Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants.

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    Lasse Folkersen

    Full Text Available BACKGROUND: Several genome-wide association studies have recently linked a group of single nucleotide polymorphisms in the 9p21 region with cardiovascular disease. The molecular mechanisms of this link are not fully understood. We investigated five different expression microarray datasets in order to determine if the genotype had effect on expression of any gene transcript in aorta, mammary artery, carotid plaque and lymphoblastoid cells. METHODOLOGY/PRINCIPAL FINDINGS: After multiple testing correction, no genes were found to have relation to the rs2891168 risk genotype, either on a genome-wide scale or on a regional (8 MB scale. The neighbouring ANRIL gene was found to have eight novel transcript variants not previously known from literature and these varied by tissue type. We therefore performed a detailed probe-level analysis and found small stretches of significant relation to genotype but no consistent associations. In all investigated tissues we found an inverse correlation between ANRIL and the MTAP gene and a positive correlation between ANRIL and CDKN2A and CDKN2B. CONCLUSIONS/SIGNIFICANCE: Investigation of relation of the risk genotype to gene expression is complicated by the transcript complexity of the locus. With our investigation of a range of relevant tissue we wish to underscore the need for careful attention to the complexity of the alternative splicing issues in the region and its implications to the design of future gene expression studies.

  11. A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

    Directory of Open Access Journals (Sweden)

    Pei-Jen Hsu

    Full Text Available Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4 promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

  12. A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4

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    Ando, Hitoshi [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Department of Neurosurgery, Fukushima Medical University School of Medicine, Fukushima (Japan); Natsume, Atsushi, E-mail: anatsume@med.nagoya-u.ac.jp [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Iwami, Kenichiro; Ohka, Fumiharu [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Kuchimaru, Takahiro; Kizaka-Kondoh, Shinae [Department of Biomolecular Engineering, Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Yokohama (Japan); Ito, Kengo [National Center for Geriatrics and Gerontology, Aichi (Japan); Saito, Kiyoshi [Department of Neurosurgery, Fukushima Medical University School of Medicine, Fukushima (Japan); Sugita, Sachi; Hoshino, Tsuneyoshi [MICRON Inc.Medical Facilities Support Department, Aichi (Japan); Wakabayashi, Toshihiko [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan)

    2013-03-29

    Highlights: ► HIF-3α4 is silenced by DNA methylation in meningiomas. ► Induction of HIF-3α4 impaired angiogenesis in meningiomas. ► Induction of HIF-3α4 impaired proliferation and oxygen-dependent metabolism. -- Abstract: Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas.

  13. Cell surface expression of MR1B, a splice variant of the MHC class I-related molecule MR1, revealed with antibodies.

    Science.gov (United States)

    Yamaguchi, Hisateru; Tsukamoto, Kentaro; Hashimoto, Keiichiro

    2014-01-10

    The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Multiple Superoxide Dismutase 1/Splicing Factor Serine Alanine 15 Variants Are Associated With the Development and Progression of Diabetic Nephropathy

    Science.gov (United States)

    Al-Kateb, Hussam; Boright, Andrew P.; Mirea, Lucia; Xie, Xinlei; Sutradhar, Rinku; Mowjoodi, Alireza; Bharaj, Bhupinder; Liu, Michelle; Bucksa, Jean M.; Arends, Valerie L.; Steffes, Michael W.; Cleary, Patricia A.; Sun, Wanjie; Lachin, John M.; Thorner, Paul S.; Ho, Michael; McKnight, Amy Jayne; Maxwell, A. Peter; Savage, David A.; Kidd, Kenneth K.; Kidd, Judith R.; Speed, William C.; Orchard, Trevor J.; Miller, Rachel G.; Sun, Lei; Bull, Shelley B.; Paterson, Andrew D.

    2008-01-01

    BACKGROUND Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS We observed association between rs17880135 in the 3′ region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64– 4.18], P = 5.6 × 10−5, q = 0.06) and persistent microalbuminuria (1.82 [1.29 –2.57], P = 6.4 × 10−4, q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10−3) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study. PMID:17914031

  15. Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Mark Schütte

    Full Text Available BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16 which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

  16. Identification of Splice Variants, Targeted MicroRNAs and Functional Single Nucleotide Polymorphisms of the BOLA-DQA2 Gene in Dairy Cattle

    Science.gov (United States)

    Hou, Qinlei; Huang, Jinming; Ju, Zhihua; Li, Qiuling; Li, Liming; Wang, Changfa; Sun, Tao; Wang, Lingling; Hou, Minghai

    2012-01-01

    Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3′-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3′-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (pmastitis-infected mammary tissues (n=5) compared with the healthy mammary gland mammary tissues (n=5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (pmastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation. PMID:22084936

  17. Natural splice variant of MHC class I cytoplasmic tail enhances dendritic cell-induced CD8+ T-cell responses and boosts anti-tumor immunity.

    Directory of Open Access Journals (Sweden)

    Tania G Rodríguez-Cruz

    Full Text Available Dendritic cell (DC-mediated presentation of MHC class I (MHC-I/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7, including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-K(b generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-D(b-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1 T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-D(b DCs were superior to WT-D(b DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.

  18. The Mouse-Specific Splice Variant mRAGE_v4 Encodes a Membrane-Bound RAGE That Is Resistant to Shedding and Does Not Contribute to the Production of Soluble RAGE

    Science.gov (United States)

    Liu, Jaron; Bertolotti, Matteo; Fritz, Günter; Bianchi, Marco E.; Raucci, Angela

    2016-01-01

    The receptor for advanced glycation end-products (RAGE) is involved in the onset and progression of several inflammatory diseases. The RAGE primary transcript undergoes numerous alternative splicing (AS) events, some of which are species-specific. Here, we characterize the mouse-specific mRAGE_v4 splice variant, which is conserved in rodents and absent in primates. mRAGE_v4 derives from exon 9 skipping and encodes a receptor (M-RAGE) that lacks 9 amino acids between the transmembrane and the immunoglobulin (Ig) domains. RNA-Seq data confirm that in mouse lung mRAGE_v4 is the most abundant RAGE mRNA isoform after mRAGE, which codes for full-length RAGE (FL-RAGE), while in heart all RAGE variants are almost undetectable. The proteins M-RAGE and FL-RAGE are roughly equally abundant in mouse lung. Contrary to FL-RAGE, M-RAGE is extremely resistant to shedding because it lacks the peptide motif recognized by both ADAM10 and MMP9, and does not contribute significantly to soluble cRAGE formation. Thus, a cassette exon in RAGE corresponds to a specific function of the RAGE protein–the ability to be shed. Given the differences in RAGE AS variants between rodents and humans, caution is due in the interpretation of results obtained in mouse models of RAGE-dependent human pathologies. PMID:27655137

  19. A novel splice variant in the N-propeptide of COL5A1 causes an EDS phenotype with severe kyphoscoliosis and eye involvement.

    Directory of Open Access Journals (Sweden)

    Sofie Symoens

    Full Text Available BACKGROUND: The Ehlers-Danlos Syndrome (EDS is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2. Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. METHODOLOGY/PRINCIPAL FINDINGS: We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. CONCLUSIONS/SIGNIFICANCE: We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site

  20. Targeting of a CCK{sub 2} receptor splice variant with {sup 111}In-labelled cholecystokinin-8 (CCK8) and {sup 111}In-labelled minigastrin

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Gotthardt, Martin; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Center, Department of Nuclear Medicine, PO Box 9101, HB Nijmegen (Netherlands); Roosenburg, Susan [Radboud University Nijmegen Medical Center, Department of Nuclear Medicine, PO Box 9101, HB Nijmegen (Netherlands); Radboud University Nijmegen, Institute for Molecules and Materials, Nijmegen (Netherlands); Park, Jeseong; Hellmich, Mark R. [University of Texas Medical Branch, Department of Surgery and the Sealy Center for Cancer Cell Biology, Galveston, TX (United States); Jong, Marion de [Erasmus Medical Center, Department of Nuclear Medicine, Rotterdam (Netherlands); Rutjes, Floris P.J.T.; Delft, Floris L. van [Radboud University Nijmegen, Institute for Molecules and Materials, Nijmegen (Netherlands)

    2008-02-15

    Radiolabelled cholecystokinin (CCK) and gastrin-derived peptides potentially can be used for peptide receptor radionuclide therapy (PRRT). Recently, a splice variant version of the CCK2R has been identified, designated CCK2i4svR. Constitutive expression of this receptor has been demonstrated in human colorectal cancer and in pancreatic cancer, but not in normal tissue. So far, it has never been shown whether radiolabelled peptides can target the CCK2i4svR in vivo. In this paper, we investigated the potential of sulfated {sup 111}In-labelled DOTA-CCK8 (sCCK8), a pan-CCKR-binding peptide, and [{sup 111}In]DOTA-minigastrin (MG0), a CCK2R selective peptide, for the targeting of the CCK2i4svR. The receptor binding affinity of [{sup 111}In]DOTA-sCCK8 and [{sup 111}In]DOTA-MG0 for the CCK2R and CCK2i4svR was determined using stably transfected HEK293 cell lines, expressing either CCK2R or CCK2i4svR. Tumour targeting was studied in HEK293-CCK2i4svR tumour-bearing athymic mice. [{sup 111}In]DOTA-sCCK8 as well as [{sup 111}In]DOTA-MG0 specifically bound both CCK2R and CCK2i4svR with affinities in the low nanomolar range. In vivo experiments revealed that accumulation of both peptides in CCK2i4svR-positive tumours was similar (3.21 {+-} 0.77 and 3.01 {+-} 0.67%ID/g, sCCK8 and MG0, respectively, 24 h p.i.). Kidney retention of [{sup 111}In]DOTA-MG0 (32.4 {+-} 7.5%ID/g, 24 h p.i.) was markedly higher than that of [{sup 111}In]DOTA-sCCK8 (2.75 {+-} 0.31%ID/g, 24 h p.i.). We demonstrated that the CCK2i4svR is a potential target for PRRT using a radiolabelled sulfated CCK8 peptide. As this receptor is expressed on colorectal and pancreatic tumours, but not in normal tissue, these tumours are potentially new targets for PRRT with CCK8 and gastrin analogs. (orig.)

  1. A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3.

    Science.gov (United States)

    Chen, Tao; Tompkins, Leslie M; Li, Linhao; Li, Haishan; Kim, Gregory; Zheng, Yuxin; Wang, Hongbing

    2010-01-01

    The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of five amino acids (APYLT), exhibits low basal but xenobiotic-inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the five amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1+A) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic-sensitive hCAR3. It is noteworthy that hCAR1+A was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3. Mammalian two-hybrid and glutathione S-transferase pull-down assays further demonstrated that hCAR1+A interacts with the coactivator SRC-1 and GRIP-1 at low level before activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1+A may represent a sensitive avenue for the identification of hCAR activators.

  2. Soluble forms of CD46 are detected in Bos taurus plasma and neutralize BVDV, the bovine pestivirus.

    Science.gov (United States)

    Alzamel, Nidal; Bayrou, Calixte; Decreux, Annabelle; Desmecht, Daniel

    2016-12-01

    The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal new CD46-encoding transcripts that are predicted to encode CCP1-containing soluble forms. Further, we show that the serum of most adult cattle contains soluble CD46 (sCD46) and that a recombinant soluble isoform neutralizes BVDV infectivity in an in vitro assay. We have then established an ELISA for determination of plasma sCD46 in a large cohort of animals. Overall, serum sCD46 amounts to 8±18ng/mL (mean±SD, n=440), with a IC [95-105] ranging from 6,4 to 9,8ng/mL and extreme values ​​between 0 and 178ng/mL. We found that sCD46 is not detectable in fetal and neonatal sera and that its plasma concentration increases progressively up to adulthood. We also detected high- and low-sCD46 performers and show that this phenotype does not depend of environment. As modern rearing techniques make it possible to disseminate genetically-determined phenotypes very quickly in a population, a large-scale study examining whether high-sCD46 animals provide epidemiological protection against BVDV infection and transmission should be undertaken. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

    Czech Academy of Sciences Publication Activity Database

    Frolíková, Michaela; Šebková, Nataša; Děd, Lukáš; Dvořáková-Hortová, Kateřina

    2016-01-01

    Roč. 6, Sep 26 (2016), s. 1-15 ISSN 2045-2322 R&D Projects: GA ČR GA14-05547S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : CD46 * β1 integrin * acrosome reaction * actin * SIM * latrunculin A Subject RIV: EC - Immunology Impact factor: 4.259, year: 2016

  4. Recurrent atypical haemolytic uraemic syndrome post kidney transplant due to a CD46 mutation in the setting of SMARCAL1-mediated inherited kidney disease.

    Science.gov (United States)

    Chan, Samuel; Mallett, Andrew J; Patel, Chirag; Francis, Ross S; Johnson, David W; Mudge, David W; Isbel, Nicole M

    2017-02-01

    Disorders in the regulation of the alternate complement pathway often result in complement-mediated damage to the microvascular endothelium and can be associated with both glomerulonephritis and atypical haemolytic uraemic syndrome. Inherited defects in complement regulatory genes or autoantibodies against complement regulatory proteins are predictive of the severity of the disease and the risk of recurrence post kidney transplantation. Heterozygous mutations in CD46, which codes for a transmembrane cofactor glycoprotein membrane cofactor protein, usually have a lower incidence of end-stage kidney disease and decreased risk of recurrent disease post transplant, as wild-type membrane cofactor protein is present in the transplanted kidney. However, some patients with CD46 mutations have a second variant in other complement regulatory genes increasing the severity of disease. The following case report illustrates the course of a young adult patient with end-stage kidney disease initially ascribed to seronegative systemic lupus erythematosus, who presented with biopsy-proven thrombotic microangiopathy following kidney transplantation. It highlights the complexity associated with disorders of complement regulation and the need for a high index of suspicion and genetic testing in patients who present with thrombotic microangiopathy post-transplant. © 2017 Asian Pacific Society of Nephrology.

  5. Significant changes in ITIH4, AHSG, ORM1, and CD46 content in milk fat globule membrane proteins of ketotic dairy cows.

    Science.gov (United States)

    Gao, Sansi; Yang, Wei; Yu, Hongjiang; Liu, Runqi; Dong, Zhihao; Zhang, Hongyou; Xia, Cheng; Xu, Chuang

    2017-11-01

    High concentrations of non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) in cows' blood caused by ketosis are associated with inflammatory states. We hypothesised that ketosis in postparturient dairy cows would result in altered levels on inflammation-related proteins not only in plasma but also in the milk fat globule membranes (MFGM). Thirty cows were selected from a dairy farm in Heilongjiang, China. Inflammatory milk fat globule membrane proteins were detected using ELISA kits, and a fully automatic biochemical analyser was used to measure the concentrations of BHBA, NEFA, glucose (GLU) and triglyceride (TG) in plasma. MFGM protein from milk of ketotic cows contained significantly different concentrations of acute-phase response proteins (complement C3 (C3), prothrombin (F2), alpha-1-acid glycoprotein (ORM1), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-HS-glycoprotein (AHSG), complement C9 (C9), complement regulatory protein variant 4 (CD46)) in comparison with milk from non-ketotic cows. Blood concentrations of C3, complement C9 (C9), tumour necrosis factor α (TNFα), MFGM C3, monocyte differentiation antigen CD14 (CD14) and ORM1 levels were correlated with energy balance. ITIH4 and CD46 increased, and AHSG and ORM1 decreased before the onset of ketosis. These biomarkers offer potential as predictors and monitors of ketosis in at-risk cows.

