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Sample records for splice variant expression

  1. Postnatal Expression of V2 Vasopressin Receptor Splice Variants in the Rat Cerebellum

    Science.gov (United States)

    Vargas, Karina J.; Sarmiento, José M.; Ehrenfeld, Pamela; Añazco, Carolina C.; Villanueva, Carolina I.; Carmona, Pamela L.; Brenet, Marianne; Navarro, Javier; Müller-Esterl, Werner; Figueroa, Carlos D.; González, Carlos B.

    2010-01-01

    The V2 vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V2a and V2b) first identified in the kidney. The open reading frame of the alternatively spliced V2b transcripten codes a truncated receptor, showing the same amino acid sequence as the canonical V2a receptor up to the 6th transmembrane segment, but displaying a distinct sequence to the corresponding 7th transmembrane segment and C-terminal domain relative to the V2a receptor. Here, we demonstrate the postnatal expression of V2a and V2b variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V2 splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V2 receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 .expressing similar amounts of both V2 splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V2a receptors, suggesting that the differential expression of the V2 splice variants regulate the vasopressin signaling in the cerebellum. PMID:19281786

  2. 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

  3. A family of splice variants of CstF-64 expressed in vertebrate nervous systems

    Science.gov (United States)

    Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda; MacDonald, Clinton C

    2009-01-01

    Background Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. Results We discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene

  4. Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

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    Hatakeyama Keiichi

    2012-12-01

    Full Text Available Abstract Background Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated. Results Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue. Conclusions Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

  5. Expression of TRAIL-splice variants in gastric carcinomas: identification of TRAIL-γ as a prognostic marker

    International Nuclear Information System (INIS)

    Krieg, Andreas; Mahotka, Csaba; Mersch, Sabrina; Wolf, Nadine; Stoecklein, Nikolas H; Verde, Pablo E; Schulte am Esch, Jan; Heikaus, Sebastian; Gabbert, Helmut E; Knoefel, Wolfram T

    2013-01-01

    TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF-superfamily that induces apoptotic cell death in a wide range of neoplastic cells in vivo as well as in vitro. We identified two alternative TRAIL-splice variants, i.e. TRAIL-β and TRAIL-γ that are characterized by the loss of their proapoptotic properties. Herein, we investigated the expression and the prognostic values of the TRAIL-splice variants in gastric carcinomas. Real time PCR for amplification of the TRAIL-splice variants was performed in tumour tissue specimens and corresponding normal tissues of 41 consecutive patients with gastric carcinoma. Differences on mRNA-expression levels of the TRAIL-isoforms were compared to histo-pathological variables and correlated with survival data. All three TRAIL-splice variants could be detected in both non-malignant and malignant tissues, irrespective of their histological staging, grading or tumour types. However, TRAIL-β exhibited a higher expression in normal gastric tissue. The proapoptotic TRAIL-α expression was increased in gastric carcinomas when compared to TRAIL-β and TRAIL-γ. In addition, overexpression of TRAIL-γ was associated with a significant higher survival rate. This is the first study that investigated the expression of TRAIL-splice variants in gastric carcinoma tissue samples. Thus, we provide first data that indicate a prognostic value for TRAIL-γ overexpression in this tumour entity

  6. 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

  7. PGC1α −1 Nucleosome Position and Splice Variant Expression and Cardiovascular Disease Risk in Overweight and Obese Individuals

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    Tara M. Henagan

    2014-01-01

    Full Text Available PGC1α, a transcriptional coactivator, interacts with PPARs and others to regulate skeletal muscle metabolism. PGC1α undergoes splicing to produce several mRNA variants, with the NTPGC1α variant having a similar biological function to the full length PGC1α (FLPGC1α. CVD is associated with obesity and T2D and a lower percentage of type 1 oxidative fibers and impaired mitochondrial function in skeletal muscle, characteristics determined by PGC1α expression. PGC1α expression is epigenetically regulated in skeletal muscle to determine mitochondrial adaptations, and epigenetic modifications may regulate mRNA splicing. We report in this paper that skeletal muscle PGC1α  −1 nucleosome (−1N position is associated with splice variant NTPGC1α but not FLPGC1α expression. Division of participants based on the −1N position revealed that those individuals with a −1N phased further upstream from the transcriptional start site (UP expressed lower levels of NTPGC1α than those with the −1N more proximal to TSS (DN. UP showed an increase in body fat percentage and serum total and LDL cholesterol. These findings suggest that the −1N may be a potential epigenetic regulator of NTPGC1α splice variant expression, and −1N position and NTPGC1α variant expression in skeletal muscle are linked to CVD risk. This trial is registered with clinicaltrials.gov, identifier NCT00458133.

  8. 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

  9. Osteopontin and splice variant expression level in human malignant glioma: Radiobiologic effects and prognosis after radiotherapy

    International Nuclear Information System (INIS)

    Güttler, Antje; Giebler, Maria; Cuno, Peter; Wichmann, Henri; Keßler, Jacqueline; Ostheimer, Christian; Söling, Ariane; Strauss, Christian; Illert, Jörg; Kappler, Matthias; Vordermark, Dirk; Bache, Matthias

    2013-01-01

    Background and purpose: We investigated the role of the hypoxia-associated secreted glycoprotein osteopontin (OPN) in the response of malignant glioma to radiotherapy by characterizing OPN and its splice variants in vitro and in patient material. Material and methods: The effect of siRNA knockdown of OPN splice variants on cellular and radiobiologic behavior was analyzed in U251MG cells using OpnS siRNA (inhibition of all OPN splice variants) and OpnAC siRNA (knockdown only of OPNa and OPNc). OPN and splice variant mRNA levels were quantified in archival material of 41 glioblastoma tumor samples. Plasma OPN was prospectively measured in 33 malignant glioma patients. Results: Inhibition of OPNa and OPNc (OpnAC) reduced clonogenic survival in U251MG cells but did not affect proliferation, migration or apoptosis. Knockdown of all OPN splice variants (OpnS) resulted in an even stronger inhibition of clonogenic survival, while cell proliferation and migration were reduced and rate of apoptosis was increased. Additional irradiation had additive effects with both siRNAs. Plasma OPN increased continuously in malignant glioma patients and was associated with poor survival. Conclusions: OPNb is partially able to compensate the effects of OPNa and OPNc knockdown in U251MG cells. High OPN plasma levels at the end of radiotherapy are associated with poor survival

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

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    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. Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

    OpenAIRE

    Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

    2012-01-01

    The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

  12. PGC1? ?1 Nucleosome Position and Splice Variant Expression and Cardiovascular Disease Risk in Overweight and Obese Individuals

    OpenAIRE

    Henagan, Tara M.; Stewart, Laura K.; Forney, Laura A.; Sparks, Lauren M.; Johannsen, Neil; Church, Timothy S.

    2014-01-01

    PGC1α, a transcriptional coactivator, interacts with PPARs and others to regulate skeletal muscle metabolism. PGC1α undergoes splicing to produce several mRNA variants, with the NTPGC1α variant having a similar biological function to the full length PGC1α (FLPGC1α). CVD is associated with obesity and T2D and a lower percentage of type 1 oxidative fibers and impaired mitochondrial function in skeletal muscle, characteristics determined by PGC1α expression. PGC1α expression is epigenetically re...

  13. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

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    John S Y Park

    Full Text Available CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2 or an alternatively spliced variant form (hCDK5RAP2-V1 that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

  14. A statistical method for predicting splice variants between two groups of samples using GeneChip® expression array data

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    Olson James M

    2006-04-01

    Full Text Available Abstract Background Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip® that uses multiple oligonucleotide probes (i.e. probe set, since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip® was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip® gene expression array data. Results We developed a two-step approach to predict alternative splicing from GeneChip® data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip® Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic

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

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    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.

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

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    Schober, Douglas A; Croy, Carrie H; Ruble, Cara L; Tao, Ran; Felder, Christian C

    2017-01-01

    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.

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

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    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.

  18. Profiling Lgals9 splice variant expression at the fetal-maternal interface: implications in normal and pathological human pregnancy.

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    Heusschen, Roy; Freitag, Nancy; Tirado-González, Irene; Barrientos, Gabriela; Moschansky, Petra; Muñoz-Fernández, Raquel; Leno-Durán, Ester; Klapp, Burghard F; Thijssen, Victor L J L; Blois, Sandra M

    2013-01-01

    Disruption of fetal-maternal tolerance mechanisms can contribute to pregnancy complications, including spontaneous abortion. Galectin-9 (LGALS9), a tandem repeat lectin associated with immune modulation, is expressed in the endometrium during the mid and late secretory phases and in decidua during human early pregnancy. However, the role of LGALS9 during pregnancy remains poorly understood. We used real-time PCR and immunohistochemical staining to analyze the expression of Lgals9/LGALS9 during mouse gestation as well as in human tissues obtained from normal pregnancy and spontaneous abortions. In mice, three Lgals9 splice variants were detected, the expression of which was differentially regulated during gestation. Furthermore, decidual Lgals9 expression was deregulated in a mouse model of spontaneous abortion, whereas placental levels did not change. We further found that the LGALS9 D5 isoform suppresses interferon gamma production by decidual natural killer cells. In human patients, six Lgals9 splice variants were detected, and a decrease in Lgals9 D5/10 was associated with spontaneous abortion. Altogether, these results show a differential regulation of Lgals9 isoform expression during normal and pathological pregnancies and designate Lgals9 as a potential marker for adverse pregnancy outcomes.