  6. Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms.

    Science.gov (United States)

    de la Hoya, Miguel; Soukarieh, Omar; López-Perolio, Irene; Vega, Ana; Walker, Logan C; van Ierland, Yvette; Baralle, Diana; Santamariña, Marta; Lattimore, Vanessa; Wijnen, Juul; Whiley, Philip; Blanco, Ana; Raponi, Michela; Hauke, Jan; Wappenschmidt, Barbara; Becker, Alexandra; Hansen, Thomas V O; Behar, Raquel; Investigators, KConFaB; Niederacher, Diether; Arnold, Norbert; Dworniczak, Bernd; Steinemann, Doris; Faust, Ulrike; Rubinstein, Wendy; Hulick, Peter J; Houdayer, Claude; Caputo, Sandrine M; Castera, Laurent; Pesaran, Tina; Chao, Elizabeth; Brewer, Carole; Southey, Melissa C; van Asperen, Christi J; Singer, Christian F; Sullivan, Jan; Poplawski, Nicola; Mai, Phuong; Peto, Julian; Johnson, Nichola; Burwinkel, Barbara; Surowy, Harald; Bojesen, Stig E; Flyger, Henrik; Lindblom, Annika; Margolin, Sara; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Galastri, Laura; Olson, Janet E; Hallberg, Emily; Giles, Graham G; Milne, Roger L; Andrulis, Irene L; Glendon, Gord; Hall, Per; Czene, Kamila; Blows, Fiona; Shah, Mitul; Wang, Qin; Dennis, Joe; Michailidou, Kyriaki; McGuffog, Lesley; Bolla, Manjeet K; Antoniou, Antonis C; Easton, Douglas F; Couch, Fergus J; Tavtigian, Sean; Vreeswijk, Maaike P; Parsons, Michael; Meeks, Huong D; Martins, Alexandra; Goldgar, David E; Spurdle, Amanda B

    2016-06-01

    A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10(-8) Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. RNA-Binding Proteins: Splicing Factors and Disease

    Directory of Open Access Journals (Sweden)

    Alger M. Fredericks

    2015-05-01

    Full Text Available Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions.

  8. Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

    Science.gov (United States)

    Suwanmanee, Siros; Buckingham, Steven David; Biggin, Philip; Sattelle, David

    2014-01-01

    Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides. PMID:24823815

  9. Actions of agonists, fipronil and ivermectin on the predominant in vivo splice and edit variant (RDLbd, I/V of the Drosophila GABA receptor expressed in Xenopus laevis oocytes.

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    Kristin Lees

    Full Text Available Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin, originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides.

  10. GLIS3, a susceptibility gene for type 1 and type 2 diabetes, modulates pancreatic beta cell apoptosis via regulation of a splice variant of the BH3-only protein Bim.

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    Tatiane C Nogueira

    2013-05-01

    Full Text Available Mutations in human Gli-similar (GLIS 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1β + interferon-γ or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS.

  11. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

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    B David Persson

    2010-09-01

    Full Text Available The human membrane cofactor protein (MCP, CD46 is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6, Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4 that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

  12. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: A Pipeline for the Development of Antisense Oligonucleotides

    NARCIS (Netherlands)

    A.J. Bergsma (Atze); S.L.M. in 't Groen (Stijn); F.W. Verheijen (Frans); A.T. van der Ploeg (Ans); W.W.M.P. Pijnappel (Pim)

    2016-01-01

    textabstractWhile 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening

  13. CD46-mediated transduction of a species D adenovirus vaccine improves mucosal vaccine efficacy.

    Science.gov (United States)

    Camacho, Zenaido T; Turner, Mallory A; Barry, Michael A; Weaver, Eric A

    2014-04-01

    The high levels of preexisting immunity against Adenovirus type 5 (Ad5) have deemed Ad5 unusable for translation as a human vaccine vector. Low seroprevalent alternative viral vectors may be less impacted by preexisting immunity, but they may also have significantly different phenotypes from that of Ad5. In this study we compare species D Ads (26, 28, and 48) to the species C Ad5. In vitro transduction studies show striking differences between the species C and D viruses. Most notably, Ad26 transduced human dendritic cells much more effectively than Ad5. In vivo imaging studies showed strikingly different transgene expression profiles. The Ad5 virus was superior to the species D viruses in BALB/c mice when delivered intramuscularly. However, the inverse was true when the viruses were delivered mucosally via the intranasal epithelia. Intramuscular transduction was restored in mice that ubiquitously expressed human CD46, the primary receptor for species D viruses. We analyzed both species C and D Ads for their ability to induce prophylactic immunity against influenza in the CD46 transgenic mouse model. Surprisingly, the species D vaccines again failed to induce greater levels of protective immunity as compared with the species C Ad5 when delivered intramuscularly. However, the species D Ad vaccine vector, Ad48, induced significantly greater protection as compared with Ad5 when delivered mucosally via the intranasal route in CD46 transgenic mice. These data shed light on the complexities between the species and types of Ad. Our findings indicate that more research will be required to identify the mechanisms that play a key role in the induction of protective immunity induced by species D Ad vaccines.

  14. Capillary Electrophoresis Analysis of Conventional Splicing Assays

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    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation...... of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...... International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c...

  15. Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts

    NARCIS (Netherlands)

    R. Kraaij (Robert); M. Verhoef-Post (Miriam); J.A. Grootegoed (Anton); A.P.N. Themmen (Axel)

    1998-01-01

    textabstractGlycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating

  16. Shaping the Arabidopsis Transcriptome through Alternative Splicing

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    Dorothee Staiger

    2015-01-01

    Full Text Available Alternative splicing is a molecular tool of the cell to generate more than one messenger RNA from the same gene. Through variable combinations of exons blueprints for different proteins are assembled from one and the same pre-messenger RNA, thus increasing the complexity of the proteome. Moreover, through alternative splicing different transcript variants with different stabilities and different regulatory motifs can be generated, leading to variation in the transcriptome. The importance of alternative splicing in plants has been increasingly recognized in the last decade. Alternative splicing has been found during abiotic and biotic stress and during development. Here, recent advancements in the understanding of alternative splicing in higher plants are presented. Mechanistic details and functional consequences of alternative splicing are discussed with a focus on the model plant Arabidopsis thaliana.

  17. Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

    DEFF Research Database (Denmark)

    Bendová, Z; Sumová, A; Mikkelsen, Jens D.

    2009-01-01

    The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase...... shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties...... of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis...

  18. Comparative study on the immunogenicity between an HLA-A24-restricted cytotoxic T-cell epitope derived from survivin and that from its splice variant survivin-2B in oral cancer patients

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    Yamaguchi Akira

    2009-01-01

    Full Text Available Abstract Background We previously reported an HLA-A24-restricted cytotoxic T-cell epitope, Survivin-2B80-88, derived from a splice variant of survivin, survivin-2B. In this report, we show a novel HLA-A24-restricted T-cell epitope, Survivin-C58, derived from a wild type survivin, and compared their immunogenicity in oral cancer patients. Methods By stimulating peripheral blood lymphocytes of HLA-A24-positive cancer patients with Survivin-C58 peptide in vitro, the peptide-specific CTLs were induced. In order to compare the immunogenic potential between C58 peptide and 2B80-88 peptide, peripheral blood T-cells from thirteen HLA-A24-positive oral cancer patients were stimulated with either or both of these two peptides. Results Survivin-2B80-88 peptide-specific CTLs were induced from four patients, and C58 peptide-specific CTLs were induced from three out of eight patients with over stage II progression. The CTLs exerted cytotoxicity against HLA-A24-positive tumor cells. In contrast, CTL induction failed from a healthy volunteer and all four patients with cancer stage I. Conclusion It was indicated that a splicing variant-derived peptide and wild type survivin-derived peptide might have a comparable potency of CTL induction, and survivin targeting immunotherapy using survivin-2B80-88 and C58 peptide cocktail should be suitable for HLA-A24+ oral cancer patients.

  19. Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

    Science.gov (United States)

    Brummell, David A; Chen, Ronan K Y; Harris, John C; Zhang, Huaibi; Hamiaux, Cyril; Kralicek, Andrew V; McKenzie, Marian J

    2011-06-01

    Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.

  20. Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

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    Helen M Wise

    Full Text Available Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42 with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

  1. Role of Splice Variants of Gtf2i, a Transcription Factor Localizing at Postsynaptic Sites, and Its Relation to Neuropsychiatric Diseases

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    Yoshinori Shirai

    2017-02-01

    Full Text Available We previously reported that various mRNAs were associated with postsynaptic density (PSD purified from rat forebrain. Among the thousands of PSD-associated mRNAs, we highlight the biology of the general transcription factor II-I (Gtf2i mRNA, focusing on the significance of its versatile splicing for targeting its own mRNA into dendrites, regulation of translation, and the effects of Gtf2i expression level as well as its relationship with neuropsychiatric disorders.

  2. Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

    Science.gov (United States)

    del Pilar Jiménez-A, M; Viriyakosol, S; Walls, L; Datta, S K; Kirkland, T; Heinsbroek, S E M; Brown, G; Fierer, J

    2008-06-01

    Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

  3. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

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    Atze J Bergsma

    2016-01-01

    Full Text Available While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.

  4. C-terminal splice variants of P/Q-type Ca(2+) channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins.

    Science.gov (United States)

    Hirano, Mitsuru; Takada, Yoshinori; Wong, Chee Fah; Yamaguchi, Kazuma; Kotani, Hiroshi; Kurokawa, Tatsuki; Mori, Masayuki X; Snutch, Terrance P; Ronjat, Michel; De Waard, Michel; Mori, Yasuo

    2017-06-02

    Voltage-dependent Ca(2+) channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. 1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

    Science.gov (United States)

    Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

    2017-10-01

    Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D3, the biologically active form of vitamin D3, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. An Interspecific Plant Hybrid Shows Novel Changes in Parental Splice Forms of Genes for Splicing Factors

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    Scascitelli, Moira; Cognet, Marie; Adams, Keith L.

    2010-01-01

    Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization. PMID:20100939

  7. A novel human TPIP splice-variant (TPIP-C2 mRNA, expressed in human and mouse tissues, strongly inhibits cell growth in HeLa cells.

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    Rasmi Rekha Mishra

    Full Text Available Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10, TPTE (Transmembrane Phosphatase with TEnsin homology and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2 from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5'-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85% inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.

  8. Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling.

    Science.gov (United States)

    Zhu, Yi; Zhang, Jing-Jing; Xie, Kun-Ling; Tang, Jie; Liang, Wen-Biao; Zhu, Rong; Zhu, Yan; Wang, Bin; Tao, Jin-Qiu; Zhi, Xiao-Fei; Li, Zheng; Gao, Wen-Tao; Jiang, Kui-Rong; Miao, Yi; Xu, Ze-Kuan

    2014-11-04

    MUC4 plays important roles in the malignant progression of human pancreatic cancer. But the huge length of MUC4 gene fragment restricts its functional and mechanism research. As one of its splice variants, MUC4/Y with coding sequence is most similar to that of the full-length MUC4 (FL-MUC4), together with alternative splicing of the MUC4 transcript has been observed in pancreatic carcinomas but not in normal pancreas. So we speculated that MUC4/Y might be involved in malignant progression similarly to FL-MUC4, and as a research model of MUC4 in pancreatic cancer. The conjecture was confirmed in the present study. MUC4/Y expression was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific probe in the clinic samples. The effects of MUC4/Y were observed by serial in vitro and in vivo experiments based on stable over-expressed cell model. The underlying mechanisms were investigated by sequence-based transcriptome analysis and verified by qRT-PCR, Western blot and enzyme-linked immunosorbent assays. The detection of clinical samples indicates that MUC4/Y is significantly positive-correlated with tumor invasion and distant metastases. Based on stable forced-expressed pancreatic cancer PANC-1 cell model, functional studies show that MUC4/Y enhances malignant activity in vitro and in vivo, including proliferation under low-nutritional-pressure, resistance to apoptosis, motility, invasiveness, angiogenesis, and distant metastasis. Mechanism studies indicate the novel finding that MUC4/Y triggers malignancy-related positive feedback loops for concomitantly up-regulating the expression of survival factors to resist adverse microenvironment and increasing the expression of an array of cytokines and adhesion molecules to affect the tumor milieu. In light of the enormity of the potential regulatory circuitry in cancer afforded by MUC4 and/or MUC4/Y, repressing MUC4 transcription, inhibiting post

  9. COMPARISON OF CD46 EXPRESSION ON THE INNER ACROSOMAL MEMBRANE OF SPERMS FROM NORMOSPERMIC AND ASTHENOSPERMIC INDIVIDUALS

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    M NASR ESFAHANI

    2003-03-01

    Full Text Available Introduction: CD46 is a membrane cofactor protein (MCP of complement system wich is present on the membrane of all somatic cells except RBC. It is also present on the inner acrosmal membrane of human sperm. Thus, the aim of this study was to compare the expression of this prote, in on the inner acrosmal membrane of sperms from normospermic and asthenospermic individuals. Method: Semen from 6 normospermic and 17 asthenospermic individuals were examined for CD46 expression. After solublization of sperms in solublizing detergent, the solublized sperm membrane was separated from the rest of cell organelles by centrifugation. Solublized sperm membrane were divide to equal parts and SOS-PAGE gel was canied out in paired on the same gel for each sample. Western blot was carried out on half of the gel and then the nitrocellose papers were stained by a monocolonal Ab and HRP conjugate Ab. The other half were stained by silver stain for identification of MW. Results: After scoring the stained nitrocellose papen in each groups, no statistical significant difference was observed for C046 expression between the two groups. However, a significant Spearmen correlation was observed between CD46 expression and sperm motility (r=0.597, P=0.003. The MW of C046 was between 36 to 45 KD. with a mean of 42 KD. Discussion: This is the first report of a positive Spearmen correlation between sperm CD46 expression and sperm motility which suggest that there might be relation between CD46 expression and sperm motility.

  10. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Aik Jiang; Chang, Thomas K.H., E-mail: thomas.chang@ubc.ca

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  11. Alternative splicing of Spg7, a gene involved in hereditary spastic paraplegia, encodes a variant of paraplegin targeted to the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Giuseppe Mancuso

    Full Text Available BACKGROUND: Hereditary spastic paraplegia defines a group of genetically heterogeneous diseases characterized by weakness and spasticity of the lower limbs owing to retrograde degeneration of corticospinal axons. One autosomal recessive form of the disease is caused by mutation in the SPG7 gene. Paraplegin, the product of SPG7, is a component of the m-AAA protease, a high molecular weight complex that resides in the mitochondrial inner membrane, and performs crucial quality control and biogenesis functions in mitochondria. PRINCIPAL FINDINGS: Here we show the existence in the mouse of a novel isoform of paraplegin, which we name paraplegin-2, encoded by alternative splicing of Spg7 through usage of an alternative first exon. Paraplegin-2 lacks the mitochondrial targeting sequence, and is identical to the mature mitochondrial protein. Remarkably, paraplegin-2 is targeted to the endoplasmic reticulum. We find that paraplegin-2 exposes the catalytic domains to the lumen of the endoplasmic reticulum. Moreover, endogenous paraplegin-2 accumulates in microsomal fractions prepared from mouse brain and retina. Finally, we show that the previously generated mouse model of Spg7-linked hereditary spastic paraplegia is an isoform-specific knock-out, in which mitochondrial paraplegin is specifically ablated, while expression of paraplegin-2 is retained. CONCLUSIONS/SIGNIFICANCE: These data suggest a possible additional role of AAA proteases outside mitochondria and open the question of their implication in neurodegeneration.