  19. RAGE splicing variants in mammals.

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    Sterenczak, Katharina Anna; Nolte, Ingo; Murua Escobar, Hugo

    2013-01-01

    The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimer's disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

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

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    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.

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

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    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.

  2. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants.

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    Griffith, Jason S; Liu, Ya-Guang; Tekmal, Rajeshwar R; Binkley, Peter A; Holden, Alan E C; Schenken, Robert S

    2010-04-01

    We previously demonstrated that adherence of endometrial epithelial (EECs) and stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) is partly regulated by ESC/EEC CD44 interactions with PMC associated hyaluronan. CD44, a transmembrane glycoprotein and major ligand for hyaluronan, has numerous splice variants which may impact hyaluronan binding. Here, we assessed whether ESCs and EECs from women with endometriosis demonstrate increased adherence to PMCs and examined CD44 splice variants' potential role in this process. In vitro study. Academic medical center. Fertility patients with and without endometriosis. Menstrual endometrium was collected from women with and without endometriosis confirmed surgically. The adherence of ESC/EECs to PMCs was measured. The ESC/EEC CD44 splice variants were assessed using dot-blot analysis. The ESCs and EECs from women with endometriosis demonstrated increased adherence to PMCs. The predominant CD44 splice variants expressed by ESCs and EECs from women with and without endometriosis were v3, v6, v7, v8, v9, and v10. The ESCs and EECs from women with endometriosis were more likely to express v6, v7, v8, and v9. Increased eutopic endometrial-PMC adherence and CD44 splice variant expression may contribute to the histogenesis of endometriotic lesions. Elucidation of factors controlling this expression may lead to novel endometriosis therapies. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. 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

    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....... 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...

  4. 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...... 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......, and tyrosine kinase activity toward the exogenous substrate poly(Glu-Tyr(4:1)). Furthermore, no significant relationship was demonstrated between the glucose disposal rate and the relative expression of insulin receptor splice variants. In conclusion, in skeletal muscle from both normal control subjects...

  5. Systematic Identification of Genes Required for Expression of Androgen Receptor Splice Variants

    Science.gov (United States)

    2016-08-01

    cells using a packaging system from SBI per the manufacturer’s protocol, as described previously [33]. For infection, exponentially growing cells were...30. Kashima T, Rao N, Manley JL. An intronic element con- tributes to splicing repression in spinal muscular atrophy. Proceedings of the National

  6. The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

    Science.gov (United States)

    Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

    2015-01-01

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

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

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    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

  8. Over-expression of the splice variant of CONSTANS enhances the in vitro synthesis of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Abhishek Kumar

    2017-10-01

    Full Text Available Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement in bio- nanotechnology and has been successfully attempted in more than 41 plant species. However, an established model plant system for unravelling the biochemical pathways of silver nanoparticle (AgNPs production is lacking. Here we have shown in Arabidopsis thaliana a genetic model plant and in its misexpressing lines of splice variant CONSTANS (COβ for the silver nanoparticle biosynthesis in vitro. Employing the biochemical, spectroscopic, Transmission Electron Microscopy (TEM, Raman spectroscopy, Nuclear Magnetic Resonance (NMR and powder x-rays diffraction (Powder XRD methods and using selected mutants and over- expressing line of Arabidopsis thaliana involved in sugar homeostasis. Additionally, a comparative analysis of AgNPs synthesis using different transgenic lines of Arabidopsis was explored. Here we have shown that plant extract of COβ and gi-100 (mutant line of GIGANTEA showed the highest potential of nanoparticle production as comparable to Col-0 and over- expressing line of GIGANTEA (35SGi. Silver nanoparticles production in the Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemical pathways, but also could address the mechanism behind different shapes of AgNPs produced using plant extracts.

  9. 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

  10. Changes in localization of human discs large (hDlg) during keratinocyte differentiation is associated with expression of alternatively spliced hDlg variants

    International Nuclear Information System (INIS)

    Roberts, S.; Calautti, E.; Vanderweil, S.; Nguyen, H.O.; Foley, A.; Baden, H.P.; Viel, A.

    2007-01-01

    Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation

  11. Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

    International Nuclear Information System (INIS)

    Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald; Roetger, Antje; Kemming, Dirk; Schier, Katrin; Sauter, Guido; Buerger, Horst

    2005-01-01

    By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employed to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer

  12. 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.

  13. Human CRF2 α and β splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

    International Nuclear Information System (INIS)

    Ardati, A.; Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J.; Valdenaire, O.

    1999-01-01

    Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF 1 and CRF 2 ). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF 2 subtype receptors, CRF 2α and CRF 2β , have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF 2β receptor. We have used radioligand binding with [ 125 I]-tyr 0 -sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [ 125 I]-tyr 0 -sauvagine binding to the hCRF 2β receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF 2α receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF 2α receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [ 125 I]-tyr 0 -sauvagine to both hCRF 2 receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF 2 α-helical CRF (9-41) oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist α-helical CRF (9-41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF 2 receptor isoforms. Taken together, these results indicate that the pharmacological profiles of the CRF 2 splice variants are identical. This indicates that the region of the N-terminus that varies

  14. New candidate tumor-suppressor gene KLF6 and its splice variant KLF6 SV2 counterbalancing expression in primary hepatocarcinoma.

    Science.gov (United States)

    Zhenzhen, Zhou; De'an, Tian; Limin, Xia; Wei, Yan; Min, Luo

    2012-01-01

    This study aimed to detect the expression of newly discovered zinc finger transcriptional factor KLF6 and its splice variant KLF6 SV2 in primary hepatocarcinoma (PHC) tissues and hepatoma cell strains, and to evaluate their clinicopathologic relationship with PHC. Wild-type KLF6 and KLF6 SV2 mRNA expression was determined by RTPCR in 27 cases of PHC tissues and cell strains of HepG2, SMMC7721 and LO2. Western blotting and immunohistochemical staining were adopted to detect KLF6 protein expression. Positive area ratio of wild-type KLF6 protein expression and its relationship with clinicopathological parameters of PHC was analyzed. Wild-type KLF6 expression in PHC tissues was lower than that in paracancerous tissues. In contrast, KLF6 SV2 mRNA expression was higher in PHC tissues and hepatoma cell strains (p<0.05). Positive area ratio of wild-type KLF6 protein expression was positively correlated with cellular differentiation degree of PHC (p<0.01), but negatively correlated not only with liver cirrhosis, tumor size and extrahepatic metastases (p<0.01), but also with portal vein thrombus and the number of lymph nodes with metastasis (p<0.05). Wild-type KLF6 deletion and inactivation was involved in the growth, cell differentiation and other physiological processes of PHC. The upregulation of KLF6 splice variant might counterbalance the wildtype KLF6 and contribute to the occurrence and development of PHC.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant-negative effect of the AF splice variant.

    Science.gov (United States)

    Hannemann, Anke; Christie, Jenny K; Flatman, Peter W

    2009-12-18

    The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive (86)Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.

  18. Characterization of a cancer cell line that expresses a splicing variant form of 53BP1: Separation of checkpoint and repair functions in 53BP1

    International Nuclear Information System (INIS)

    Iwabuchi, Kuniyoshi; Matsui, Tadashi; Hashimoto, Mitsumasa; Matsumoto, Yoshihisa; Kurihara, Takayuki; Date, Takayasu

    2008-01-01

    53BP1 plays important roles in checkpoint signaling and repair for DNA double-strand breaks. We found that a colon cancer cell line, SW48, expressed a splicing variant form of 53BP1, which lacks the residues corresponding to exons 10 and 11. Activation of ATM and phosphorylation of ATM and ATR targets occurred in SW48 cells in response to X-irradiation, and these X-ray-induced responses were not enhanced by expression of full-length 53BP1 in SW48 cells, indicating that this splicing variant fully activates the major checkpoint signaling in SW48 cells. In contrast, the expression of full-length 53BP1 in SW48 cells promoted the repair of X-ray-induced DNA damage, evidenced by faster disappearance of X-ray-induced γ-H2AX foci, a marker for DNA damage, and less residual chromosomal aberrations after X-irradiation. We conclude that the two major roles of 53BP1, the checkpoint signaling and repair for DNA damage, can be functionally separated

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

    International Nuclear Information System (INIS)

    Li, YanHua; Li, AiHua; Yang, Z.Q.

    2016-01-01

    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

  20. 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

  1. Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

    Science.gov (United States)

    Ji, Xia-Jie; Mao, Xue; Hao, Qing-Ting; Liu, Bao-Ling; Xue, Jin-Ai; Li, Run-Zhi

    2018-01-05

    The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean ( Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1 , designated as RcWRI1-A and RcWRI1-B . The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A . Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

  2. Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves

    Directory of Open Access Journals (Sweden)

    Xia-Jie Ji

    2018-01-01

    Full Text Available The plant-specific WRINKLED1 (WRI1 is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L., an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp and RcWRI1-B (1332 bp differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues “VYL” that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3–4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

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

    Directory of Open Access Journals (Sweden)

    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

  4. A Splice Variant of HER2 Corresponding to Herstatin Is Expressed in the Noncancerous Breast and in Breast Carcinomas

    Directory of Open Access Journals (Sweden)

    Triantafyllia Koletsa

    2008-07-01

    Full Text Available Herstatin (HST is an alternatively spliced HER2 product with growth-inhibitory properties in experimental cancer systems. The role of HST in adult human tissues and disease remains unexplored. Here, we investigated HST expression at the mRNA and protein (immunohistochemistry [IHC] level in parallel with parameters reflecting HER activation in 187 breast carcinomas and matched noncancerous breast tissues (NCBT. Noncancerous breast tissues demonstrated the highest HST/HER2 transcript ratios corresponding to a few positive epithelial and stromal cells by IHC. Although HST/HER2 transcript ratios in tumors were inversely associated with HER2 IHC grading (P = .0048 for HER2 IHC-1+ and P = .0006 for HER2 IHC-2+ vs HER2-negative tumors, relative HST expression within the same tumor/NCBT system remained constant. HST/HER2 ratios did not predict the presence of HST protein, which was found in 46 (25% of 187 tumors. A subgroup of HER2 IHC-3+ tumors exhibited high HST/HER2 transcript ratios, strong HST protein positivity, and cytoplasmic phospho-Akt/PKB and p21CIP1/WAF1 localization. In conclusion, HST may act as a paracrine factor in the adult breast. Because HST is described as an endogenous pan-HER inhibitor, the presence of this protein in breast carcinomas may portent the inefficiency of exogenous efforts to block HER2 dimerization, whereas its absence may justify such interventions.

  5. Rare Drosha Splice Variants Are Deficient in MicroRNA Processing but Do Not Affect General MicroRNA Expression in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Stefanie E. Grund

    2012-03-01

    Full Text Available Drosha is a key enzyme in microRNA biogenesis, generating the precursor miRNA (pre-miRNA by excising the stem-loop embedded in the primary transcripts (pri-miRNA. The specificity for the pri-miRNAs and determination of the cleavage site are provided by its binding partner DGCR8, which is necessary for efficient processing. The crucial Drosha domains for pri-miRNA cleavage are the middle part, the two enzymatic RNase III domains (RIIID, and the dsRNA binding domain (dsRBD in the C-terminus. Here, we identify alternatively spliced transcripts in human melanoma and NT2 cell lines, encoding C-terminally truncated Drosha proteins lacking part of the RIIIDb and the entire dsRBD. Proteins generated from these alternative splice variants fail to bind to DGCR8 but still interact with Ewing sarcoma protein (EWS. In vitro as well as in vivo, the Drosha splice variants are deficient in pri-miRNA processing. However, the aberrant transcripts in melanoma cells do not consistently reduce mature miRNA levels compared with melanoma cell lines lacking those splice variants, possibly owing to their limited abundance. Our findings show that alternative processing-deficient Drosha splice variants exist in melanoma cells. In elevated amounts, these alternatively spliced transcripts could provide one potential mechanism accounting for the deregulation of miRNAs in cancer cells. On the basis of our results, the search for alternative inactive splice variants might be fruitful in different tumor entities to unravel the molecular basis of the previously observed decreased microRNA processing efficiency in cancer.

  6. Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis.

    Science.gov (United States)

    Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

    2012-01-01

    Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

  7. Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

    Science.gov (United States)

    Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

    2016-10-01

    The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; pprostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  8. Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns.

    Science.gov (United States)

    De Laere, Bram; van Dam, Pieter-Jan; Whitington, Tom; Mayrhofer, Markus; Diaz, Emanuela Henao; Van den Eynden, Gert; Vandebroek, Jean; Del-Favero, Jurgen; Van Laere, Steven; Dirix, Luc; Grönberg, Henrik; Lindberg, Johan

    2017-08-01

    Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of

  9. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2017-10-01

    resistant prostate cancer ; docetaxel; cabazitaxel; chemotherapy; androgen receptor splice variants; microtubule; ligand-binding domain; microtubule... receptor splice variants (AR-Vs) are associated with resistance to taxane chemotherapy in castration- resistant prostate cancer (CRPC). However, this...androgen receptor inhibitors in prostate cancer . Nat Rev Cancer . 2015;15:701–11.

  10. Undernutrition regulates the expression of a novel splice variant of myostatin and insulin-like growth factor 1 in ovine skeletal muscle.

    Science.gov (United States)

    Jeanplong, F; Osepchook, C C; Falconer, S J; Smith, H K; Bass, J J; McMahon, C D; Oldham, J M

    2015-07-01

    Undernutrition suppresses the growth of skeletal muscles and alters the expression of insulin-like growth factor 1 (IGF1), a key mitogen, and myostatin, a potent inhibitor of myogenesis. These changes can explain, at least in part, the reduced growth of skeletal muscles in underfed lambs. We have recently identified a myostatin splice variant (MSV) that binds to and antagonizes the canonical signaling of myostatin. In the present study, we hypothesized that the expression of MSV would be reduced in conjunction with myostatin and IGF1 in response to underfeeding in skeletal muscles of sheep. Young growing ewes were fed either ad libitum or an energy-restricted diet (30% of maintenance requirements) for 28 d. This regime of underfeeding resulted in a 24% reduction in body mass (P myostatin mRNA was not altered in semitendinosus muscles. Unlike the reduced expression of mRNA, the abundance of MSV protein was increased (P myostatin protein. Our results suggest that undernutrition for 28 d decreases the signaling of myostatin by increasing the abundance of MSV protein. Although this action may reduce the growth inhibitory activity of myostatin, it cannot prevent the loss of growth of skeletal muscles during undernutrition. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    Rocafull, Miguel A.; Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H + ), have been reported. They include reticulum and plasma-membrane Ca 2+ -ATPases, Na + /K + -ATPase and H + /K + -ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K + -ATPase α1-isoform, H + /K + -ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K + -ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  12. Characterization of a novel splicing variant in the RAPTOR gene

    International Nuclear Information System (INIS)

    Sun Chang; Southard, Catherine; Di Rienzo, Anna

    2009-01-01

    The mammalian target of rapamycin (mTOR) plays an essential role in the regulation of cell growth, proliferation and apoptosis. Raptor, the regulatory associated protein of mTOR, is an important member in this signaling pathway. In the present report, we identified and characterized a novel splicing variant of this gene, RAPTOR v 2, in which exons 14-17, 474 bp in total, are omitted from the mRNA. This deletion does not change the open reading frame, but causes a nearly complete absence of HEAT repeats, which were shown to be involved in the binding of mTOR substrates. Real time PCR performed on 48 different human tissues demonstrated the ubiquitous presence of this splice variant. Quantification of mRNA levels in lymphoblastoid cell lines (LCL) from 56 unrelated HapMap individuals revealed that the expression of this splicing form is quite variable. One synonymous SNP, rs2289759 in exon 14, was predicted by ESEfinder to cause a significant gain/loss of SRp55 and/or SF2/ASF binding sites, and thus potentially influence splicing. This prediction was confirmed by linear regression analysis between the ratio of RAPTOR v 2 to total RAPTOR mRNA levels and the SNP genotype in the above 56 individuals (r = 0.281 and P = 0.036). Moreover, the functional evaluation indicated that this splicing isoform is expected to retain the ability to bind mTOR, but is unlikely to bind mTOR substrates, hence affecting signal transduction and further cell proliferation

  13. GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Holst, Birgitte; Petersen, Pia S

    2007-01-01

    nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being......G protein-coupled receptor 39 (GPR39) is a constitutively active, orphan member of the ghrelin receptor family that is activated by zinc ions. GPR39 is here described to be expressed in a full-length, biologically active seven-transmembrane form, GPR39-1a, as well as in a truncated splice variant...... five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas...

  14. Osteopontin splice variants are differential predictors of breast cancer treatment responses

    OpenAIRE

    Zduniak, Krzysztof; Agrawal, Anil; Agrawal, Siddarth; Hossain, Md Monir; Ziolkowski, Piotr; Weber, Georg F.

    2016-01-01

    Background Osteopontin is a marker for breast cancer progression, which in previous studies has also been associated with resistance to certain anti-cancer therapies. It is not known which splice variants may mediate treatment resistance. Methods Here we analyze the association of osteopontin variant expression before treatment, differentiated according to immunohistochemistry with antibodies to exon 4 and to the osteopontin-c splice junction respectively, with the ensuing therapy responses i...