  12. The emerging role of alternative splicing in senescence and aging.

    Science.gov (United States)

    Deschênes, Mathieu; Chabot, Benoit

    2017-10-01

    Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  13. Alternative BCR/ABL splice variants in Philadelphia chromosome-positive leukemias result in novel tumor-specific fusion proteins that may represent potential targets for immunotherapy approaches.

    Science.gov (United States)

    Volpe, Gisella; Cignetti, Alessandro; Panuzzo, Cristina; Kuka, Mirela; Vitaggio, Katiuscia; Brancaccio, Mara; Perrone, Giuseppe; Rinaldi, Monica; Prato, Giuseppina; Fava, Milena; Geuna, Massimo; Pautasso, Marisa; Casnici, Claudia; Signori, Emanuela; Tonon, Giancarlo; Tarone, Guido; Marelli, Ornella; Fazio, Vito M; Saglio, Giuseppe

    2007-06-01

    Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

  14. Crystallization and preliminary X-ray analysis of the splice variant of human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9-2).

    Science.gov (United States)

    Fei, Xiangwei; Zhang, Yong; Gu, Xing; Qiu, Rui; Mao, Yumin; Ji, Chaoneng

    2008-01-01

    Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9), a subunit of an Elongin C-cullin-SOCS box (ECS) E3 ubiquitin ligase complex, is believed to be involved in specific substrate-recognition for ubiquitination and degradation. In fact, this specific substrate-recognition is determined by the ankyrin repeats of hASB9 protein. Here, we have cloned and overexpressed the hASB9-2, the splice variant of hASB9 with only one ankyrin repeat domain, as a 6His-tagged recombinant protein in Escherichia coli. The purified hASB9-2 protein was crystallized by the hanging-drop vapor-diffusion technique and diffracted to 2.2A resolution. The data showed that the cubic hASB9-2 crystal belongs to space group P4(3)32 with unit-cell parameters (a=b=c=129.25A, alpha=beta=gamma=90 degrees ). An asymmetric unit in the crystal was assumed to contain one protein molecule giving the Matthews Coefficient factor of 2.81 and the solvent content of 56.3%.

  15. Complement regulatory proteins (CD46, 55 and 59) expressed on Schwann cells: immune targets in demyelinating neuropathies?

    Science.gov (United States)

    Miyaji, Kazuki; Paul, Friedemann; Shahrizaila, Nortina; Umapathi, Thirugnanam; Yuki, Nobuhiro

    2014-11-15

    Given their localization and important role in regulating complement, complement regulatory proteins may act as target antigens and their antibodies as biomarkers in demyelinating neuropathies. We investigated the binding of autoantibodies to complement regulatory proteins (CD46, 55 and 59) in demyelinating diseases. In 42 acute inflammatory demyelinating polyneuropathy, 23 chronic inflammatory demyelinating polyneuropathy, 13 acute motor axonal neuropathy, 71 multiple sclerosis, and 19 neuromyelitis optica patients as well as 55 healthy controls, we were unable to detect significant titers of antibodies to CD46, CD55 and CD59. These autoantibodies are unlikely to be biomarkers in acute and chronic inflammatory demyelinating polyneuropathies. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. NT5C2 novel splicing variant expands the phenotypic spectrum of Spastic Paraplegia (SPG45): case report of a new member of thin corpus callosum SPG-Subgroup.

    Science.gov (United States)

    Elsaid, Mahmoud F; Ibrahim, Khalid; Chalhoub, Nader; Elsotouhy, Ahmed; El Mudehki, Noora; Abdel Aleem, Alice

    2017-03-21

    Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative diseases. Thin Corpus Callosum (TCC) associated HSP is a distinguished subgroup of complex forms. Purines and pyrimidine, the basic DNA and RNA components, are regulating the cell metabolism, having roles in signal transduction, energy preservation and cellular repair. Genetic defects in nucleotide metabolism related genes have been only recently implicated in brain and neurodegenerative diseases' pathogenesis. We present a consanguineous Qatari family with two brothers, 9 and 3 years, who displayed a characteristic phenotype of early onset and markedly-severe spasticity with tiptoe walking, delayed dysarthric speech, persistent truncal hypotonia, and multiple variable-sized areas of brownish skin discoloration appearing at different places on the body. A clinical diagnosis suggestive of complex hereditary spastic paraplegia (HSP) was set after the family had the second affected child. Whole genome sequencing identified a novel homozygous NT5C2 splice site mutation (NM_012229.4/NM_001134373.2: c.1159 + 1G > T) that recessively segregated in family members. Brain MRI revealed dysgenic and thin corpus callosum (TCC) with peri-trigonal white matter cystic changes in both affected boys, whereas a well-developed corpus callosum with normal white matter was shown in their apparently normal brother, who found to be a carrier for the mutant variant. This mutation led to skipping of exon 14 with removal of 58 amino acid residues at the C-terminal half. The aberrantly spliced NT5C2 showed substantial reduction in expression level in the in-vitro study, indicating marked instability of the mutant NT5C2 protein. The present report expands the phenotypic spectrum of SPG45 and confirms NT5C2-SPG45 as a member of the rare TCC SPG-subtypes. Homozygous alteration in NT5C2 seems essential to produce central white matter developmental defects. The study highlights the importance of

  17. A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients.

    Science.gov (United States)

    Aran, Veronica; Masson Domingues, Pedro; Carvalho de Macedo, Fabiane; Moreira de Sousa, Carlos Augusto; Caldas Montella, Tatiane; de Souza Accioly, Maria Theresa; Ferreira, Carlos Gil

    2018-02-01

    Mammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. The present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. Our results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. Our results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer

  18. Presence of sst5TMD4, a truncated splice variant of the somatostatin receptor subtype 5, is associated to features of increased aggressiveness in pancreatic neuroendocrine tumors.

    Science.gov (United States)

    Sampedro-Núñez, Miguel; Luque, Raúl M; Ramos-Levi, Ana M; Gahete, Manuel D; Serrano-Somavilla, Ana; Villa-Osaba, Alicia; Adrados, Magdalena; Ibáñez-Costa, Alejandro; Martín-Pérez, Elena; Culler, Michael D; Marazuela, Mónica; Castaño, Justo P

    2016-02-09

    Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous tumors, and their biological behavior is not well known. We studied the presence and potential functional roles of somatostatin receptors (sst1-5), focusing particularly on the truncated variants (sst5TMD4, sst5TMD5) and on their relationships with the angiogenic system (Ang/Tie-2 and VEGF) in human GEP-NETs. We evaluated 42 tumor tissue samples (26 primary/16 metastatic) from 26 patients with GEP-NETs, and 30 non-tumoral tissues (26 from adjacent non-tumor regions and 4 from normal controls) from a single center. Expression of sst1-5, sst5TMD4, sst5TMD5, Ang1-2, Tie-2 and VEGF was analyzed using real-time qPCR, immunofluorescence and immunohistochemistry. Expression levels were associated with tumor characteristics and clinical outcomes. Functional role of sst5TMD4 was analyzed in GEP-NET cell lines. sst1 exhibited the highest expression in GEP-NET, whilst sst2 was the most frequently observed sst-subtype (90.2%). Expression levels of sst1, sst2, sst3, sst5TMD4, and sst5TMD5 were significantly higher in tumor tissues compared to their adjacent non-tumoral tissue. Lymph-node metastases expressed higher levels of sst5TMD4 than in its corresponding primary tumor tissue. sst5TMD4 was also significantly higher in intestinal tumor tissues from patients with residual disease of intestinal origin compared to those with non-residual disease. Functional assays demonstrated that the presence of sst5TMD4 was associated to enhanced malignant features in GEP-NET cells. Angiogenic markers correlated positively with sst5TMD4, which was confirmed by immunohistochemical/fluorescence studies. sst5TMD4 is overexpressed in GEP-NETs and is associated to enhanced aggressiveness, suggesting its potential value as biomarker and target in GEP-NETs.

  19. Alternative Splicing in CKD

    OpenAIRE

    Stevens, Megan; Oltean, Sebastian

    2016-01-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ���94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion o...

  20. Neuron-specific splicing.

    Science.gov (United States)

    Hakim, Nor Hakimah Ab; Majlis, Burhanuddin Yeop; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2017-03-22

    During pre-mRNA splicing events, introns are removed from the pre-mRNA, and the remaining exons are connected together to form a single continuous molecule. Alternative splicing is a common mechanism for the regulation of gene expression in eukaryotes. More than 90% of human genes are known to undergo alternative splicing. The most common type of alternative splicing is exon skipping, which is also known as cassette exon. Other known alternative splicing events include alternative 5' splice sites, alternative 3' splice sites, intron retention, and mutually exclusive exons. Alternative splicing events are controlled by regulatory proteins responsible for both positive and negative regulation. In this review, we focus on neuronal splicing regulators and discuss several notable regulators in depth. In addition, we have also included an example of splicing regulation mediated by the RBFox protein family. Lastly, as previous studies have shown that a number of splicing factors are associated with neuronal diseases such as Alzheime's disease (AD) and Autism spectrum disorder (ASD), here we consider their importance in neuronal diseases wherein the underlying mechanisms have yet to be elucidated.

  1. Alternative splicing in ascomycetes.

    Science.gov (United States)

    Kempken, Frank

    2013-05-01

    Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation.

  2. Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer

    Directory of Open Access Journals (Sweden)

    Yongsheng Li

    2017-10-01

    Full Text Available It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

  3. Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

    Science.gov (United States)

    Emery, Ann; Zhou, Shuntai; Pollom, Elizabeth; Swanstrom, Ronald

    2017-03-15

    Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of

  4. Resolving deconvolution ambiguity in gene alternative splicing

    Directory of Open Access Journals (Sweden)

    Hubbell Earl

    2009-08-01

    Full Text Available Abstract Background For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution. Results In this paper, we analyze the situations under which the problem occurs and perform a rigorous mathematical study which gives necessary and sufficient conditions on how many and what type of constraints are needed to resolve all ambiguity. This analysis is generally applicable to matrix models of splice variants. We explore the proposal that probe sequence information may provide sufficient additional constraints to resolve real-world instances. However, probe behavior cannot be predicted with sufficient accuracy by any existing probe sequence model, and so we present a Bayesian framework for estimating variant abundances by incorporating the prediction uncertainty from the micro-model of probe responsiveness into the macro-model of probe intensities. Conclusion The matrix analysis of constraints provides a tool for detecting real-world instances in which additional constraints may be necessary to resolve splice variants. While purely mathematical constraints can be stated without error, real-world constraints may themselves be poorly resolved. Our Bayesian framework provides a generic solution to the problem of uniquely estimating transcript abundances given additional constraints that themselves may be uncertain, such as regression fit to probe sequence models. We demonstrate the efficacy of it by extensive simulations as well as various biological data.

  5. Identification of common genetic variation that modulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Jeremy Hull

    2007-06-01

    Full Text Available Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs. In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  6. Splicing Regulation in Neurologic Disease

    National Research Council Canada - National Science Library

    Licatalosi, Donny D; Darnell, Robert B

    2006-01-01

    .... It is becoming evident that alternative splicing plays a particularly important role in neurologic disease, which is perhaps not surprising given the important role splicing plays in generating...

  7. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  8. Decreased alternative splicing of estrogen receptor-α mRNA in the Alzheimer's disease brain

    NARCIS (Netherlands)

    Ishunina, Tatjana A.; Swaab, Dick F.

    2012-01-01

    In this study we identified 62 estrogen receptor alpha (ERα) mRNA splice variants in different human brain areas of Alzheimer's disease (AD) and control cases and classified them into 12 groups. Forty-eight of these splice forms were identified for the first time. The distribution of alternatively

  9. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  10. Chromatin and alternative splicing.

    Science.gov (United States)

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  11. The UDP-Glucuronosyltransferase (UGT) 1A Polymorphism c.2042C>G (rs8330) Is Associated with Increased Human Liver Acetaminophen Glucuronidation, Increased UGT1A Exon 5a/5b Splice Variant mRNA Ratio, and Decreased Risk of Unintentional Acetaminophen-Induced Acute Liver FailureS⃞

    Science.gov (United States)

    Freytsis, Marina; Wang, Xueding; Peter, Inga; Guillemette, Chantal; Hazarika, Suwagmani; Duan, Su X.; Greenblatt, David J.; Lee, William M.

    2013-01-01

    Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury after acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen-glucuronidating enzymes (UGTs 1A1, 1A6, 1A9, and 2B15). Of these, only three linked single nucleotide polymorphisms (SNPs) located in the shared UGT1A-3′UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all the tested concentrations of acetaminophen. Mechanistic studies using luciferase-UGT1A-3′UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (P = 0.027, χ2 test) in patients who had acute liver failure from unintentional acetaminophen overdose compared with patients with acute liver failure from other causes or a race- or ethnicity-matched population. Together, these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation and could affect an individual’s risk for acetaminophen-induced liver injury. PMID:23408116

  12. Genome-wide survey of allele-specific splicing in humans

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

    data, including several examples for which there is experimental evidence of polymorphisms affecting splicing in the literature. We also present a set of novel allele-specific splicing candidates and discuss the strengths and weaknesses of alternative technologies for inferring the effect of sequence variants on mRNA splicing.

  13. Aberrant and alternative splicing in skeletal system disease.

    Science.gov (United States)

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  14. Specific acquisition of functional CD59 but not CD46 or CD55 by hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Asim Ejaz

    Full Text Available Viruses of different families encode for regulators of the complement system (RCAs or acquire such RCAs from the host to get protection against complement-mediated lysis (CML. As hepatitis C virus (HCV shares no genetic similarity to any known RCA and is detectable at high titers in sera of infected individuals, we investigated whether HCV has adapted host-derived RCAs to resist CML. Here we report that HCV selectively incorporates CD59 while neither CD55, nor CD46 are associated with the virus. The presence of CD59 was shown by capture assays using patient- and cell culture-derived HCV isolates. Association of CD59 with HCV was further confirmed by Western blot analysis using purified viral supernatants from infected Huh 7.5 cells. HCV captured by antibodies specific for CD59 remained infectious for Huh 7.5 cells. In addition, blocking of CD59 in the presence of active complement reduced the titer of HCV most likely due to CML. HCV produced in CD59 knock-down cells were more significantly susceptible to CML compared to wild type virus, but neither replication, assembly nor infectivity of the virus seemed to be impaired in the absence of CD59. In summary our data indicate that HCV incorporates selectively CD59 in its envelope to gain resistance to CML in serum of infected individuals.