  15. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

    Science.gov (United States)

    Gorgoglione, Bartolomeo; Carpio, Yamila; Secombes, Christopher J; Taylor, Nick G H; Lugo, Juana María; Estrada, Mario Pablo

    2015-12-01

    Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens. Copyright © 2015

  18. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    Energy Technology Data Exchange (ETDEWEB)

    Rocafull, Miguel A., E-mail: mrocaful@ivic.ve [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca{sup 2+}, Na{sup +}, K{sup +} and H{sup +}), have been reported. They include reticulum and plasma-membrane Ca{sup 2+}-ATPases, Na{sup +}/K{sup +}-ATPase and H{sup +}/K{sup +}-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg{sup 2+}ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na{sup +}/K{sup +}-ATPase {alpha}1-isoform, H{sup +}/K{sup +}-ATPase {alpha}2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H{sup +}/K{sup +}-ATPase {alpha}2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  19. Human CRF{sub 2} {alpha} and {beta} splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

    Energy Technology Data Exchange (ETDEWEB)

    Ardati, A. [Rhone-Poulenc Rorer, Cardiovascular Biology, NW4, 500 Arcola Road, Collegeville, PA (United States); Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J. [Central Nervous System, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland); Valdenaire, O. [Cardiovascular Research, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland)

    1999-03-14

    pharmacological profiles of the CRF{sub 2} splice variants are identical. This indicates that the region of the N-terminus that varies between the receptors is probably not important in the binding of peptide CRF receptor ligands or functional activation of the receptor. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Early diagnostic value of survivin and its alternative splice variants in breast cancer

    International Nuclear Information System (INIS)

    Khan, Salma; Bennit, Heather Ferguson; Turay, David; Perez, Mia; Mirshahidi, Saied; Yuan, Yuan; Wall, Nathan R

    2014-01-01

    The inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues. Our previous work showed Survivin is released from tumor cells via small membrane-bound vesicles called exosomes. We, therefore, hypothesize that analysis of serum exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer. We collected sera from forty breast cancer patients and ten control patients who were disease free for 5 years after treatment. In addition, twenty-three paired breast cancer tumor tissues from those same 40 patients were analyzed for splice variants. Serum levels of Survivin were analyzed using ELISA and exosomes were isolated from this serum using the commercially available ExoQuick kit, with subsequent Western blots and immunohistochemistry performed. Survivin levels were significantly higher in all the breast cancer samples compared to controls (p < 0.05) with exosome amounts significantly higher in cancer patient sera compared to controls (p < 0.01). While Survivin and Survivin-∆Ex3 splice variant expression and localization was identical in serum exosomes, differential expression of Survivin-2B protein existed in the exosomes. Similarly, Survivin and Survivin-∆Ex3 proteins were the predominant forms detected in all of the breast cancer tissues evaluated in this study, whereas a more variable expression of Survivin-2B level was found at different cancer stages. In this study we show for the first time that like Survivin, the Survivin splice variants are also exosomally packaged in the breast cancer patients’ sera, mimicking the survivin splice variant pattern that we also report in breast cancer tissues. Differential expression of exosomal-Survivin, particularly Survivin-2B, may serve as a diagnostic and/or prognostic marker, a “liquid biopsy” if you will, in early breast cancer patients. Furthermore, a more thorough understanding of the role of this

  2. Two new splice variants in porcine PPARGC1A

    Directory of Open Access Journals (Sweden)

    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.

  3. 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

    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......Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently...... 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...

  4. Osteopontin splice variants are differential predictors of breast cancer treatment responses.

    Science.gov (United States)

    Zduniak, Krzysztof; Agrawal, Anil; Agrawal, Siddarth; Hossain, Md Monir; Ziolkowski, Piotr; Weber, Georg F

    2016-07-11

    Osteopontin is a marker for breast cancer progression, which in previous studies has also been associated with resistance to certain anti-cancer therapies. It is not known which splice variants may mediate treatment resistance. Here we analyze the association of osteopontin variant expression before treatment, differentiated according to immunohistochemistry with antibodies to exon 4 and to the osteopontin-c splice junction respectively, with the ensuing therapy responses in 119 Polish breast cancer patients who presented between 1995 and 2008. We found from Cox hazard models, logrank test and Wilcoxon test that osteopontin exon 4 was associated with a favorable response to tamoxifen, but a poor response to chemotherapy with CMF (cyclophosphamide, methotrexate, fluorouracil). Osteopontin-c is prognostic, but falls short of being a significant predictor for sensitivity to treatment. The addition of osteopontin splice variant immunohistochemistry to standard pathology work-ups has the potential to aid decision making in breast cancer treatment.

  5. Osteopontin splice variants are differential predictors of breast cancer treatment responses

    International Nuclear Information System (INIS)

    Zduniak, Krzysztof; Agrawal, Anil; Agrawal, Siddarth; Hossain, Md Monir; Ziolkowski, Piotr; Weber, Georg F.

    2016-01-01

    Osteopontin is a marker for breast cancer progression, which in previous studies has also been associated with resistance to certain anti-cancer therapies. It is not known which splice variants may mediate treatment resistance. Here we analyze the association of osteopontin variant expression before treatment, differentiated according to immunohistochemistry with antibodies to exon 4 and to the osteopontin-c splice junction respectively, with the ensuing therapy responses in 119 Polish breast cancer patients who presented between 1995 and 2008. We found from Cox hazard models, logrank test and Wilcoxon test that osteopontin exon 4 was associated with a favorable response to tamoxifen, but a poor response to chemotherapy with CMF (cyclophosphamide, methotrexate, fluorouracil). Osteopontin-c is prognostic, but falls short of being a significant predictor for sensitivity to treatment. The addition of osteopontin splice variant immunohistochemistry to standard pathology work-ups has the potential to aid decision making in breast cancer treatment

  6. Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

    Directory of Open Access Journals (Sweden)

    Ulrika Lind

    Full Text Available The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU up to fully marine conditions (35 PSU and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.

  7. Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

    Science.gov (United States)

    Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

    2015-10-01

    Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. 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

  9. Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

    Science.gov (United States)

    Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

    2016-06-01

    The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

  10. 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...... and RCK2 (4 aa at SS1) and upstream of the calcium "bowl" (27 aa at SS4). Two other truncated variants, X2(92) and X2(188), lacking the intracellular C-terminal (stop downstream of S6), were cloned from cerebral vascular/meningeal tissue. They appear non-functional as no current expression was observed...

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

    Directory of Open Access Journals (Sweden)

    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.

  12. A novel splice variant of supervillin, SV5, promotes carcinoma cell proliferation and cell migration

    International Nuclear Information System (INIS)

    Chen, Xueran; Yang, Haoran; Zhang, Shangrong; Wang, Zhen; Ye, Fang; Liang, Chaozhao; Wang, Hongzhi; Fang, Zhiyou

    2017-01-01

    Supervillin is an actin-associated protein that regulates actin dynamics by interacting with Myosin II, F-actin, and Cortactin to promote cell contractility and cell motility. Two splicing variants of human Supervillin (SV1 and SV4) have been reported in non-muscle cells; SV1 lacks 3 exons present in the larger isoform SV4. SV2, also called archvillin, is present in striated muscle; SV3, also called smooth muscle archvillin or SmAV, was cloned from smooth muscle. In the present study, we identify a novel splicing variant of Supervillin (SV5). SV5 contains a new splicing pattern. In the mouse tissues and cell lines examined, SV5 was predominantly expressed in skeletal and cardiac muscles and in proliferating cells, but was virtually undetectable in most normal tissues. Using RNAi and rescue experiments, we show here that SV5 displays altered functional properties in cancer cells, and regulates cell proliferation and cell migration.

  13. Differential Expression Profile of ZFX Variants Discriminates Breast Cancer Subtypes

    Science.gov (United States)

    Pourkeramati, Fatemeh; Asadi, Malek Hossein; Shakeri, Shahryar; Farsinejad, Alireza

    2018-05-13

    ZFX is a transcriptional regulator in embryonic stem cells that plays an important role in pluripotency and self-renewal. ZFX is widely expressed in pluripotent stem cells and is down-regulated during differentiation of embryonic stem cells. ZFX has five different variants that encode three different protein isoforms. While several reports have determined the overexpression of ZFX in a variety of somatic cancers, the expression of ZFX-spliced variants in cancer cells is not well-understood. We investigated the expression of ZFX variants in a series of breast cancer tissues and cell lines using quantitative PCR. The expression of ZFX variant 1/3 was higher in tumor tissue compared to marginal tissue. In contrast, the ZFX variant 5 was down-regulated in tumor tissues. While the ZFX variant 1/3 and ZFX variant 5 expression significantly increased in low-grade tumors, ZFX variant 4 was strongly expressed in high-grade tumors and demonstrating lymphatic invasion. In addition, our result revealed a significant association between the HER2 status and the expression of ZFX-spliced variants. Our data suggest that the expression of ZFX-spliced transcripts varies between different types of breast cancer and may contribute to their tumorigenesis process. Hence, ZFX-spliced transcripts could be considered as novel tumor markers with a probable value in diagnosis, prognosis, and therapy of breast cancer.

  14. 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/ .

  15. Detection of alternative splice variants at the proteome level in Aspergillus flavus.

    Science.gov (United States)

    Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

    2010-03-05

    Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

  16. 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.

  17. Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

    Science.gov (United States)

    Huang, J M; Wang, Z Y; Ju, Z H; Wang, C F; Li, Q L; Sun, T; Hou, Q L; Hang, S Q; Hou, M H; Zhong, J F

    2011-12-21

    Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

  18. Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

    Science.gov (United States)

    Jeanplong, Ferenc; Falconer, Shelley J.; Oldham, Jenny M.; Thomas, Mark; Gray, Tarra S.; Hennebry, Alex; Matthews, Kenneth G.; Kemp, Frederick C.; Patel, Ketan; Berry, Carole; Nicholas, Gina; McMahon, Christopher D.