  15. Rules and tools to predict the splicing effects of exonic and intronic mutations.

    Science.gov (United States)

    Ohno, Kinji; Takeda, Jun-Ichi; Masuda, Akio

    2017-09-26

    Development of next generation sequencing technologies has enabled detection of extensive arrays of germline and somatic single nucleotide variations (SNVs) in human diseases. SNVs affecting intronic GT-AG dinucleotides invariably compromise pre-mRNA splicing. Most exonic SNVs introduce missense/nonsense codons, but some affect auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. Similarly, most intronic SNVs are silent, but some affect canonical and auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. However, prediction of the splicing effects of SNVs is challenging. The splicing effects of SNVs generating cryptic AG or disrupting canonical AG can be inferred from the AG-scanning model. Similarly, the splicing effects of SNVs affecting the first nucleotide G of an exon can be inferred from AG-dependence of the 3' splice site (ss). A variety of tools have been developed for predicting the splicing effects of SNVs affecting the 5' ss, as well as exonic and intronic splicing enhancers/silencers. In contrast, only two tools, the Human Splicing Finder and the SVM-BP finder, are available for predicting the position of the branch point sequence. Similarly, IntSplice and Splicing based Analysis of Variants (SPANR) are the only tools to predict the splicing effects of intronic SNVs. The rules and tools introduced in this review are mostly based on observations of a limited number of genes, and no rule or tool can ensure 100% accuracy. Experimental validation is always required before any clinically relevant conclusions are drawn. Development of efficient tools to predict aberrant splicing, however, will facilitate our understanding of splicing pathomechanisms in human diseases. For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

  16. Identification and characterization of aberrant GAA pre-mRNA splicing in pompe disease using a generic approach.

    Science.gov (United States)

    Bergsma, Atze J; Kroos, Marian; Hoogeveen-Westerveld, Marianne; Halley, Dicky; van der Ploeg, Ans T; Pijnappel, W W

    2015-01-01

    Identification of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre-mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize effects of sequence variants on splicing would improve current diagnostic practice. Here, it is shown that such approach is feasible by combining flanking exon RT-PCR, sequence analysis of PCR products, and exon-internal quantitative RT-PCR for all coding exons. Application of this approach to one novel and six previously published variants in the acid-alpha glucosidase (GAA) gene causing Pompe disease enabled detection of a total of 11 novel splicing events. Aberrant splicing included cryptic splice-site usage, intron retention, and exon skipping. Importantly, the extent of leaky wild-type splicing correlated with disease onset and severity. These results indicate that this approach enables sensitive detection and in-depth characterization of variants affecting splicing, many of which are still unrecognized or poorly understood. The approach is generic and should be adaptable for application to other monogenic diseases to aid in improved diagnostics. © 2014 WILEY PERIODICALS, INC.

  17. Alternative splicing in colon, bladder, and prostate cancer identified by exon-array analysis

    DEFF Research Database (Denmark)

    Thorsen, Kasper; Sørensen, Karina D.; Brems-Eskildsen, Anne Sofie

    2008-01-01

    from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19......, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2....... In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications....

  18. TGF-β regulates the expression of transcription factor KLF6 and its splice variants and promotes co-operative transactivation of common target genes through a Smad3–Sp1–KLF6 interaction

    Science.gov (United States)

    BOTELLA, Luisa M.; SANZ-RODRIGUEZ, Francisco; KOMI, Yusuke; FERNANDEZ-L, Africa; VARELA, Elisa; GARRIDO-MARTIN, Eva M.; NARLA, Goutham; FRIEDMAN, Scott L.; KOJIMA, Soichi

    2010-01-01

    KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-β1 (transforming growth factor-β1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-β auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-β on KLF6 expression and splicing, and to define the contribution of TGF-β on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-β1 through at least two mechanisms. First, in specific cell types, TGF-β1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6–Sp1 co-operation is further enhanced by the TGF-β–Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6–Sp1–Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-β to regulate target genes. PMID:19076057

  19. Variants affecting exon skipping contribute to complex traits.

    Directory of Open Access Journals (Sweden)

    Younghee Lee

    Full Text Available DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1 experimentally validate our in silico predictions of skipped exons and 2 characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits.

  20. Expressiveness of basic Splice

    NARCIS (Netherlands)

    J.C. van de Pol (Jaco)

    2000-01-01

    textabstractWe study a simple software architecture, in which application processes are coordinated by writing into and reading from a global set. This architecture underlies Splice, which is developed and used at the company Hollandse Signaalapparaten. Our approach is distinguished by viewing the

  1. Detecting splicing patterns in genes involved in hereditary breast and ovarian cancer.

    Science.gov (United States)

    Davy, Grégoire; Rousselin, Antoine; Goardon, Nicolas; Castéra, Laurent; Harter, Valentin; Legros, Angelina; Muller, Etienne; Fouillet, Robin; Brault, Baptiste; Smirnova, Anna S; Lemoine, Fréderic; de la Grange, Pierre; Guillaud-Bataille, Marine; Caux-Moncoutier, Virginie; Houdayer, Claude; Bonnet, Françoise; Blanc-Fournier, Cécile; Gaildrat, Pascaline; Frebourg, Thierry; Martins, Alexandra; Vaur, Dominique; Krieger, Sophie

    2017-10-01

    Interpretation of variants of unknown significance (VUS) is a major challenge for laboratories performing molecular diagnosis of hereditary breast and ovarian cancer (HBOC), especially considering that many genes are now known to be involved in this syndrome. One important way these VUS can have a functional impact is through their effects on RNA splicing. Here we present a custom RNA-Seq assay plus bioinformatics and biostatistics pipeline to analyse specifically alternative and abnormal splicing junctions in 11 targeted HBOC genes. Our pipeline identified 14 new alternative splices in BRCA1 and BRCA2 in addition to detecting the majority of known alternative spliced transcripts therein. We provide here the first global splicing pattern analysis for the other nine genes, which will enable a comprehensive interpretation of splicing defects caused by VUS in HBOC. Previously known splicing alterations were consistently detected, occasionally with a more complex splicing pattern than expected. We also found that splicing in the 11 genes is similar in blood and breast tissue, supporting the utility and simplicity of blood splicing assays. Our pipeline is ready to be integrated into standard molecular diagnosis for HBOC, but it could equally be adapted for an integrative analysis of any multigene disorder.

  2. Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

    Science.gov (United States)

    Layden, R E; Eisen, H

    1988-03-01

    We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

  3. Width of gene expression profile drives alternative splicing.

    Directory of Open Access Journals (Sweden)

    Daniel Wegmann

    Full Text Available Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection.

  4. Genome-wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron-based splicing and its possible function through network analysis.

    Science.gov (United States)

    Panahi, Bahman; Mohammadi, Seyed Abolghasem; Ebrahimi Khaksefidi, Reyhaneh; Fallah Mehrabadi, Jalil; Ebrahimie, Esmaeil

    2015-11-30

    In this study, using homology mapping of assembled expressed sequence tags against the genomic data, we identified alternative splicing events in barley. Results demonstrated that intron retention is frequently associated with specific abiotic stresses. Network analysis resulted in discovery of some specific sub-networks between miRNAs and transcription factors in genes with high number of alternative splicing, such as cross talk between SPL2, SPL10 and SPL11 regulated by miR156 and miR157 families. To confirm the alternative splicing events, elongation factor protein (MLOC_3412) was selected followed by experimental verification of the predicted splice variants by Semi quantitative Reverse Transcription PCR (qRT-PCR). Our novel integrative approach opens a new avenue for functional annotation of alternative splicing through regulatory-based network discovery. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Exon first nucleotide mutations in splicing: evaluation of in silico prediction tools.

    Science.gov (United States)

    Grodecká, Lucie; Lockerová, Pavla; Ravčuková, Barbora; Buratti, Emanuele; Baralle, Francisco E; Dušek, Ladislav; Freiberger, Tomáš

    2014-01-01

    Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

  6. Alternatively spliced Spalax heparanase inhibits extracellular matrix degradation, tumor growth, and metastasis

    Science.gov (United States)

    Nasser, Nicola J.; Avivi, Aaron; Shafat, Itay; Edovitsky, Evgeny; Zcharia, Eyal; Ilan, Neta; Vlodavsky, Israel; Nevo, Eviatar

    2009-01-01

    Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16–BL6 lung colonization in a mouse model. Intriguingly, Spalax splice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic–hypoxic subterranean environment that Spalax experiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis. PMID:19164514

  7. Binding of cell-surface expressed CD44 to hyaluronate is dependent on splicing and cell type

    NARCIS (Netherlands)

    van der Voort, R.; Manten-Horst, E.; Smit, L.; Ostermann, E.; van den Berg, F.; Pals, S. T.

    1995-01-01

    CD44 is a major cell-surface receptor for hyaluronate (HA). By alternative RNA-splicing a large number of CD44 variants are generated. To explore the role of CD44 splicing in the regulation of cell binding to HA, three different isoforms of CD44 were transfected in the CD44 negative B-cell lymphoma

  8. Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes

    Science.gov (United States)

    Auboeuf, Didier; Dowhan, Dennis H.; Kang, Yun Kyoung; Larkin, Kimberly; Lee, Jae Woon; Berget, Susan M.; O'Malley, Bert W.

    2004-01-01

    The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones. PMID:14982999

  9. Spliceman2: a computational web server that predicts defects in pre-mRNA splicing.

    Science.gov (United States)

    Cygan, Kamil Jan; Sanford, Clayton Hendrick; Fairbrother, William Guy

    2017-09-15

    Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online.

  10. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

    Science.gov (United States)

    Vega, Yolanda; Delgado, Elena; de la Barrera, Jorge; Carrera, Cristina; Zaballos, Ángel; Cuesta, Isabel; Mariño, Ana; Ocampo, Antonio; Miralles, Celia; Pérez-Castro, Sonia; Álvarez, Hortensia; López-Miragaya, Isabel; García-Bodas, Elena; Díez-Fuertes, Francisco; Thomson, Michael M.

    2016-01-01

    HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known. PMID:27355361

  11. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes.

    Directory of Open Access Journals (Sweden)

    Yolanda Vega

    Full Text Available HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS, and doubly spliced (DS. Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G, were identified. In four samples, three of non-B subtypes, five 3' splice sites (3'ss mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3'ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h and a subtype G (A7i viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3'ss. Usage of unusual 3'ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known.

  12. High incidence of functional ion-channel abnormalities in a consecutive Long QT cohort with novel missense genetic variants of unknown significance

    DEFF Research Database (Denmark)

    Steffensen, Annette Buur; Refaat, Marwan M; David, Jens-Peter

    2015-01-01

    and variants. Seven variants of unknown significance were identified, six were missense variants and one was a splice site variant. We investigated the six novel missense VUS in five patients; three missense variants in KCNQ1 (L236R, W379R, Y522S) and three missense variants in KCNH2 (R35W, S620G, V491I). We...

  13. A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5' and 3' splice sites.

    Science.gov (United States)

    Machinaga, Akihito; Takase-Yoden, Sayaka

    2014-01-01

    The genome of the Friend murine leukemia virus (Fr-MLV) contains a 5' splice site (5'ss) located at 205 nt and a 3'ss located at 5489 nt. In our previous studies, it was shown that if the HindIII-BglII (879-1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr-MLV sequence, then cryptic splicing of env-mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII-BglII fragment were constructed. The vector, in which a 38 bp fragment (1612-1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183-1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI-NdeI (5140-5400 bp) fragment just upstream of the 3'ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140-5400 nt region located just upstream of the 3'ss is required for the splicing function of the 38 nt fragment and its flanking sequences. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  14. High affinity complexes of pannexin channels and L-type calcium channel splice-variants in human lung: Possible role in clevidipine-induced dyspnea relief in acute heart failure

    Directory of Open Access Journals (Sweden)

    Gerhard P. Dahl

    2016-08-01

    Research in Context: Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure.

  15. Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

    DEFF Research Database (Denmark)

    Agerholm, Jørgen Steen; Menzi, Fiona; McEvoy, Fintan

    2016-01-01

    genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant...... to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring...

  16. Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue

    DEFF Research Database (Denmark)

    Usher, Pernille Autzen; Sieuwerts, A.M.; Bartels, Annette

    2007-01-01

    TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT......-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP...

  17. Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

    DEFF Research Database (Denmark)

    Fackenthal, James D; Yoshimatsu, Toshio; Zhang, Bifeng

    2016-01-01

    BACKGROUND: BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing...... patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium...

  18. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  19. Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

    Science.gov (United States)

    Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Hervé; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis

    2012-01-01

    Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-xS splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level. PMID:22203037

  20. Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica

    Science.gov (United States)

    Kabran, Philomène; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuvéglise, Cécile

    2012-01-01

    Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3′-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment. PMID:22368181

  1. A structured RNA in hepatitis B virus post-transcriptional regulatory element represses alternative splicing in a sequence-independent and position-dependent manner.

    Science.gov (United States)

    Huang, Chen; Xie, Mao-Hua; Liu, Wei; Yang, Bo; Yang, Fan; Huang, Jingang; Huang, Jie; Wu, Qijia; Fu, Xiang-Dong; Zhang, Yi

    2011-05-01

    Hepatitis B virus (HBV) transcripts are subjected to multiple splicing decisions, but the mechanism of splicing regulation remains poorly understood. In this study, we used a well-investigated alternative splicing reporter to dissect splicing regulatory elements residing in the post-transcriptional regulatory element (PRE) of HBV. A strong intronic splicing silencer (ISS) with a minimal functional element of 105 nucleotides (referred to as PRE-ISS) was identified and, interestingly, both the sense and antisense strands of the element were found to strongly suppress alternative splicing in multiple human cell lines. PRE-ISS folds into a double-hairpin structure, in which substitution mutations disrupting the double-hairpin structure abolish the splicing silencer activity. Although it harbors two previously identified binding sites for polypyrimidine tract binding protein, PRE-ISS represses splicing independent of this protein. The silencing function of PRE-ISS exhibited a strong position dependence, decreasing with the distance from affected splice sites. PRE-ISS does not belong to the intronic region of any HBV splicing variants identified thus far, preventing the testing of this intronic silencer function in the regulation of HBV splicing. These findings, together with the identification of multiple sense-antisense ISSs in the HBV genome, support the hypothesis that a sequence-independent and structure-dependent regulatory mechanism may have evolved to repress cryptic splice sites in HBV transcripts, thereby preventing their aberrant splicing during viral replication in the host. © 2011 The Authors Journal compilation © 2011 FEBS.

  2. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

    Full Text Available BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM, clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val, identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1 a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU, which resulted in the deletion of 4 base pairs and presumably protein truncation; 2 a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3 partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

  3. Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

    Science.gov (United States)

    Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

    2016-05-27

    Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus

  4. Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

    Science.gov (United States)

    Guiliano, David B; Blaxter, Mark L

    2006-11-24

    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

  5. Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

    Directory of Open Access Journals (Sweden)

    David B Guiliano

    2006-11-01

    Full Text Available The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

  6. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2008-09-01

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

  7. Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.

    Directory of Open Access Journals (Sweden)

    Barbara Wappenschmidt

    Full Text Available Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

  8. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    Directory of Open Access Journals (Sweden)

    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  9. Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Haroon Kalam

    2017-03-01

    Full Text Available Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb infection is widely studied; however, the significance of alternate splicing (AS in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the

  10. Fine-tuning and autoregulation of the intestinal determinant and tumor suppressor homeobox gene CDX2 by alternative splicing

    NARCIS (Netherlands)

    Balbinot, Camille; Vanier, Marie; Armant, Olivier; Nair, Asmaa; Penichon, Julien; Soret, Christine; Martin, Elisabeth; Saandi, Thoueiba; Reimund, Jean-Marie; Deschamps, Jacqueline; Beck, Felix; Domon-Dell, Claire; Gross, Isabelle; Duluc, Isabelle; Freund, Jean-Noël

    2017-01-01

    On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation,

  11. Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid.

    Science.gov (United States)

    Lara, Beatriz; Martínez, Maria Teresa; Blanco, Ignacio; Hernández-Moro, Cristina; Velasco, Eladio A; Ferrarotti, Ilaria; Rodriguez-Frias, Francisco; Perez, Laura; Vazquez, Irene; Alonso, Javier; Posada, Manuel; Martínez-Delgado, Beatriz

    2014-10-07

    Severe Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema. A family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process. We report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity. Finding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.

  12. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

    Clearly written and amply illustrated with 208 figures, this classic guide ranges from simple and useful knots to complex varieties. Additional topics include rope splicing, working cordage, hammock making, more.

  13. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Directory of Open Access Journals (Sweden)

    Omar Soukarieh

    2016-01-01

    Full Text Available The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants, including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs. We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  14. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Science.gov (United States)

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  15. Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

    Directory of Open Access Journals (Sweden)

    Lorena Suarez-Artiles

    2018-01-01

    Full Text Available Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr and c.2581G>C; p.(Ala861Pro abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

  16. Design of RNA-Binding Proteins: Manipulate Alternative Splicing in Human Cells with Artificial Splicing Factors.

    Science.gov (United States)

    Wang, Yang; Wang, Zefeng

    2016-01-01

    The majority of human genes undergo alternative splicing to produce multiple isoforms with distinct functions. The dysregulations of alternative splicing have been found to be closely associated with various human diseases; thus new approaches to modulate disease-associated splicing events will provide great therapeutic potentials. Here we report protocols for constructing novel artificial splicing factors that can be designed to specifically modulate alternative splicing of target genes. By following the method outlined in this protocol, it is possible to design and generate artificial splicing factors with diverse activities in regulating different types of alternative splicing. The artificial splicing factors can be used to change splicing of either minigenes or endogenous genes in cultured human cells, providing a new strategy to study the regulation of alternative splicing and function of alternatively spliced products.

  17. A synonymous polymorphic variation in ACADM exon 11 affects splicing efficiency and may affect fatty acid oxidation

    DEFF Research Database (Denmark)

    Bruun, Gitte Hoffmann; Doktor, Thomas Koed; Andresen, Brage Storstein

    2013-01-01

    beta-oxidation of medium-chain fatty acids. We examined the functional basis for this association and identified linkage between rs211718 and the intragenic synonymous polymorphic variant c.1161A>G in ACADM exon 11 (rs1061337). Employing minigene studies we show that the c.1161A allele is associated...... with exon 11 missplicing, and that the c.1161G allele corrects this missplicing. This may result in production of more full length MCAD protein from the c.1161G allele. Our analysis suggests that the improved splicing of the c.1161G allele is due to changes in the relative binding of splicing regulatory......, perhaps due to improved splicing. This study is a proof of principle that synonymous SNPs are not neutral. By changing the binding sites for splicing regulatory proteins they can have significant effects on pre-mRNA splicing and thus protein function. In addition, this study shows that for a sequence...

  18. Tissue-specific alternative splicing and expression of ATP1B2 gene ...

    African Journals Online (AJOL)

    The Na+-K+-ATPase is an essential transport enzyme expressed in all animal tissues, where it generates ion gradients to maintain membrane potential and drive the transport of other solutes. It also balances metabolism and body temperature. In this study, the characterization of three novel bovine ATP1B2 splice variants, ...

  19. A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.

    Science.gov (United States)

    Blanco, Ana M; Rausell, Laura; Aguado, Begoña; Perez-Alonso, Manuel; Artero, Rubén

    2009-09-01

    We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5-11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA-RNA interactions.

  20. Splice testing for LHC quadrupole magnets

    CERN Document Server

    Barzi, E; Fehér, S; Kashikhin, V V; Kerby, J S; Lamm, M J; Orris, D; Ray, G; Tartaglia, M; Zlobin, A V

    2003-01-01

    Electrical splices between NbTi Rutherford type cables need to be made for the LHC IR inner triplet quadrupoles. Splices between magnets as well as internal to the magnets are necessary. Various splice configurations, solders, and fluxes have been considered. Testing of these splices at cryogenic temperatures and at various currents has been completed. The results were satisfactory; Fermilab is capable of making excellent low resistance (<1n Omega ) solder joints for the LHC project. (4 refs).

  1. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2015-10-01

    membranes. After blocking in TBS buffer (150 mM NaCl, 10 mM Tris, pH 7.4) containing 5% nonfat milk , the blots were incubated with a primary antibody...blot analysis The procedure was described previously (29). The anti-GAPDH (Millipore), anti-AR (N-20, Santa Cruz Biotechnology ), anti- HSP70 (Abcam

  2. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2016-10-01

    recruitment and consent Funding support DOD-PCRP, American Cancer Society DOD-PCRP American Cancer Society DOD-PCRP, American Cancer...study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene...enriched by depleting hematopoietic cells. For this purpose CD45, which is expressed on the surface of all leukocytes and their progenitors,9 is commonly

  3. Splice Variant Biomarkers for Parkinson’s Disease

    Science.gov (United States)

    2014-05-01

    slowing PD. NUTRIENTS THAT MAY BE ASSOCIATED WITH AN INCREASED RISK OR PROGRESSION OF PD DAIRY PRODUCTS Dairy product consumption and drinking milk may...controls. • A subcontract was established with Stephen Wood, National Centre for Adult Stem Cell Research, Eskitis Institute, Griffith University...particularly in men (Chen et al., 2007a). Nonetheless, a positive association between milk consumption and PD risk was also observed in women in one study

  4. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...... cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5;- and the 3;-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3;-splice site of intron3 with a complementarity of 4 bases to intron...... method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs....

  5. PIN1 gene variants in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Siedlecki Janusz

    2009-11-01

    Full Text Available Abstract Background Peptidyl-prolyl isomerase, NIMA-interacting 1 (PIN1 plays a significant role in the brain and is implicated in numerous cellular processes related to Alzheimer's disease (AD and other neurodegenerative conditions. There are confounding results concerning PIN1 activity in AD brains. Also PIN1 genetic variation was inconsistently associated with AD risk. Methods We performed analysis of coding and promoter regions of PIN1 in early- and late-onset AD and frontotemporal dementia (FTD patients in comparison with healthy controls. Results Analysis of eighteen PIN1 common polymorphisms and their haplotypes in EOAD, LOAD and FTD individuals in comparison with the control group did not reveal their contribution to disease risk. In six unrelated familial AD patients four novel PIN1 sequence variants were detected. c.58+64C>T substitution that was identified in three patients, was located in an alternative exon. In silico analysis suggested that this variant highly increases a potential affinity for a splicing factor and introduces two intronic splicing enhancers. In the peripheral leukocytes of one living patient carrying the variant, a 2.82 fold decrease in PIN1 expression was observed. Conclusion Our data does not support the role of PIN1 common polymorphisms as AD risk factor. However, we suggest that the identified rare sequence variants could be directly connected with AD pathology, influencing PIN1 splicing and/or expression.

  6. Human peptidylglycine alpha-amidating monooxygenase transcripts derived by alternative mRNA splicing of an unreported exon.

    Science.gov (United States)

    Vos, M D; Jones, J E; Treston, A M

    1995-10-03

    We are characterizing the alternatively spliced human peptidylglycine alpha-amidating monooxygenase (hPAM)-encoding mRNA transcripts expressed by human cells. Reverse transcription coupled to the polymerase chain reaction (RT-PCR) has been used to identify four alternatively spliced variants that differ in the region joining the two catalytic domains. Two of the transcripts represent previously reported splice variants differentiated by the presence (hPAM-A) or absence (hPAM-B) of a 321-nucleotide (nt) linker (optional exon A) which in the rat produce functionally distinct enzymes. Different mRNAs represent two splice variants, hPAM-C and hPAM-D, that show the presence of an exon unreported for PAM in any other species. This new exon, designated exon C, is 54 nt in length, encodes an 18-amino-acid (aa) peptide containing a conserved dibasic aa endoproteolytic processing motif, and is located 3' of exon A in human genomic DNA. We propose that cell-specific regulation of mRNA splicing would provide a mechanism for control of prohormone activation by these variants of the PAM enzyme.

  7. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); Redal, María Ana [Instituto de Ciencias Básicas y Medicina Experimental, Hospital Italiano de Buenos Aires (Argentina); Alghamdi, Mansour A. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Khoder, Mamdouh I. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah (Saudi Arabia); Shamy, Magdy [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Muñoz, Manuel J., E-mail: mmunoz@fbmc.fcen.uba.ar [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); and others

    2015-07-15

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.

  8. Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

    Directory of Open Access Journals (Sweden)

    Ulrich F. Müller

    2017-01-01

    Full Text Available Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

  9. Alternative Splicing of Fibroblast Growth Factor Receptor IgIII Loops in Cancer

    Directory of Open Access Journals (Sweden)

    Klaus Holzmann

    2012-01-01

    Full Text Available Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs 1–3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1–3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application.

  10. A novel splice-site mutation in the ASPM gene underlies autosomal recessive primary microcephaly.

    Science.gov (United States)

    Hashmi, Jamil A; Al-Harbi, Khalid M; Ramzan, Khushnooda; Albalawi, Alia M; Mehmood, Amir; Samman, Mohammed I; Basit, Sulman

    2016-01-01

    Autosomal recessive primary microcephaly (MCPH) is a clinically and genetically heterogeneous disorder. Patients with MCPH exhibit reduced occipito-frontal head circumference and non-progressive intellectual disability. To date, 17 genes have been known as an underlying cause of MCPH in humans. ASPM (abnormal spindle-like, microcephaly associated) is the most commonly mutated MCPH gene. Identify the genetic defect underlying MCPH in a Saudi family. A cross-sectional clinical genetic study of a Saudi family. Madinah Maternity and Children Hospital and Centre for Genetics and Inherited Diseases, Taibah University. A molecular analysis was carried out on DNA samples from 10 individuals of a Saudi family segregating MCPH. DNA was isolated from the peripheral blood of 10 individuals, including 2 patients, and whole exome sequencing was performed using the Nextera Rapid Capture kit and NextSeq500 instrument. VariantStudio was used to filter and prioritize variants. Detection of mutation in the ASPM gene in a family segregating autoso- mal recessive primary microcephaly. A novel homozygous splice-site variant (c.3742-1G > C) in the ASPM gene was identified. The variant is predicted to have an effect on splicing. Human Splice Finder, an in silico tool, predicted skipping of exon 16 due to this variant. Skipping of exon 16 may change the order and number of IQ motifs in the ASPM protein leading to typical MCPH phenotype. Single family study.

  11. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  12. Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools

    Directory of Open Access Journals (Sweden)

    Nancy Martínez-Montiel

    2017-10-01

    Full Text Available Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

  13. Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

    Science.gov (United States)

    Avin, Brittany A; Umbricht, Christopher B; Zeiger, Martha A

    2016-12-01

    The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.

  14. Titin Diversity—Alternative Splicing Gone Wild

    Directory of Open Access Journals (Sweden)

    Wei Guo

    2010-01-01

    Full Text Available Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

  15. LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding

    Directory of Open Access Journals (Sweden)

    Ji Won Um

    2016-02-01

    Full Text Available The four members of the LRRTM family (LRRTM1-4 are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.

  16. Conserved and species-specific alternative splicing in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Favorov Alexander V

    2007-12-01

    Full Text Available Abstract Background Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants. Results We analyzed conservation of human alternative splice sites and cassette exons in the mouse and dog genomes. Alternative exons, especially minor-isofom ones, were shown to be less conserved than constitutive exons. Frame-shifting alternatives in the protein-coding regions are less conserved than frame-preserving ones. Similarly, the conservation of alternative sites is highest for evenly used alternatives, and higher when the distance between the sites is divisible by three. The rate of alternative-exon and site loss in mouse is slightly higher than in dog, consistent with faster evolution of the former. The evolutionary dynamics of alternative sites was shown to be consistent with the model of random activation of cryptic sites. Conclusion Consistent with other studies, our results show that minor cassette exons are less conserved than major-alternative and constitutive exons. However, our study provides evidence that this is caused not only by exon birth, but also lineage-specific loss of alternative exons and sites, and it depends on exon functionality.

  17. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

    Directory of Open Access Journals (Sweden)

    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  18. Splicing and alternative splicing in rice and humans

    Directory of Open Access Journals (Sweden)

    Zhiguo E

    2013-09-01

    Full Text Available Rice is a monocot gramineous crop, and one of the mostimportant staple foods. Rice is considered a model species formost gramineous crops. Extensive research on rice hasprovided critical guidance for other crops, such as maize andwheat. In recent years, climate change and exacerbated soildegradation have resulted in a variety of abiotic stresses, suchas greenhouse effects, lower temperatures, drought, floods,soil salinization and heavy metal pollution. As such, there isan extremely high demand for additional research, in order toaddress these negative factors. Studies have shown that thealternative splicing of many genes in rice is affected by stressconditions, suggesting that manipulation of the alternativesplicing of specific genes may be an effective approach for riceto adapt to abiotic stress. With the advancement ofmicroarrays, and more recently, next generation sequencingtechnology, several studies have shown that more than half ofthe genes in the rice genome undergo alternative splicing. Thismini-review summarizes the latest progress in the research ofsplicing and alternative splicing in rice, compared to splicingin humans. Furthermore, we discuss how additional studiesmay change the landscape of investigation of rice functionalgenomics and genetically improved rice. [BMB Reports 2013;46(9: 439-447

  19. Alteration of branch site consensus sequence and enhanced pre-mRNA splicing of an NMDAR1 intron not associated with schizophrenia.

    Science.gov (United States)

    Hammond, Linda; Castanotto, Daniela; Rice, Sharmon R; Nimgaonkar, Vishwajit L; Wirshing, Donna A; Rossi, John J; Heston, Leonard L; Sobell, Janet L

    2002-08-08

    Aberrant splicing of pre-mRNA is recognized to account for a significant minority of disease-causing mutations. The N-methyl-D-aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African-American male near a weak putative branch-site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in-frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer-extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African-American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease. Copyright 2002 Wiley-Liss, Inc.

  20. Safely splicing glass optical fibers

    Science.gov (United States)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  1. Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

    Science.gov (United States)

    Chapat, Clément; Chettab, Kamel; Simonet, Pierre; Wang, Peng; De La Grange, Pierre; Le Romancer, Muriel; Corbo, Laura

    2017-08-21

    The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1

    Directory of Open Access Journals (Sweden)

    Zaphiropoulos Peter G

    2010-04-01

    Full Text Available Abstract Background Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. Results We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. Conclusions Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.

  3. SplicePlot: a utility for visualizing splicing quantitative trait loci.

    Science.gov (United States)

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B

    2014-04-01

    RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

  4. The Cancer Exome Generated by Alternative mRNA Splicing Dilutes Predicted HLA Class I Epitope Density

    DEFF Research Database (Denmark)

    Stranzl, Thomas; Larsen, Mette Voldby; Lund, Ole

    2012-01-01

    is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based......Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes...... on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I...