    2013-01-01

    Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (Pmyostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product. PMID:24312578

  19. Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Zhao, Shan; Lu, Xin; Zhang, Yueling; Zhao, Xianliang; Zhong, Mingqi; Li, Shengkang; Lun, Jingsheng

    2013-01-01

    Recent evidences suggest that invertebrates express families of immune molecules with high levels of sequence diversity. Hemocyanin is an important non-specific immune molecule present in the hemolymph of both mollusks and arthropods. In the present study, we characterized a novel alternative splicing variant of hemocyanin (cHE1) from Litopenaeus vannamei that produced mRNA transcript of 2579 bp in length. The isoform contained two additional sequences of 296 and 267 bp in the 5'- and 3'-terminus respectively, in comparison to that of wild type hemocyanin (cHE). Sequence of cHE1 shows 100% identity to that of hemocyanin genomic DNA (HE, which does not form an open reading frame), suggesting that cHE1 might be an alternative splicing variant due to intron retention. Moreover, cHE1 could be detected by RT-PCR from five tissues (heart, gill, stomach, intestine and brain), and from shrimps at stages from nauplius to mysis larva. Further, cHE1 mRNA transcripts were significantly increased in hearts after 12h of infection with Vibrio parahemolyticus or poly I: C, while no significant difference in the transcript levels of hepatopancreas cHE was detected in the pathogen-treated shrimps during the period. In summary, these studies suggested a novel splicing variant of hemocyanin in shrimp, which might be involved in shrimp resistance to pathogenic infection. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

    2016-01-01

    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.

  1. 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.

  2. 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

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

    International Nuclear Information System (INIS)

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

    2014-01-01

    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

  4. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

    Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

  5. α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

    Science.gov (United States)

    Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

    2018-05-02

    α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

  6. 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...... and flPD-1 upon activation suggests an important interplay between the putative soluble PD-1 and flPD-1 possibly involved in maintenance of peripheral self-tolerance and prevention of autoimmunity....

  7. Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model.

    Science.gov (United States)

    Nakata, Daisuke; Nakayama, Kazuhide; Masaki, Tsuneo; Tanaka, Akira; Kusaka, Masami; Watanabe, Tatsuya

    2016-12-01

    Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the

  8. Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

    Science.gov (United States)

    Tran, Trung T; Bollineni, Ravi C; Strozynski, Margarita; Koehler, Christian J; Thiede, Bernd

    2017-07-07

    Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

  9. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

    Directory of Open Access Journals (Sweden)

    Ferenc Jeanplong

    Full Text Available Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05, which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a

  10. 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

  11. 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.

  12. 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.

  13. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  14. An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

    Science.gov (United States)

    Saraco, Nora; Nesi-Franca, Suzana; Sainz, Romina; Marino, Roxana; Marques-Pereira, Rosana; La Pastina, Julia; Perez Garrido, Natalia; Sandrini, Romolo; Rivarola, Marco Aurelio; de Lacerda, Luiz; Belgorosky, Alicia

    2015-01-01

    Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient. © 2015 S. Karger AG, Basel.

  15. 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.

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

    International Nuclear Information System (INIS)

    Zhang, Xinshang; Zhao, Heng; Chen, Yeyu; Luo, Huiying; Yao, Bin

    2014-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    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.

  19. 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

  20. 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...... 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...

  1. Co-expression networks reveal the tissue-specific regulation of transcription and splicing.

    Science.gov (United States)

    Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D H; Jo, Brian; Gao, Chuan; McDowell, Ian C; Engelhardt, Barbara E; Battle, Alexis

    2017-11-01

    Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. © 2017 Saha et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury.

    Science.gov (United States)

    Lana, Beatrice; Schlick, Bettina; Martin, Stuart; Pratt, Wendy S; Page, Karen M; Goncalves, Leonor; Rahman, Wahida; Dickenson, Anthony H; Bauer, Claudia S; Dolphin, Annette C

    2014-03-01

    The α2δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α2δ-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3' splice acceptor site. Here we have examined the presence of α2δ-1 splice variants in DRG neurons, and have found that although the main α2δ-1 splice variant in DRG is the same as that in brain (α2δ-1 ΔA+B+C), there is also another α2δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant α2δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The α2δ-1 ΔA+BΔC splice variant supports CaV2 calcium currents with unaltered properties compared to α2δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  3. 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.

  4. 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.

  5. Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts

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    Brewster Brooke L

    2010-05-01

    Full Text Available Abstract Background Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs remains a challenge. Methods This study used a combination of RT-PCR analysis and splicing reporter minigene assays to assess five unclassified variants in the BRCA2 gene that we had previously predicted to disrupt an ESE using bioinformatic approaches. Results Analysis of BRCA2 c.8308 G > A (p.Ala2770Thr by mRNA analysis, and BRCA2 c.8962A > G (p.Ser2988Gly, BRCA2 c.8972G > A (p.Arg2991His, BRCA2 c.9172A > G (p.Ser3058Gly, and BRCA2 c.9213G > T (p.Glu3071Asp by a minigene assay, revealed no evidence for aberrant splicing. Conclusions These results illustrate the need for improved methods for predicting functional ESEs and the potential consequences of sequence variants contained therein.

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

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    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.

  7. 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

    BRCA1 and 176 BRCA2 unique variants, from 77 publications. At least six independent reviewers from research and/or clinical settings comprehensively examined splicing assay methods and data reported for 22 variant assays of 21 variants in four publications, and classified the variants using the 5-tier......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...... of results, and the lack of quantitative data for the aberrant transcripts. We propose suggestions for minimum reporting guidelines for splicing assays, and improvements to the 5-tier splicing classification system to allow future evaluation of its performance as a clinical tool....

  8. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

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    Sugnet Charles

    2006-12-01

    Full Text Available Abstract Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic

  9. Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

    Science.gov (United States)

    Tee, Meng Kian; Speek, Mart; Legeza, Balázs; Modi, Bhavi; Teves, Maria Eugenia; McAllister, Janette M; Strauss, Jerome F; Miller, Walter L

    2016-10-15

    Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

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    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.

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

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    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.

  12. Alternative Splice Variants Modulates Dominant-Negative Function of Helios in T-Cell Leukemia.

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    Shaorong Zhao

    Full Text Available The molecular defects which lead to multistep incidences of human T-cell leukemia have yet to be identified. The DNA-binding protein Helios (known as IKZF2, a member of the Ikaros family of Krüppel-like zinc-finger proteins, functions pivotally in T-cell differentiation and activation. In this study, we identify three novel short Helios splice variants which are T-cell leukemic specific, and demonstrate their dominant-negative function. We then test the cellular localization of distinct Helios isoforms, as well as their capability to form heterodimer with Ikaros, and the association with complexes comprising histone deacetylase (HDAC. In addition, the ectopic expression of T-cell leukemic Helios isoforms interferes with T-cell proliferation and apoptosis. The gene expression profiling and pathway analysis indicated the enrichment of signaling pathways essential for gene expression, translation, cell cycle checkpoint, and response to DNA damage stimulus. These data indicate the molecular function of Helios to be involved in the leukemogenesis and phenotype of T-cell leukemia, and also reveal Helios deregulation as a novel marker for T-cell leukemia.

  13. Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events.

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    Baudhuin, Linnea M; Kotzer, Katrina E; Lagerstedt, Susan A

    2015-03-01

    Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome. Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed. A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria-positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177-187.

  14. Identification of alternative splice variants in Aspergillus flavus through comparison of multiple tandem MS search algorithms

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    Chang Kung-Yen

    2011-07-01

    Full Text Available Abstract Background Database searching is the most frequently used approach for automated peptide assignment and protein inference of tandem mass spectra. The results, however, depend on the sequences in target databases and on search algorithms. Recently by using an alternative splicing database, we identified more proteins than with the annotated proteins in Aspergillus flavus. In this study, we aimed at finding a greater number of eligible splice variants based on newly available transcript sequences and the latest genome annotation. The improved database was then used to compare four search algorithms: Mascot, OMSSA, X! Tandem, and InsPecT. Results The updated alternative splicing database predicted 15833 putative protein variants, 61% more than the previous results. There was transcript evidence for 50% of the updated genes compared to the previous 35% coverage. Database searches were conducted using the same set of spectral data, search parameters, and protein database but with different algorithms. The false discovery rates of the peptide-spectrum matches were estimated Conclusions We were able to detect dozens of new peptides using the improved alternative splicing database with the recently updated annotation of the A. flavus genome. Unlike the identifications of the peptides and the RefSeq proteins, large variations existed between the putative splice variants identified by different algorithms. 12 candidates of putative isoforms were reported based on the consensus peptide-spectrum matches. This suggests that applications of multiple search engines effectively reduced the possible false positive results and validated the protein identifications from tandem mass spectra using an alternative splicing database.

  15. Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells.