  5. Integrating splice-isoform expression into genome-scale models characterizes breast cancer metabolism.

    Science.gov (United States)

    Angione, Claudio

    2018-02-01

    Despite being often perceived as the main contributors to cell fate and physiology, genes alone cannot predict cellular phenotype. During the process of gene expression, 95% of human genes can code for multiple proteins due to alternative splicing. While most splice variants of a gene carry the same function, variants within some key genes can have remarkably different roles. To bridge the gap between genotype and phenotype, condition- and tissue-specific models of metabolism have been constructed. However, current metabolic models only include information at the gene level. Consequently, as recently acknowledged by the scientific community, common situations where changes in splice-isoform expression levels alter the metabolic outcome cannot be modeled. We here propose GEMsplice, the first method for the incorporation of splice-isoform expression data into genome-scale metabolic models. Using GEMsplice, we make full use of RNA-Seq quantitative expression profiles to predict, for the first time, the effects of splice isoform-level changes in the metabolism of 1455 patients with 31 different breast cancer types. We validate GEMsplice by generating cancer-versus-normal predictions on metabolic pathways, and by comparing with gene-level approaches and available literature on pathways affected by breast cancer. GEMsplice is freely available for academic use at https://github.com/GEMsplice/GEMsplice_code. Compared to state-of-the-art methods, we anticipate that GEMsplice will enable for the first time computational analyses at transcript level with splice-isoform resolution. https://github.com/GEMsplice/GEMsplice_code. c.angione@tees.ac.uk. Supplementary data are available at Bioinformatics online.

  6. A novel AVPR2 splice site mutation leads to partial X-linked nephrogenic diabetes insipidus in two brothers.

    Science.gov (United States)

    Schernthaner-Reiter, Marie Helene; Adams, David; Trivellin, Giampaolo; Ramnitz, Mary Scott; Raygada, Margarita; Golas, Gretchen; Faucz, Fabio R; Nilsson, Ola; Nella, Aikaterini A; Dileepan, Kavitha; Lodish, Maya; Lee, Paul; Tifft, Cynthia; Markello, Thomas; Gahl, William; Stratakis, Constantine A

    2016-05-01

    X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain. We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis. • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

  7. Loss-of-function variants in ADCY3 increase risk of obesity and type 2 diabetes

    DEFF Research Database (Denmark)

    Grarup, Niels; Moltke, Ida; Andersen, Mette K

    2018-01-01

    We have identified a variant in ADCY3 (encoding adenylate cyclase 3) associated with markedly increased risk of obesity and type 2 diabetes in the Greenlandic population. The variant disrupts a splice acceptor site, and carriers have decreased ADCY3 RNA expression. Additionally, we observe an enr...

  8. Ecotype dependent expression and alternative splicing of epithiospecifier protein (ESP) in Arabidopsis thaliana.

    Science.gov (United States)

    Kissen, R; Hyldbakk, E; Wang, C-W V; Sørmo, C G; Rossiter, J T; Bones, A M

    2012-03-01

    Epithiospecifier protein (ESP) is responsible for diverting glucosinolate hydrolysis from the generation of isothiocyanates to that of epithionitriles or nitriles, and thereby negatively affects the ability of the plant to defend itself against certain insects. Despite this important role of ESP, little is known about its expression in plant tissues and the regulation thereof. We therefore investigated ESP expression by qPCR and Western blot in different organs during the growth cycle of the two Arabidopsis thaliana ecotypes Col-0 and Mt-0. Besides the fact that ESP transcript and protein levels were revealed to be much higher in Mt-0 than in Col-0 in all cases, our qPCR results also indicated that ESP expression is regulated differently in the two A. thaliana ecotypes. No ESP protein was detected by Western blot in any organ or developmental stage for Col-0. During the assays an alternative splice variant of ESP was identified in Col-0, but not Mt-0, leading to a mis-spliced transcript which could explain the low expression levels of ESP in the former ecotype. Analysis of genomic sequences containing the ESP splice sites, of ESP protein level and ESP activity from seven A. thaliana ecotypes showed a positive correlation between the presence of a non-canonical 5' splice site for ESP and the absence of detectable ESP protein levels and ESP activity. When analysing the expression of both transcript variants in Col-0 after treatment with methyl jasmonate, a condition known to "induce ESP", it was indeed the alternative splice variant that was preferentially induced.

  9. Impaired RNA splicing of 5'-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma

    NARCIS (Netherlands)

    Münch, C.; Penndorf, A.; Schwalenstöcker, B.; Troost, D.; Ludolph, A. C.; Ince, P.; Meyer, T.

    2001-01-01

    A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5'-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human

  10. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  11. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    OpenAIRE

    Churbanov, Alexander; Vo?echovsk?, Igor; Hicks, Chindo

    2009-01-01

    Abstract Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from t...

  12. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    Science.gov (United States)

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  13. Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

    Science.gov (United States)

    Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

    2014-04-01

    Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

  14. Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.

    Science.gov (United States)

    Movassat, Maliheh; Shenasa, Hossein; Hertel, Klemens J

    2017-01-01

    The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.

  15. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    been initiated. In this article I present a brief history of HGT, showing how the ethical and practical viability of the field was achieved by key scientific and regulatory actors. These parties carefully articulated gene therapy’s scope, limiting it to therapeutic interventions on somatic cells......, and cultivated alliances and divisions that bolstered the field’s legitimacy. At times these measures faltered, and then practitioners and sometimes patients would invoke an ethical imperative, posing gene therapy as the best solution to life and death problems. I suggest that we consider how boundary...

  16. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer.

    Science.gov (United States)

    Inoue, Kazushi; Fry, Elizabeth A

    2015-01-01

    Breast cancer (BC) is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor) and human epidermal growth factor receptor 2 (HER2), play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER, HER2, and CD44 genes from the viewpoint of BC development. ERα36, a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2) has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms (CD44s, CD44v) play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci will provide keys

  17. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Kazushi Inoue

    2015-01-01

    Full Text Available Breast cancer (BC is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor and human epidermal growth factor receptor 2 (HER2, play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER , HER2 , and CD44 genes from the viewpoint of BC development. ERα36 , a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2 has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms ( CD44s , CD44v play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci

  18. Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

    Science.gov (United States)

    Hong, Yoonki; Kim, Woo Jin; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

    2016-04-01

    Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

  19. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    Science.gov (United States)

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program. © 2016 S. Karger AG, Basel.

  20. A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Sailesh Gopalakrishna-Pillai

    Full Text Available A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs relative to spontaneously differentiated cells (SDCs. Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

  1. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  2. Alternatively spliced neuronal nitric oxide synthase mediates penile erection

    Science.gov (United States)

    Hurt, K. Joseph; Sezen, Sena F.; Champion, Hunter C.; Crone, Julie K.; Palese, Michael A.; Huang, Paul L.; Sawa, Akira; Luo, Xiaojiang; Musicki, Biljana; Snyder, Solomon H.; Burnett, Arthur L.

    2006-01-01

    A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOS−/− animals might suffice for normal physiologic function. We show that the β splice variant of nNOS elicits normal erection despite a decrease in stimulus-response characteristics and a 5-fold increased sensitivity to the NOS inhibitor, l-NAME. Residual nNOSβ generates only 10% of the normal NO level in vitro but produces citrulline and diaphorase staining reflecting in vivo NOS activity in pelvic ganglion nerves that is comparable to WT animals. Thus, alternatively spliced forms of nNOS are major mediators of penile erection and so may be targets for therapeutic intervention. PMID:16488973

  3. Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

    Science.gov (United States)

    Zhang, Heng Yu; Liao, Ping; Wang, Jue Jin; Yu, De Jie; Soong, Tuck Wah

    2010-01-01

    Background and purpose: As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Cav1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Cav1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Cav1.2 channels, predominant in cardiac and vascular smooth muscles. Experimental approach: Cardiac and smooth muscle isoforms of Cav1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques. Key results: Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Cav1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Cav1.2SM and Cav1.2b. Cav1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Cav1.2CM channels. When we modified alternatively spliced exons of cardiac Cav1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms. Conclusions and implications: Alternative splicing of Cav1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels. PMID:20649567

  4. Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

    Science.gov (United States)

    Tan, Gregory Ming Yeong; Yu, Dejie; Wang, Juejin; Soong, Tuck Wah

    2012-01-01

    The CaV1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type CaV1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native CaV1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the CaV1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The CaV1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length CaV1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native CaV1.4 channels. PMID:22069316

  5. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_pattern...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  6. Regular languages, regular grammars and automata in splicing systems

    Science.gov (United States)

    Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

    2013-04-01

    Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

  7. CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

    Science.gov (United States)

    Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

    2009-12-01

    Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

  8. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid conjugates targeting intron-exon junctions

    Directory of Open Access Journals (Sweden)

    Nielsen Peter E

    2010-06-01

    Full Text Available Abstract Background Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. Methods We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512 targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT. Results We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406 targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512 targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. Conclusion We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.

  9. Heat shock activates splicing at latent alternative 5' splice sites in nematodes.

    Science.gov (United States)

    Nevo, Yuval; Sperling, Joseph; Sperling, Ruth

    2015-01-01

    Pre-mRNA splicing is essential for the regulation of gene expression in eukaryotes and is fundamental in development and cancer, and involves the selection of a consensus sequence that defines the 5' splice site (5'SS). Human introns harbor multiple sequences that conform to the 5'SS consensus, which are not used under normal growth conditions. Under heat shock conditions, splicing at such intronic latent 5'SSs occurred in thousands of human transcripts, resulting in pre-maturely terminated aberrant proteins. Here we performed a survey of the C. elegans genome, showing that worm's introns contain latent 5'SSs, whose use for splicing would have resulted in pre-maturely terminated mRNAs. Splicing at these latent 5'SSs could not be detected under normal growth conditions, while heat shock activated latent splicing in a number of tested C. elegans transcripts. Two scenarios could account for the lack of latent splicing under normal growth conditions (i) Splicing at latent 5'SSs do occur, but the nonsense mRNAs thus formed are rapidly and efficiently degraded (e.g. by NMD); and (ii) Splicing events at intronic latent 5'SSs are suppressed. Here we support the second scenario, because, nematode smg mutants that are devoid of NMD-essential factors, did not show latent splicing under normal growth conditions. Hence, these experiments together with our previous experiments in mammalian cells, indicate the existence of a nuclear quality control mechanism, termed Suppression Of Splicing (SOS), which discriminates between latent and authentic 5'SSs in an open reading frame dependent manner, and allows splicing only at the latter. Our results show that SOS is an evolutionary conserved mechanism, probably shared by most eukaryotes.

  10. Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF.

    Science.gov (United States)

    Piekielko-Witkowska, Agnieszka; Kedzierska, Hanna; Poplawski, Piotr; Wojcicka, Anna; Rybicka, Beata; Maksymowicz, Maria; Grajkowska, Wieslawa; Matyja, Ewa; Mandat, Tomasz; Bonicki, Wieslaw; Nauman, Pawel

    2013-06-01

    Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. SpliceAid-F: a database of human splicing factors and their RNA-binding sites.

    Science.gov (United States)

    Giulietti, Matteo; Piva, Francesco; D'Antonio, Mattia; D'Onorio De Meo, Paolo; Paoletti, Daniele; Castrignanò, Tiziana; D'Erchia, Anna Maria; Picardi, Ernesto; Zambelli, Federico; Principato, Giovanni; Pavesi, Giulio; Pesole, Graziano

    2013-01-01

    A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called 'SpliceAid-F' (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA-SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA-SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 'no-binding' sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements.

  12. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  13. Thermopriming Triggers Splicing Memory in Arabidopsis

    KAUST Repository

    Ling, Yu

    2018-02-20

    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

  14. Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome

    DEFF Research Database (Denmark)

    Krejsgaard, T; Gjerdrum, L M; Ralfkiaer, E

    2008-01-01

    Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3 i...... different from wt FOXP3 and not involved in the execution of the suppressive function. Thus, this is the first description of FOXP3 splice forms in human disease.Leukemia advance online publication, 4 September 2008; doi:10.1038/leu.2008.224....... in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-beta and suppress...... the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-kappaB (NF...

  15. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

    Directory of Open Access Journals (Sweden)

    Daw-Yang Hwang

    Full Text Available 17α-Hydroxylase/17, 20-lyase deficiency (17OHD is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90% of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

  16. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Directory of Open Access Journals (Sweden)

    Clemens Hüttner

    2016-09-01

    Full Text Available RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM. We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.

  17. Alternative Splicing of P/Q-Type Ca2+ Channels Shapes Presynaptic Plasticity

    Directory of Open Access Journals (Sweden)

    Agnes Thalhammer

    2017-07-01

    Full Text Available Alternative splicing of pre-mRNAs is prominent in the mammalian brain, where it is thought to expand proteome diversity. For example, alternative splicing of voltage-gated Ca2+ channel (VGCC α1 subunits can generate thousands of isoforms with differential properties and expression patterns. However, the impact of this molecular diversity on brain function, particularly on synaptic transmission, which crucially depends on VGCCs, is unclear. Here, we investigate how two major splice isoforms of P/Q-type VGCCs (Cav2.1[EFa/b] regulate presynaptic plasticity in hippocampal neurons. We find that the efficacy of P/Q-type VGCC isoforms in supporting synaptic transmission is markedly different, with Cav2.1[EFa] promoting synaptic depression and Cav2.1[EFb] synaptic facilitation. Following a reduction in network activity, hippocampal neurons upregulate selectively Cav2.1[EFa], the isoform exhibiting the higher synaptic efficacy, thus effectively supporting presynaptic homeostatic plasticity. Therefore, the balance between VGCC splice variants at the synapse is a key factor in controlling neurotransmitter release and presynaptic plasticity.

  18. Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

    Science.gov (United States)

    Wang, Xiuge; Cui, Xiaohui; Zhang, Yan; Hao, Haisheng; Ju, Zhihua; Liu, Deyu; Jiang, Qiang; Yang, Chunhong; Sun, Yan; Wang, Changfa; Huang, Jinming; Zhu, Huabin

    2017-11-01

    RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (Pquality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

  19. Alternative Splicing of the RAGE Cytoplasmic Domain Regulates Cell Signaling and Function

    Science.gov (United States)

    Jules, Joel; Maiguel, Dony; Hudson, Barry I.

    2013-01-01

    The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling. PMID:24260107

  20. Tools to covisualize and coanalyze proteomic data with genomes and transcriptomes: validation of genes and alternative mRNA splicing

    DEFF Research Database (Denmark)

    Pang, Chi; Tay, Aidan; Aya, Carlos

    2014-01-01

    the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene...... contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates...... validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus...

  1. Splicing factors control C. elegans behavioural learning in a single neuron by producing DAF-2c receptor.

    Science.gov (United States)

    Tomioka, Masahiro; Naito, Yasuki; Kuroyanagi, Hidehito; Iino, Yuichi

    2016-05-20

    Alternative splicing generates protein diversity essential for neuronal properties. However, the precise mechanisms underlying this process and its relevance to physiological and behavioural functions are poorly understood. To address these issues, we focused on a cassette exon of the Caenorhabditis elegans insulin receptor gene daf-2, whose proper variant expression in the taste receptor neuron ASER is critical for taste-avoidance learning. We show that inclusion of daf-2 exon 11.5 is restricted to specific neuron types, including ASER, and is controlled by a combinatorial action of evolutionarily conserved alternative splicing factors, RBFOX, CELF and PTB families of proteins. Mutations of these factors cause a learning defect, and this defect is relieved by DAF-2c (exon 11.5+) isoform expression only in a single neuron ASER. Our results provide evidence that alternative splicing regulation of a single critical gene in a single critical neuron is essential for learning ability in an organism.

  2. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Daniel Nilsson

    2010-08-01

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  3. Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene.