    Science.gov (United States)

    Fan, Lingling; Zhang, Fengbo; Xu, Songhui; Cui, Xiaolu; Hussain, Arif; Fazli, Ladan; Gleave, Martin; Dong, Xuesen; Qi, Jianfei

    2018-05-15

    Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

  16. TAPAS: tools to assist the targeted protein quantification of human alternative splice variants.

    Science.gov (United States)

    Yang, Jae-Seong; Sabidó, Eduard; Serrano, Luis; Kiel, Christina

    2014-10-15

    In proteomes of higher eukaryotes, many alternative splice variants can only be detected by their shared peptides. This makes it highly challenging to use peptide-centric mass spectrometry to distinguish and to quantify protein isoforms resulting from alternative splicing events. We have developed two complementary algorithms based on linear mathematical models to efficiently compute a minimal set of shared and unique peptides needed to quantify a set of isoforms and splice variants. Further, we developed a statistical method to estimate the splice variant abundances based on stable isotope labeled peptide quantities. The algorithms and databases are integrated in a web-based tool, and we have experimentally tested the limits of our quantification method using spiked proteins and cell extracts. The TAPAS server is available at URL http://davinci.crg.es/tapas/. luis.serrano@crg.eu or christina.kiel@crg.eu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. A novel splice variant of the Fas gene in patients with cutaneous T-cell lymphoma.

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    van Doorn, Remco; Dijkman, Remco; Vermeer, Maarten H; Starink, Theo M; Willemze, Rein; Tensen, Cornelis P

    2002-10-01

    Defective apoptosis signaling has been implicated in the pathogenesis of primary cutaneous T-cell lymphomas (CTCLs), a group of malignancies derived from skin-homing T cells. An important mediator of apoptosis in T cells is the Fas receptor. We identified a novel splice variant of the Fas gene that displays retention of intron 5 and encodes a dysfunctional Fas protein in 13 of 22 patients (59%) in both early and advanced CTCL. Impairment of Fas-induced apoptosis resulting from aberrant splicing potentially contributes to the development and progression of CTCL by allowing continued clonal expansion of activated T cells and by reducing susceptibility to antitumor immune responses.

  18. Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

    Science.gov (United States)

    He, Xian-hui; Xu, Li-hui; Liu, Yi

    2005-04-01

    To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  19. Differential HFE gene expression is regulated by alternative splicing in human tissues.

    Science.gov (United States)

    Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula

    2011-03-03

    The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

  20. Differential HFE gene expression is regulated by alternative splicing in human tissues.

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    Rute Martins

    Full Text Available BACKGROUND: The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. METHODOLOGY/PRINCIPAL FINDINGS: Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE that is mostly secreted by cells to the medium in association with β2M. CONCLUSIONS/SIGNIFICANCE: HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

  1. 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

    , 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...... dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories...

  2. Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.

    Science.gov (United States)

    Zhou, Jie J; Wang, Feng; Xu, Zhiwen; Lo, Wing-Sze; Lau, Ching-Fun; Chiang, Kyle P; Nangle, Leslie A; Ashlock, Melissa A; Mendlein, John D; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

    2014-07-11

    Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

  6. 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.

  7. Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

    Science.gov (United States)

    Roundhill, E; Burchill, S

    2013-07-09

    Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (PMRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

  8. LOX-1 and Its Splice Variants: A New Challenge for Atherosclerosis and Cancer-Targeted Therapies

    Science.gov (United States)

    Rizzacasa, Barbara; Morini, Elena; Pucci, Sabina; Murdocca, Michela; Novelli, Giuseppe; Amati, Francesca

    2017-01-01

    Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both “risk” and “protective” splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target. PMID:28146073

  9. SUN1 splice variants, SUN1_888, SUN1_785, and predominant SUN1_916, variably function in directional cell migration

    OpenAIRE

    Nishioka, Yu; Imaizumi, Hiromasa; Imada, Junko; Katahira, Jun; Matsuura, Nariaki; Hieda, Miki

    2016-01-01

    The LINC complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, such as nuclear migration, mechanotransduction and chromatin tethering in the meiotic phase. However, it remains unknown how these functions are regulated in different cell contexts. An inner nuclear membrane component of the LINC complex, SUN1, is ubiquitously expressed. The human SUN1 gene produces over 10 variants by alternative splicing. Although functions of SUN1 are relat...

  10. Mechano growth factor, a splice variant of IGF-1, promotes neurogenesis in the aging mouse brain.

    Science.gov (United States)

    Tang, Jason J; Podratz, Jewel L; Lange, Miranda; Scrable, Heidi J; Jang, Mi-Hyeon; Windebank, Anthony J

    2017-07-07

    Mechano growth factor (MGF) is a splice variant of IGF-1 first described in skeletal muscle. MGF induces muscle cell proliferation in response to muscle stress and injury. In control mice we found endogenous expression of MGF in neurogenic areas of the brain and these levels declined with age. To better understand the role of MGF in the brain, we used transgenic mice that constitutively overexpressed MGF from birth. MGF overexpression significantly increased the number of BrdU+ proliferative cells in the dentate gyrus (DG) of the hippocampus and subventricular zone (SVG). Although MGF overexpression increased the overall rate of adult hippocampal neurogenesis at the proliferation stage it did not alter the distribution of neurons at post-mitotic maturation stages. We then used the lac-operon system to conditionally overexpress MGF in the mouse brain beginning at 1, 3 and 12 months with histological and behavioral observation at 24 months of age. With conditional overexpression there was an increase of BrdU+ proliferating cells and BrdU+ differentiated mature neurons in the olfactory bulbs at 24 months when overexpression was induced from 1 and 3 months of age but not when started at 12 months. This was associated with preserved olfactory function. In vitro, MGF increased the size and number of neurospheres harvested from SVZ-derived neural stem cells (NSCs). These findings indicate that MGF overexpression increases the number of neural progenitor cells and promotes neurogenesis but does not alter the distribution of adult newborn neurons at post-mitotic stages. Maintaining youthful levels of MGF may be important in reversing age-related neuronal loss and brain dysfunction.

  11. Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels*

    Science.gov (United States)

    Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E.; Sinnegger-Brauns, Martina J.; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

    2011-01-01

    An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes. PMID:21998310

  12. Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

    Science.gov (United States)

    Tuluc, Petronel; Flucher, Bernhard E

    2011-12-01

    Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

  13. Distinctive expression pattern of OCT4 variants in different types of breast cancer.

    Science.gov (United States)

    Soheili, Saamaaneh; Asadi, Malek Hossein; Farsinejad, Alireza

    2017-01-01

    OCT4 is a key regulator of self-renewal and pluripotency in embryonic stem cells which can potentially encode three spliced variants designated OCT4A, OCT4B and OCT4B1. Based on cancer stem cell concept, it is suggested that the stemness factors misexpressed in cancer cells and potentially is involved in tumorigenesis. Accordingly, in this study, we investigated the potential expression of OCT4 variants in breast cancer tissues. A total of 94 tumoral and peritumoral breast specimens were evaluated with respect to the expression of OCT4 variants using quantitative RT-PCR and immunohistochemical (IHC) analysis. We detected the expression of OCT4 variants in breast tumor tissues with no or very low levels of expression in peritumoral samples of the same patients. While OCT4B was highly expressed in lobular type of breast cancer, OCT4A and OCTB1 variants are highly expressed in low grade (I and II) ductal tumors. Furthermore, the results of this study revealed a considerable association between the expression level of OCT4 variants and the expression of ER, PR, Her2 and P53 factors. All data demonstrated a distinctive expression pattern of OCT4 spliced variants in different types of breast cancer and provide further evidence for the involvement of embryonic genes in carcinogenesis.

  14. A Loss-of-Function Splice Acceptor Variant in IGF2 Is Protective for Type 2 Diabetes.

    Science.gov (United States)

    Mercader, Josep M; Liao, Rachel G; Bell, Avery D; Dymek, Zachary; Estrada, Karol; Tukiainen, Taru; Huerta-Chagoya, Alicia; Moreno-Macías, Hortensia; Jablonski, Kathleen A; Hanson, Robert L; Walford, Geoffrey A; Moran, Ignasi; Chen, Ling; Agarwala, Vineeta; Ordoñez-Sánchez, María Luisa; Rodríguez-Guillen, Rosario; Rodríguez-Torres, Maribel; Segura-Kato, Yayoi; García-Ortiz, Humberto; Centeno-Cruz, Federico; Barajas-Olmos, Francisco; Caulkins, Lizz; Puppala, Sobha; Fontanillas, Pierre; Williams, Amy L; Bonàs-Guarch, Sílvia; Hartl, Chris; Ripke, Stephan; Tooley, Katherine; Lane, Jacqueline; Zerrweck, Carlos; Martínez-Hernández, Angélica; Córdova, Emilio J; Mendoza-Caamal, Elvia; Contreras-Cubas, Cecilia; González-Villalpando, María E; Cruz-Bautista, Ivette; Muñoz-Hernández, Liliana; Gómez-Velasco, Donaji; Alvirde, Ulises; Henderson, Brian E; Wilkens, Lynne R; Le Marchand, Loic; Arellano-Campos, Olimpia; Riba, Laura; Harden, Maegan; Gabriel, Stacey; Abboud, Hanna E; Cortes, Maria L; Revilla-Monsalve, Cristina; Islas-Andrade, Sergio; Soberon, Xavier; Curran, Joanne E; Jenkinson, Christopher P; DeFronzo, Ralph A; Lehman, Donna M; Hanis, Craig L; Bell, Graeme I; Boehnke, Michael; Blangero, John; Duggirala, Ravindranath; Saxena, Richa; MacArthur, Daniel; Ferrer, Jorge; McCarroll, Steven A; Torrents, David; Knowler, William C; Baier, Leslie J; Burtt, Noel; González-Villalpando, Clicerio; Haiman, Christopher A; Aguilar-Salinas, Carlos A; Tusié-Luna, Teresa; Flannick, Jason; Jacobs, Suzanne B R; Orozco, Lorena; Altshuler, David; Florez, Jose C

    2017-11-01

    Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage-dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction. © 2017 by the American Diabetes Association.