    Science.gov (United States)

    Valacca, Cristina; Bonomi, Serena; Buratti, Emanuele; Pedrotti, Simona; Baralle, Francisco Ernesto; Sette, Claudio; Ghigna, Claudia; Biamonti, Giuseppe

    2010-10-04

    Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell-derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.

  4. Differential splicing using whole-transcript microarrays

    Directory of Open Access Journals (Sweden)

    Robinson Mark D

    2009-05-01

    Full Text Available Abstract Background The latest generation of Affymetrix microarrays are designed to interrogate expression over the entire length of every locus, thus giving the opportunity to study alternative splicing genome-wide. The Exon 1.0 ST (sense target platform, with versions for Human, Mouse and Rat, is designed primarily to probe every known or predicted exon. The smaller Gene 1.0 ST array is designed as an expression microarray but still interrogates expression with probes along the full length of each well-characterized transcript. We explore the possibility of using the Gene 1.0 ST platform to identify differential splicing events. Results We propose a strategy to score differential splicing by using the auxiliary information from fitting the statistical model, RMA (robust multichip analysis. RMA partitions the probe-level data into probe effects and expression levels, operating robustly so that if a small number of probes behave differently than the rest, they are downweighted in the fitting step. We argue that adjacent poorly fitting probes for a given sample can be evidence of differential splicing and have designed a statistic to search for this behaviour. Using a public tissue panel dataset, we show many examples of tissue-specific alternative splicing. Furthermore, we show that evidence for putative alternative splicing has a strong correspondence between the Gene 1.0 ST and Exon 1.0 ST platforms. Conclusion We propose a new approach, FIRMAGene, to search for differentially spliced genes using the Gene 1.0 ST platform. Such an analysis complements the search for differential expression. We validate the method by illustrating several known examples and we note some of the challenges in interpreting the probe-level data. Software implementing our methods is freely available as an R package.

  5. Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

    Directory of Open Access Journals (Sweden)

    Olgica Trenchevska

    Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

  6. Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles.

    Science.gov (United States)

    Hill, Jennifer K; Williams, Diane E; LeMasurier, Meredith; Dumont, Rachel A; Strehler, Emanuel E; Gillespie, Peter G

    2006-06-07

    Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport.

  7. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    Science.gov (United States)

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  8. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  9. Identification of novel alternative splicing events in the huntingtin gene and assessment of the functional consequences using structural protein homology modelling.

    Science.gov (United States)

    Hughes, Alis C; Mort, Matthew; Elliston, Lyn; Thomas, Rhian M; Brooks, Simon P; Dunnett, Stephen B; Jones, Lesley

    2014-04-03

    Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by a pathological CAG trinucleotide repeat expansion in the large multi-exon gene, huntingtin (HTT). Although multiple pathogenic mechanisms have been proposed for HD, there is increasing interest in the RNA processing of the HTT gene. In mammals, most multi-exon genes are alternatively spliced; however, few alternative transcripts have been described for HTT. Given the numerous protein bands detected in mouse and human brain tissue by Western blotting using anti-huntingtin antibodies, we examined whether alternative splicing of HTT may account for some of these fragments. Using RT-PCR in mouse brain, we detected two novel splice variants of Htt that lacked the 111-bp exon 29 (Htt∆ex29) or retained a 57-bp portion of intron 28 (Htt(+57)in28) via use of a cryptic splice site. The alternative transcripts were present in wild-type and homozygous Hdh(Q150/Q150) mouse brain at all ages and in all brain regions and peripheral tissues studied. Differential splicing of Htt∆ex29 was found in the cerebellum of Hdh(Q150/Q150) mice with a significant reduction in transcript levels in mutant animals. In human brain, we detected similar splice variants lacking exons 28 and 29. The ability of alternatively spliced transcripts to encode different protein isoforms with individual functions in the cell, combined with the known role of splicing in disease, renders these novel transcripts of interest in the context of HD pathogenesis. Copyright © 2013. Published by Elsevier Ltd.

  10. Design of a high-throughput assay for alternative splicing using polymerase colonies.

    Science.gov (United States)

    Buhler, J D; Souvenir, R M; Zhang, W; Mitra, R D

    2004-01-01

    We propose an assay to detect and quantify alternative splicing simultaneously for numerous genes in a pool of cellular mRNA. The assay exploits polymerase colonies, a recently developed method for sampling and amplifying large numbers of individual transcript molecules into discrete spots on a gel. The proposed assay combines the advantages of microarrays for transcript quantitation with the sensitivity and precision of methods based on counting single transcript molecules. Given a collection of spots s(i), each containing an unknown splice variant of some known gene G(i), we design a series of hybridizations to short oligonucleotide probes to determine in parallel which exons of G(i) are present in every spot s(i). We give algorithms to minimize the cost of such designs.

  11. Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes Uruguay Syndrome, an X-Linked Disorder With Skeletal Muscle Hypertrophy and Premature Cardiac Death

    National Research Council Canada - National Science Library

    Xue, Yuan; Schoser, Benedikt; Rao, Aliz R; Quadrelli, Roberto; Vaglio, Alicia; Rupp, Verena; Beichler, Christine; Nelson, Stanley F; Schapacher-Tilp, Gudrun; Windpassinger, Christian; Wilcox, William R

    2016-01-01

    .... FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the FHL1C isoform...

  12. Aberrant Splicing Promotes Proteasomal Degradation of L-type Ca v 1.2 Calcium Channels by Competitive Binding for CaV β Subunits in Cardiac Hypertrophy

    NARCIS (Netherlands)

    Hu, Zhenyu; Wang, Jiong Wei; Yu, Dejie; Soon, Jia Lin; De Kleijn, Dominique P V|info:eu-repo/dai/nl/30481489X; Foo, Roger; Liao, Ping; Colecraft, Henry M.; Soong, Tuck Wah

    2016-01-01

    Decreased expression and activity of Ca V1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of Ca V1.2 channel, named Ca V1.2 e21+22, that

  13. A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation.

    Science.gov (United States)

    Conn, Vanessa M; Hugouvieux, Véronique; Nayak, Aditya; Conos, Stephanie A; Capovilla, Giovanna; Cildir, Gökhan; Jourdain, Agnès; Tergaonkar, Vinay; Schmid, Markus; Zubieta, Chloe; Conn, Simon J

    2017-04-18

    Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life 1 , yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS 2-4 . This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

  14. Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

    Science.gov (United States)

    Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

    2014-03-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Understanding alternative splicing of Cav1.2 calcium channels for a new approach towards individualized medicine

    Science.gov (United States)

    Liao, Ping; Soong, Tuck Wah

    2010-01-01

    Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Cav1.2, that are expressed in the cardiovascular system. Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects. PMID:23554629

  16. Functional characterisation of an intron retaining K+ transporter of barley reveals intron-mediated alternate splicing

    KAUST Repository

    Shahzad, K.

    2015-01-01

    Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K+ transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K+ uptake restored growth in medium containing hygromycin in the presence of different concentrations of K+ and mediated hypersensitivity to Na+. HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K+ and Na+ concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

  17. Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

    Science.gov (United States)

    Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M

    2010-10-01

    CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

  18. Variants of stretch-activated two-pore potassium channel TREK-1 associated with preterm labor in humans.

    Science.gov (United States)

    Wu, Yi-Ying; Singer, Cherie A; Buxton, Iain L O

    2012-10-01

    Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.

  19. Variants of Stretch-Activated Two-Pore Potassium Channel TREK-1 Associated with Preterm Labor in Humans1

    Science.gov (United States)

    Wu, Yi-Ying; Singer, Cherie A.; Buxton, Iain L.O.

    2012-01-01

    ABSTRACT Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype. PMID:22811574

  20. Genomics of alternative splicing: evolution, development and pathophysiology.

    Science.gov (United States)

    Gamazon, Eric R; Stranger, Barbara E

    2014-06-01

    Alternative splicing is a major cellular mechanism in metazoans for generating proteomic diversity. A large proportion of protein-coding genes in multicellular organisms undergo alternative splicing, and in humans, it has been estimated that nearly 90 % of protein-coding genes-much larger than expected-are subject to alternative splicing. Genomic analyses of alternative splicing have illuminated its universal role in shaping the evolution of genomes, in the control of developmental processes, and in the dynamic regulation of the transcriptome to influence phenotype. Disruption of the splicing machinery has been found to drive pathophysiology, and indeed reprogramming of aberrant splicing can provide novel approaches to the development of molecular therapy. This review focuses on the recent progress in our understanding of alternative splicing brought about by the unprecedented explosive growth of genomic data and highlights the relevance of human splicing variation on disease and therapy.

  1. Tissue-specific splicing factor gene expression signatures

    NARCIS (Netherlands)

    A.R. Grosso; A.Q. Gomes (Anita); N.L. Barbosa-Morais (Nuno); S. Caldeira (Sandra); N.P. Thorne (Natalie); G. Grech (Godfrey); M.M. von Lindern (Marieke); M. Carmo-Fonseca (Maria)

    2008-01-01

    textabstractThe alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that

  2. Tissue-specific splicing factor gene expression signatures

    NARCIS (Netherlands)

    Grosso, Ana Rita; Gomes, Anita Q.; Barbosa-Morais, Nuno L.; Caldeira, Sandra; Thorne, Natalie P.; Grech, Godfrey; von Lindern, Marieke; Carmo-Fonseca, Maria

    2008-01-01

    The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific

  3. Prevalence of Titin Truncating Variants in General Population.

    Directory of Open Access Journals (Sweden)

    Oyediran Akinrinade

    Full Text Available Truncating titin (TTN mutations, especially in A-band region, represent the most common cause of dilated cardiomyopathy (DCM. Clinical interpretation of these variants can be challenging, as these variants are also present in reference populations. We carried out systematic analyses of TTN truncating variants (TTNtv in publicly available reference populations, including, for the first time, data from Exome Aggregation Consortium (ExAC. The goal was to establish more accurate estimate of prevalence of different TTNtv to allow better clinical interpretation of these findings.Using data from 1000 Genomes Project, Exome Sequencing Project (ESP and ExAC, we estimated the prevalence of TTNtv in the population. In the three population datasets, 52-54% of TTNtv were not affecting all TTN transcripts. The frequency of truncations affecting all transcripts in ExAC was 0.36% (0.32% - 0.41%, 95% CI and 0.19% (0.16% - 0.23%, 95% CI for those affecting the A-band. In the A-band region, the prevalences of frameshift, nonsense and essential splice site variants were 0.057%, 0.090%, and 0.047% respectively. Cga/Tga (arginine/nonsense-R/* transitional change at CpG mutation hotspots was the most frequent type of TTN nonsense mutation accounting for 91.3% (21/23 of arginine residue nonsense mutation (R/* at TTN A-band region. Non-essential splice-site variants had significantly lower proportion of private variants and higher proportion of low-frequency variants compared to essential splice-site variants (P = 0.01; P = 5.1 X 10-4, respectively.A-band TTNtv are more rare in the general population than previously reported. Based on this analysis, one in 500 carries a truncation in TTN A-band suggesting the penetrance of these potentially harmful variants is still poorly understood, and some of these variants do not manifest as autosomal dominant DCM. This calls for caution when interpreting TTNtv in individuals and families with no history of DCM. Considering the

  4. CCA1 alternative splicing as a way of linking the circadian clock to temperature response in Arabidopsis

    OpenAIRE

    Park, Mi-Jeong; Seo, Pil Joon; Park, Chung-Mo

    2012-01-01

    Most living organisms on the earth have the circadian clock to synchronize their biochemical processes and physiological activities with environmental changes to optimize their propagation and survival. CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) is one of the core clock components in Arabidopsis. Notably, it is also associated with cold acclimation. However, it is largely unknown how CCA1 activity is modulated by low temperatures. We found that the CCA1 activity is self-regulated by a splice variant ...

  5. Comparison of splice sites in mammals and chicken

    OpenAIRE

    Abril Ferrando, Josep Francesc; Castelo Valdueza, Robert; Guigó Serra, Roderic

    2005-01-01

    We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by backgroun...

  6. Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa

    Science.gov (United States)

    Pan, Xinyuan; Chen, Xue; Liu, Xiaoxing; Gao, Xiang; Kang, Xiaoli; Xu, Qihua; Chen, Xuejuan; Zhao, Kanxing; Zhang, Xiumei; Chu, Qiaomei; Wang, Xiuying

    2014-01-01

    Purpose Seven genes involved in precursor mRNA (pre-mRNA) splicing have been implicated in autosomal dominant retinitis pigmentosa (adRP). We sought to detect mutations in all seven genes in Chinese families with RP, to characterize the relevant phenotypes, and to evaluate the prevalence of mutations in splicing genes in patients with adRP. Methods Six unrelated families from our adRP cohort (42 families) and two additional families with RP with uncertain inheritance mode were clinically characterized in the present study. Targeted sequence capture with next-generation massively parallel sequencing (NGS) was performed to screen mutations in 189 genes including all seven pre-mRNA splicing genes associated with adRP. Variants detected with NGS were filtered with bioinformatics analyses, validated with Sanger sequencing, and prioritized with pathogenicity analysis. Results Mutations in pre-mRNA splicing genes were identified in three individual families including one novel frameshift mutation in PRPF31 (p.Leu366fs*1) and two known mutations in SNRNP200 (p.Arg681His and p.Ser1087Leu). The patients carrying SNRNP200 p.R681H showed rapid disease progression, and the family carrying p.S1087L presented earlier onset ages and more severe phenotypes compared to another previously reported family with p.S1087L. In five other families, we identified mutations in other RP-related genes, including RP1 p. Ser781* (novel), RP2 p.Gln65* (novel) and p.Ile137del (novel), IMPDH1 p.Asp311Asn (recurrent), and RHO p.Pro347Leu (recurrent). Conclusions Mutations in splicing genes identified in the present and our previous study account for 9.5% in our adRP cohort, indicating the important role of pre-mRNA splicing deficiency in the etiology of adRP. Mutations in the same splicing gene, or even the same mutation, could correlate with different phenotypic severities, complicating the genotype–phenotype correlation and clinical prognosis. PMID:24940031

  7. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2007-01-01

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...... expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general...

  8. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Science.gov (United States)

    2010-07-01

    ... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically efficient... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Electrical connections or splices; suitability...

  9. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam

    2007-01-01

    Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been...

  10. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Common and rare variants in SCN10A modulate the risk of atrial fibrillation

    DEFF Research Database (Denmark)

    Jabbari, Javad; Olesen, Morten S; Yuan, Lei

    2015-01-01

    of other cardiovascular disease or dysfunction (lone AF). In an association study of the rs6795970 single nucleotide polymorphism variant, we included 515 AF patients and 2 control cohorts of 730 individuals free of AF and 6161 randomly sampled individuals. Functional characterization of SCN10A variants...... is in high linkage disequilibrium with the nonsynonymous variant in SCN10A, rs6795970 (V1073A, r(2)=0.933). We therefore sought to determine whether common and rare SCN10A variants are associated with early onset AF. METHODS AND RESULTS: SCN10A was sequenced in 225 AF patients in whom there was no evidence...... was performed by whole-cell patch-clamping. In the lone AF cohort, 9 rare missense variants and 1 splice site donor variant were detected. Interestingly, AF patients were found to have higher G allele frequency of rs6795970, which encodes the alanine variant at position 1073 (described from here on as A1073...

  12. Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides.

    Science.gov (United States)

    Hemphill, James; Liu, Qingyang; Uprety, Rajendra; Samanta, Subhas; Tsang, Michael; Juliano, Rudolph L; Deiters, Alexander

    2015-03-18

    The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF → ON and ON → OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.