  15. A splice variant of RILP induces lysosomal clustering independent of dynein recruitment

    International Nuclear Information System (INIS)

    Marsman, Marije; Jordens, Ingrid; Rocha, Nuno; Kuijl, Coenraad; Janssen, Lennert; Neefjes, Jacques

    2006-01-01

    The small GTPase Rab7 controls fusion and transport of late endocytic compartments. A critical mediator is the Rab7 effector RILP that recruits the minus-end dynein-dynactin motor complex to these compartments. We identified a natural occurring splice variant of RILP (RILPsv) lacking only 27 amino acids encoded by exon VII. Both variants bind Rab7, prolong its GTP-bound state, and induce clustering of late endocytic compartments. However, RILPsv does not recruit the dynein-dynactin complex, implicating exon VII in motor recruitment. Clustering might still occur via dimerization, since both RILP and RILPsv are able to form hetero- and homo-dimers. Moreover, both effectors compete for Rab7 binding but with different outcome for dynein-dynactin recruitment and transport. Hence, RILPsv provides an extra dimension to the control of vesicle fusion and transport by the small GTPase Rab7

  16. Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain

    International Nuclear Information System (INIS)

    Yoshimoto, Nobuo; Tatematsu, Kenji; Koyanagi, Tomoyoshi; Okajima, Toshihide; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2005-01-01

    RBCC protein interacting with PKC 1 (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex

  17. Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

    Science.gov (United States)

    van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

    2015-01-01

    A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

  18. 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...

  19. Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7-Induced Nuclear Factor-Kappa B (NF-κB Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC Therapy

    Directory of Open Access Journals (Sweden)

    Vincent Wing Sun Liu

    2017-05-01

    Full Text Available A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7, nuclear factor-kappa B (NF-κB was activated and could result in up-regulated interleukin (IL-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.

  20. Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy.

    Science.gov (United States)

    Liu, Vincent Wing Sun; Yau, Wing Lung; Tam, Chun Wai; Yao, Kwok-Ming; Shiu, Stephen Yuen Wing

    2017-05-31

    A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin ( IL ) -6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT₁ receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.

  1. 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.

  2. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Yan [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Nguyen, Hong T. [Graduate Program in Biomedical Sciences, Tulane School of Medicine, New Orleans, LA 70112 (United States); Lasky, Joseph A. [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Cao, Subing [Graduate Program in Biomedical Sciences, Tulane School of Medicine, New Orleans, LA 70112 (United States); Li, Cui [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Xiangya Hospital, Central South University, Hunan 41008 (China); Hu, Jiyao; Guo, Xinyue; Burow, Matthew E. [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States); Shan, Bin, E-mail: bshan@tulane.edu [Department of Medicine, Tulane School of Medicine, New Orleans, LA 70112 (United States)

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  3. 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.

  4. 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.

  5. Sequence variants of KHDRBS1 as high penetrance susceptibility risks for primary ovarian insufficiency by mis-regulating mRNA alternative splicing.

    Science.gov (United States)

    Wang, Binbin; Li, Lin; Zhu, Ying; Zhang, Wei; Wang, Xi; Chen, Beili; Li, Tengyan; Pan, Hong; Wang, Jing; Kee, Kehkooi; Cao, Yunxia

    2017-10-01

    Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. We identified a heterozygous mutation (c.460A > G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C > T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ

  6. Cloning and characterization of human RTVP-1b, a novel splice variant of RTVP-1 in glioma cells

    International Nuclear Information System (INIS)

    Xiang Cunli; Sarid, Ronit; Cazacu, Simona; Finniss, Susan; Lee, Hae-Kyung; Ziv-Av, Amotz; Mikkelsen, Tom; Brodie, Chaya

    2007-01-01

    Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas

  7. Differential regulation of iPLA2beta splice variants by in vitro ischemia in C2C12 myotubes

    DEFF Research Database (Denmark)

    Poulsen, K. A.; Kolko, M.; Lambert, I. H.

    2006-01-01

    In this study we investigated the activity, expression and regulation of iPLA2 during ischemia in mouse C2C12 myotubes. Here, we show that in vitro ischemia, i.e. oxygen deprivation and glucose starvation, induces an iPLA2 activity that is totally reversed by siRNA knock down of iPLA2£], indicating...... preferential activation of iPLA2£]. The activity of the native iPLA2£] tetramer has in humans been proposed to be negatively regulated by interactions with catalytic inactive splice variants of the full-length protein. These variants, characterized by the presence exon 9a, have however not been identified...... of this transcript would be a C-terminally truncated î50 kDa protein lacking the catalytic site. qPCR indicated that, while the total iPLA2£] mRNA level in C2C12 myotubes increased weakly within 1-2 hours of in vitro ischemia, the transcript containing the mouse exon 9a was rapidly down regulated. In addition...

  8. Alternative Splicing and Caspase-Mediated Cleavage Generate Antagonistic Variants of the Stress Oncoprotein LEDGF/p75

    Science.gov (United States)

    Brown-Bryan, Terry A.; Leoh, Lai S.; Ganapathy, Vidya; Pacheco, Fabio J.; Mediavilla-Varela, Melanie; Filippova, Maria; Linkhart, Thomas A.; Gijsbers, Rik; Debyser, Zeger; Casiano, Carlos A.

    2009-01-01

    There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium–derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress–induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH2-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies. PMID:18708362

  9. Plasma Membrane Ca2+-ATPase 4 in Murine Epididymis: Secretion of Splice Variants in the Luminal Fluid and a Role in Sperm Maturation1

    OpenAIRE

    Patel, Ramkrishna; Al-Dossary, Amal A.; Stabley, Deborah L.; Barone, Carol; Galileo, Deni S.; Strehler, Emanuel E.; Martin-DeLeon, Patricia A.

    2013-01-01

    Plasma membrane Ca2+-ATPase isoform 4 (PMCA4) is the primary Ca2+ efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca2+/CaM-dependent serine kinase (CASK) in regulating Ca2+ homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we sho...

  10. Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4.

    Directory of Open Access Journals (Sweden)

    Eri Murakami

    Full Text Available Diacylglycerol kinase (DGK phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line, DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner.

  11. TUMOR-SPECIFIC EXPRESSION AND ALTERNATIVE SPLICING OF THE COL6A3 GENE IN PANCREATIC CANCER

    Science.gov (United States)

    Arafat, Hwyda; Lazar, Melissa; Salem, Khalifa; Chipitsyna, Galina; Gong, Qiaoke; Pan, Te-Cheng; Zhang, Rui-Zhu; Yeo, Charles J.; Chu, Mon-Li

    2011-01-01

    Introduction Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. Methods We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA. Results COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma

  12. Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

    Science.gov (United States)

    Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E; Sinnegger-Brauns, Martina J; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

    2011-12-09

    An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

  13. The Possible Role of Mena Protein and Its Splicing-Derived Variants in Embryogenesis, Carcinogenesis, and Tumor Invasion: A Systematic Review of the Literature

    Directory of Open Access Journals (Sweden)

    Simona Gurzu

    2013-01-01

    Full Text Available The Ena/VASP (enabled/vasodilator stimulated phosphoprotein family includes the binding actin proteins such as mammalian Ena (Mena, VASP, and Ena-VASP-like. It is known that the perturbation of actin cycle could determine alteration in the mobility of cells and in consequence of organogenesis. Few recent studies have revealed that Mena protein could play a role in breast or pancreatic carcinogenesis. Based on our researches, we observed that the intensity of Mena expression increased from premalignant to malignant lesions in some organs such as large bowel, stomach, cervix, and salivary glands. These findings prove that Mena could be a marker of premalignant epithelial lesions. In premalignant lesions, it could be helpful to define more accurately the risk for malignant transformation. In malignant tumors, correlation of expression of its splice variants could indicate metastatic behavior. In conclusion, we consider that it is necessary to analyze the expression of Mena splice variants in a higher number of cases, in different epithelial lesions, and also in experimental studies to define its exact role in carcinogenesis and also its possible prognostic and predictive values.

  14. The possible role of Mena protein and its splicing-derived variants in embryogenesis, carcinogenesis, and tumor invasion: a systematic review of the literature.

    Science.gov (United States)

    Gurzu, Simona; Ciortea, Diana; Ember, Istvan; Jung, Ioan

    2013-01-01

    The Ena/VASP (enabled/vasodilator stimulated phosphoprotein) family includes the binding actin proteins such as mammalian Ena (Mena), VASP, and Ena-VASP-like. It is known that the perturbation of actin cycle could determine alteration in the mobility of cells and in consequence of organogenesis. Few recent studies have revealed that Mena protein could play a role in breast or pancreatic carcinogenesis. Based on our researches, we observed that the intensity of Mena expression increased from premalignant to malignant lesions in some organs such as large bowel, stomach, cervix, and salivary glands. These findings prove that Mena could be a marker of premalignant epithelial lesions. In premalignant lesions, it could be helpful to define more accurately the risk for malignant transformation. In malignant tumors, correlation of expression of its splice variants could indicate metastatic behavior. In conclusion, we consider that it is necessary to analyze the expression of Mena splice variants in a higher number of cases, in different epithelial lesions, and also in experimental studies to define its exact role in carcinogenesis and also its possible prognostic and predictive values.