  13. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor

  14. On the Possibility of an Early Evolutionary Origin for the Spliced Leader Trans-Splicing.

    Science.gov (United States)

    Krchňáková, Zuzana; Krajčovič, Juraj; Vesteg, Matej

    2017-08-01

    Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

  15. Survey of gene splicing algorithms based on reads.

    Science.gov (United States)

    Si, Xiuhua; Wang, Qian; Zhang, Lei; Wu, Ruo; Ma, Jiquan

    2017-09-05

    Gene splicing is the process of assembling a large number of unordered short sequence fragments to the original genome sequence as accurately as possible. Several popular splicing algorithms based on reads are reviewed in this article, including reference genome algorithms and de novo splicing algorithms (Greedy-extension, Overlap-Layout-Consensus graph, De Bruijn graph). We also discuss a new splicing method based on the MapReduce strategy and Hadoop. By comparing these algorithms, some conclusions are drawn and some suggestions on gene splicing research are made.

  16. Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.

    Science.gov (United States)

    Lambolez, B; Ropert, N; Perrais, D; Rossier, J; Hestrin, S

    1996-03-05

    In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.

  17. AR-A 014418 Used against GSK3beta Downregulates Expression of hnRNPA1 and SF2/ASF Splicing Factors.

    Science.gov (United States)

    Yadav, Ajay K; Vashishta, Vidhi; Joshi, Nidhi; Taneja, Pankaj

    2014-01-01

    Glioblastoma is one of the most aggressive forms of primary brain tumors of glial cells, including aberrant regulation of glycogen synthase kinase 3 β (GSK3 β ) and splicing factors deregulation. Here, we investigate the role of small molecule AR-A014418 and Manzamine A against GSK3 kinase with factual control on splicing regulators. AR-A 014418, 48 hrs posttreatment, caused dose (25-100  μ M) dependent inhibition in U373 and U87 cell viability with also inhibition in activating tyrosine phosphorylation of GSK3alpha (Tyr 279) and beta (Tyr 216). Furthermore, inhibition of GSK3 kinase resulted in significant downregulation of splicing factors (SRSF1, SRSF5, PTPB1, and hnRNP) in U87 cells with downregulation of antiapoptotic genes such as BCL2, BCL-xL, Survivin, MCL1, and BMI1. Similarly, downregulation of splicing factors was also observed in U373 glioma cell after using SiRNA against AKT and GSK3beta kinase. In addition, potential roles of AR-A014418 in downregulation of splicing factors were reflected with decrease in Anxa7 (VA) variant and increase in Anxa7 WT tumor suppressor transcript and protein. The above results suggest that inhibition of GSK3beta kinase activation could be the beneficial strategy to inhibit the occurrence of alternative cancer escape pathway via downregulating the expression of splicing regulators as well as apoptosis.

  18. Alteration of introns in a hyaluronan synthase 1 (HAS1 minigene convert Pre-mRNA [corrected] splicing to the aberrant pattern in multiple myeloma (MM: MM patients harbor similar changes.

    Directory of Open Access Journals (Sweden)

    Jitra Kriangkum

    Full Text Available Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1 have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.

  19. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    Science.gov (United States)

    Churbanov, Alexander; Vořechovský, Igor; Hicks, Chindo

    2009-01-01

    Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research. PMID:19889216

  20. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2009-11-01

    Full Text Available Abstract Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15% were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3% cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.

  1. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms.

    Science.gov (United States)

    Churbanov, Alexander; Vorechovský, Igor; Hicks, Chindo

    2009-11-04

    Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.

  2. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

    Directory of Open Access Journals (Sweden)

    Clément Bouton

    Full Text Available The plant pararetrovirus Cauliflower mosaic virus (CaMV uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

  3. Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

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    Ferdos Alaa El Din

    Full Text Available Hereditary Hemorrhagic Telangiectasia syndrome (HHT or Rendu-Osler-Weber (ROW syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG and activin receptor-like kinase 1 (ACVRL1or ALK1 genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1, the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7 was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

  4. A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.

    Science.gov (United States)

    Crotti, Lia; Lewandowska, Marzena A; Schwartz, Peter J; Insolia, Roberto; Pedrazzini, Matteo; Bussani, Erica; Dagradi, Federica; George, Alfred L; Pagani, Franco

    2009-02-01

    Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

  5. Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer.

    Science.gov (United States)

    Blanchard, Anne A; Zelinski, Teresa; Xie, Jiuyong; Cooper, Steven; Penner, Carla; Leygue, Etienne; Myal, Yvonne

    2016-01-01

    The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (CLDN1) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for CLDN1 in human invasive breast cancer. RT-PCR analysis of CLDN1 transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR CLDN1 specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted. A number of CLDN1 transcript variants were identified in these breast tumors. All variants were shorter than the classical CLDN1 transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of CLDN1; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of CLDN1 transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how CLDN1 is reduced or silenced in human breast cancer.

  6. Genome-wide data-mining of candidate human splice translational efficiency polymorphisms (STEPs and an online database.

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    Christopher A Raistrick

    2010-10-01

    Full Text Available Variation in pre-mRNA splicing is common and in some cases caused by genetic variants in intronic splicing motifs. Recent studies into the insulin gene (INS discovered a polymorphism in a 5' non-coding intron that influences the likelihood of intron retention in the final mRNA, extending the 5' untranslated region and maintaining protein quality. Retention was also associated with increased insulin levels, suggesting that such variants--splice translational efficiency polymorphisms (STEPs--may relate to disease phenotypes through differential protein expression. We set out to explore the prevalence of STEPs in the human genome and validate this new category of protein quantitative trait loci (pQTL using publicly available data.Gene transcript and variant data were collected and mined for candidate STEPs in motif regions. Sequences from transcripts containing potential STEPs were analysed for evidence of splice site recognition and an effect in expressed sequence tags (ESTs. 16 publicly released genome-wide association data sets of common diseases were searched for association to candidate polymorphisms with HapMap frequency data. Our study found 3324 candidate STEPs lying in motif sequences of 5' non-coding introns and further mining revealed 170 with transcript evidence of intron retention. 21 potential STEPs had EST evidence of intron retention or exon extension, as well as population frequency data for comparison.Results suggest that the insulin STEP was not a unique example and that many STEPs may occur genome-wide with potentially causal effects in complex disease. An online database of STEPs is freely accessible at http://dbstep.genes.org.uk/.

  7. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

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    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  8. Deciphering the plant splicing code: Experimental and computational approaches for predicting alternative splicing and splicing regulatory elements

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    Anireddy S.N. Reddy

    2012-02-01

    Full Text Available Extensive alternative splicing (AS of precursor mRNAs (pre-mRNAs in multicellular eukaryotes increases the protein-coding capacity of a genome and allows novel ways to regulate gene expression. In fowering plants, up to 48% of intron-containing genes exhibit AS. However, the full extent of AS in plants is not yet known, as only a few high throughput RNA-Seq studies have been performed. As the cost of obtaining RNA-Seq reads continues to fall, it is anticipated that huge amounts of plant sequence data will accumulate and help in obtaining a more complete picture of AS in plants. Although it is not an onerous task to obtain hundreds of millions of reads using high throughput sequencing technologies, computational tools to accurately predict and visualize AS are still being developed and refined. This review will discuss the tools to predict and visualize transcriptome-wide AS in plants using short reads and highlight their limitations. Comparative studies of AS events between plants and animals have revealed that there are major differences in the most prevalent types of AS events, suggesting that plants and animals differ in the way they recognize exons and introns. Extensive studies have been performed in animals to identify cis-elements involved in regulating AS, especially in exon skipping. However, such studies are in their infancy in plants. Here, we review the current state of research on splicing regulatory elements (SREs and briefly discuss emerging experimental and computational tools to identify cis-elements involved in regulation of AS in plants. The availability of curated alternative splice forms in plants makes it possible to use computational tools to predict SREs involved in AS regulation, which can then be verified experimentally. Such studies will permit identification of plant-specific features involved in AS regulation and contribute to deciphering the splicing code in plants.

  9. Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing

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    Michèle Rouleau

    2016-09-01

    Full Text Available Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides, cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes.

  10. Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing.

    Science.gov (United States)

    Rouleau, Michèle; Tourancheau, Alan; Girard-Bock, Camille; Villeneuve, Lyne; Vaucher, Jonathan; Duperré, Anne-Marie; Audet-Delage, Yannick; Gilbert, Isabelle; Popa, Ion; Droit, Arnaud; Guillemette, Chantal

    2016-09-27

    Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

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    Erkelenz, Steffen; Theiss, Stephan; Otte, Marianne; Widera, Marek; Peter, Jan Otto; Schaal, Heiner

    2014-01-01

    Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

    Science.gov (United States)

    Jiménez-López, Claudia; Lorenz, Michael C.; van Hoof, Ambro

    2013-01-01

    Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes. PMID:23516382

  13. Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

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    Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

    2005-06-01

    In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed

  14. Multiple splice defects in ABCA1 cause low HDL-C in a family with Hypoalphalipoproteinemia and premature coronary disease

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    Miller Michael

    2009-01-01

    Full Text Available Abstract Background Mutations at splice junctions causing exon skipping are uncommon compared to exonic mutations, and two intronic mutations causing an aberrant phenotype have rarely been reported. Despite the high number of functional ABCA1 mutations reported to date, splice variants have been reported infrequently. We screened DNA from a 41 year-old male with low HDL-C (12 mg/dL [0.31 mmol/L] and a family history of premature coronary heart disease (CHD using polymerase chain reaction single-strand conformation polymorphism (SSCP analysis. Methods Family members with low levels of HDL-C (n = 6 were screened by SSCP for mutations in ABCA1. Samples with altered SSCP patterns were sequenced directly using either an ABI 3700 or ABI3730Xl DNA Analyzer. To screen for splicing defects, cDNA was isolated from the proband's RNA and was sequenced as above. A series of minigenes were constructed to determine the contribution of normal and defective alleles. Results Two novel splice variants in ABCA1 were identified. The first mutation was a single base pair change (T->C in IVS 7, 6 bps downstream from the exon7/intron7 junction. Amplification of cDNA and allelic subcloning identified skipping of Exon 7 that results in the elimination of 59 amino acids from the first extracellular loop of the ABCA1 protein. The second mutation was a single base pair change (G->C at IVS 31 -1, at the intron/exon junction of exon 32. This mutation causes skipping of exon 32, resulting in 8 novel amino acids followed by a stop codon and a predicted protein size of 1496 AA, compared to normal (2261 AA. Bioinformatic studies predicted an impact on splicing as confirmed by in vitro assays of constitutive splicing. Conclusion In addition to carnitine-acylcarnitine translocase (CACT deficiency and Hermansky-Pudlak syndrome type 3, this represents only the third reported case in which 2 different splice mutations has resulted in an aberrant clinical phenotype.

  15. DNA splicing by directed ligation (SDL).

    Science.gov (United States)

    Berlin, Y A

    1999-01-01

    Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

  16. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

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    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  17. Stochastic principles governing alternative splicing of RNA

    OpenAIRE

    Jianfei Hu; Eli Boritz; William Wylie; Douek, Daniel C.

    2017-01-01

    Author summary Alternative RNA splicing within eukaryotic cells enables each gene to generate multiple different mature transcripts which further encode proteins with distinct or even opposing functions. The relative frequencies of the transcript isoforms generated by a particular gene are essential to the maintenance of normal cellular physiology; however, the underlying mechanisms and principles that govern these frequencies are unknown. We analyzed the frequency distribution of all transcr...

  18. An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer

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    Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya; Grover, Himanshu; Askenazi, Manor; Teubl, Jennifer; Cao, Song; McLellan, Michael D.; Clauser, Karl R.; Tabb, David L.; Mertins, Philipp; Slebos, Robbert; Erdmann-Gilmore, Petra; Li, Shunqiang; Gunawardena, Harsha P.; Xie, Ling; Liu, Tao; Zhou, Jian-Ying; Sun, Shisheng; Hoadley, Katherine A.; Perou, Charles M.; Chen, Xian; Davies, Sherri R.; Maher, Christopher A.; Kinsinger, Christopher R.; Rodland, Karen D.; Zhang, Hui; Zhang, Zhen; Ding, Li; Townsend, R. Reid; Rodriguez, Henry; Chan, Daniel; Smith, Richard D.; Liebler, Daniel C.; Carr, Steven A.; Payne, Samuel; Ellis, Matthew J.; Fenyő, David

    2015-12-02

    Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic and germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.

  19. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  20. Tracking the evolution of alternatively spliced exons within the Dscam family

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    Vision Todd J

    2006-02-01

    Full Text Available Abstract Background The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17, potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. Results Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee, we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248–283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. Conclusion Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays

  1. The Clinical Significance of Unknown Sequence Variants in BRCA Genes

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    Calò, Valentina; Bruno, Loredana; Paglia, Laura La; Perez, Marco; Margarese, Naomi [Department of Surgery and Oncology, Regional Reference Center for the Biomolecular Characterization and Genetic Screening of Hereditary Tumors, University of Palermo, Via del Vespro 127, 90127 Palermo (Italy); Gaudio, Francesca Di [Department of Medical Biotechnologies and Legal Medicine, University of Palermo, Palermo (Italy); Russo, Antonio, E-mail: lab-oncobiologia@usa.net [Department of Surgery and Oncology, Regional Reference Center for the Biomolecular Characterization and Genetic Screening of Hereditary Tumors, University of Palermo, Via del Vespro 127, 90127 Palermo (Italy)

    2010-09-10

    Germline mutations in BRCA1/2 genes are responsible for a large proportion of hereditary breast and/or ovarian cancers. Many highly penetrant predisposition alleles have been identified and include frameshift or nonsense mutations that lead to the translation of a truncated protein. Other alleles contain missense mutations, which result in amino acid substitution and intronic variants with splicing effect. The discovery of variants of uncertain/unclassified significance (VUS) is a result that can complicate rather than improve the risk assessment process. VUSs are mainly missense mutations, but also include a number of intronic variants and in-frame deletions and insertions. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, located throughout the whole gene (Breast Cancer Information Core Database (BIC database)). Up to 10–20% of the BRCA tests report the identification of a variant of uncertain significance. There are many methods to discriminate deleterious/high-risk from neutral/low-risk unclassified variants (i.e., analysis of the cosegregation in families of the VUS, measure of the influence of the VUSs on the wild-type protein activity, comparison of sequence conservation across multiple species), but only an integrated analysis of these methods can contribute to a real interpretation of the functional and clinical role of the discussed variants. The aim of our manuscript is to review the studies on BRCA VUS in order to clarify their clinical relevance.

  2. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    Science.gov (United States)

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells.

  3. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells

    Directory of Open Access Journals (Sweden)

    Ge Changhui

    2010-04-01

    Full Text Available Abstract Background PCBP1 (or alpha CP1 or hnRNP E1, a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. Results We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. Conclusions We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

  4. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  5. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs...... and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  6. Accumulation of GC donor splice signals in mammals

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    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  7. Test and Analysis of Spliced DI-BSCCO HTS Tapes

    Science.gov (United States)

    Fetisov, S. S.; Sotnikov, D. V.; Radchenko, I. P.; Vysotsky, V. S.; Osabe, G.; Kinoshita, K.; Fujikami, J.; Kobayashi, S.; Yamazaki, K.

    For some applications, sho