  15. A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

    Science.gov (United States)

    Shin, Sangsu; Song, Yan; Ahn, Jinsoo; Kim, Eunsoo; Chen, Paula; Yang, Shujin; Suh, Yeunsu; Lee, Kichoon

    2015-11-15

    Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species. Copyright © 2015 the American Physiological Society.

  16. Reconciling newborn screening and a novel splice variant in BTD associated with partial biotinidase deficiency: A BabySeq Project case report.

    Science.gov (United States)

    Murry, Jaclyn B; Machini, Kalotina; Ceyhan-Birsoy, Ozge; Kritzer, Amy; Krier, Joel B; Lebo, Matthew S; Fayer, Shawn; Genetti, Casie A; Vannoy, Grace E; Yu, Timothy W; Agrawal, Pankaj B; Parad, Richard B; Holm, Ingrid A; McGuire, Amy L; Green, Robert C; Beggs, Alan H; Rehm, Heidi L; Project, The BabySeq

    2018-05-04

    Here, we report a newborn female infant from the well-baby cohort of the BabySeq Project who was identified with compound heterozygous BTD gene variants. The two identified variants included a well-established pathogenic variant (c.1612C>T, p.Arg538Cys) that causes profound biotinidase deficiency (BTD) in homozygosity. In addition, a novel splice variant (c.44+1G>A, p.?) was identified in the invariant splice donor region of intron 1, potentially predictive of loss of function. The novel variant was predicted to impact splicing of exon 1; however, given the absence of any reported pathogenic variants in exon 1 and the presence of alternative splicing with exon 1 absent in most tissues in the GTEx database, we assigned an initial classification of uncertain significance. Follow-up medical record review of state mandated newborn screen (NBS) results revealed an initial out-of-range biotinidase activity level. Levels from a repeat NBS sample barely passed cut-off into the normal range. To determine whether the infant was biotinidase deficient, subsequent diagnostic enzyme activity testing was performed, confirming partial BTD, and resulted in a change of management for this patient. This led to reclassification of the novel splice variant based on these results. In conclusion, combining the genetic and NBS results together prompted clinical follow-up that confirmed partial biotinidase deficiency, and informed this novel splice site's reclassification emphasizing the importance of combining iterative genetic and phenotypic evaluations. Cold Spring Harbor Laboratory Press.

  17. Characterisation of the legume SERK-NIK gene superfamily including splice variants: Implications for development and defence

    Directory of Open Access Journals (Sweden)

    Rose Ray J

    2011-03-01

    Full Text Available Abstract Background SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins function both in developmental and defence signalling pathways, which occur in response to both peptide and steroid ligands. SERKs are generally present as small gene families in plants, with five SERK genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. The SERK1 gene of the model legume, Medicago truncatula functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation. However, other SERK genes in M. truncatula and other legumes are largely unidentified and their functions unknown. Results To aid the understanding of signalling pathways in M. truncatula, we have identified and annotated the SERK genes in this species. Using degenerate PCR and database mining, eight more SERK-like genes have been identified and these have been shown to be expressed. The amplification and sequencing of several different PCR products from one of these genes is consistent with the presence of splice variants. Four of the eight additional genes identified are upregulated in cultured leaf tissue grown on embryogenic medium. The sequence information obtained from M. truncatula was used to identify SERK family genes in the recently sequenced soybean (Glycine max genome. Conclusions A total of nine SERK or SERK-like genes have been identified in M. truncatula and potentially 17 in soybean. Five M. truncatula SERK genes arose from duplication events not evident in soybean and Lotus. The presence of splice variants has not been previously reported in a SERK gene. Upregulation of four newly identified SERK genes (in addition to the

  18. ASC-J9 Suppresses Castration-Resistant Prostate Cancer Growth through Degradation of Full-length and Splice Variant Androgen Receptors

    Directory of Open Access Journals (Sweden)

    Shinichi Yamashita

    2012-01-01

    Full Text Available Early studies suggested androgen receptor (AR splice variants might contribute to the progression of prostate cancer (PCa into castration resistance. However, the therapeutic strategy to target these AR splice variants still remains unresolved. Through tissue survey of tumors from the same patients before and after castration resistance, we found that the expression of AR3, a major AR splice variant that lacks the AR ligand-binding domain, was substantially increased after castration resistance development. The currently used antiandrogen, Casodex, showed little growth suppression in CWR22Rv1 cells. Importantly, we found that AR degradation enhancer ASC-J9 could degrade both full-length (fAR and AR3 in CWR22Rv1 cells as well as in C4-2 and C81 cells with addition of AR3. The consequences of such degradation of both fAR and AR3 might then result in the inhibition of AR transcriptional activity and cell growth in vitro. More importantly, suppression of AR3 specifically by short-hairpin AR3 or degradation of AR3 by ASC-J9 resulted in suppression of AR transcriptional activity and cell growth in CWR22Rv1-fARKD (fAR knockdown cells in which DHT failed to induce, suggesting the importance of targeting AR3. Finally, we demonstrated the in vivo therapeutic effects of ASC-J9 by showing the inhibition of PCa growth using the xenografted model of CWR22Rv1 cells orthotopically implanted into castrated nude mice with undetectable serum testosterone. These results suggested that targeting both fAR- and AR3-mediated PCa growth by ASC-J9 may represent the novel therapeutic approach to suppress castration-resistant PCa. Successful clinical trials targeting both fAR and AR3 may help us to battle castration-resistant PCa in the future.

  19. A liver X receptor (LXR)-β alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-β

    International Nuclear Information System (INIS)

    Hashimoto, Koshi; Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi; Monden, Tsuyoshi; Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo

    2009-01-01

    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-β LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-β gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-β. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  20. An unusual spliced variant of DELLA protein, a negative regulator of gibberellin signaling, in lettuce.

    Science.gov (United States)

    Sawada, Yoshiaki; Umetsu, Asami; Komatsu, Yuki; Kitamura, Jun; Suzuki, Hiroyuki; Asami, Tadao; Fukuda, Machiko; Honda, Ichiro; Mitsuhashi, Wataru; Nakajima, Masatoshi; Toyomasu, Tomonobu

    2012-01-01

    DELLA proteins are negative regulators of the signaling of gibberellin (GA), a phytohormone regulating plant growth. DELLA degradation is triggered by its interaction with GID1, a soluble GA receptor, in the presence of bioactive GA. We isolated cDNA from a spliced variant of LsDELLA1 mRNA in lettuce, and named it LsDELLA1sv. It was deduced that LsDELLA1sv encodes truncated LsDELLA1, which has DELLA and VHYNP motifs at the N terminus but lacks part of the C-terminal GRAS domain. The recombinant LsDELLA1sv protein interacted with both Arabidopsis GID1 and lettuce GID1s in the presence of GA. A yeast two-hybrid assay suggested that LsDELLA1sv interacted with LsDELLA1. The ratio of LsDELLA1sv to LsDELLA1 transcripts was higher in flower samples at the late reproductive stage and seed samples (dry seeds and imbibed seeds) than in the other organ samples examined. This study suggests that LsDELLA1sv is a possible modulator of GA signaling in lettuce.

  1. 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.

  2. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    International Nuclear Information System (INIS)

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-01-01

    Highlights: → Novel role for poliovirus 2A protease as splicing modulator. → Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. → Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A pro modulating the alternative splicing of pre-mRNAs. Expression of 2A pro potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A pro abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A pro , leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A pro on splicing is to selectively block the second catalytic step.

  3. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  4. Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays

    Directory of Open Access Journals (Sweden)

    Nixon Tamara J

    2008-05-01

    Full Text Available Abstract Background Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples. Results In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2. Our data indicate that large changes (> 5-fold in alternative splicing are infrequent in gliomagenesis ( Conclusion While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.

  5. Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation.

    Science.gov (United States)

    Patel, Ramkrishna; Al-Dossary, Amal A; Stabley, Deborah L; Barone, Carol; Galileo, Deni S; Strehler, Emanuel E; Martin-DeLeon, Patricia A

    2013-07-01

    Plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is the primary Ca(2+) efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca(2+)/CaM-dependent serine kinase (CASK) in regulating Ca(2+) homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT-PCR, that both the splice variants of Pmca4 mRNA are expressed in murine testis and throughout the epididymis. Immunofluorescence localized PMCA4a to the apical membrane of the epididymal epithelium, and Western analysis not only confirmed its presence but showed for the first time that PMCA4a and PMCA4b are secreted in the epididymal luminal fluid (ELF), from which epididymosomes containing PMCA4a were isolated. Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5-fold compared to caput sperm (detected by Western blotting) and ~2-fold after incubation in ELF, revealing in vitro uptake and implicating PMCA4a in epididymal sperm maturation. Coimmunoprecipitation using pan-PMCA4 antibodies, revealed that both variants associate with CASK, suggesting their presence in a complex. Because they have different kinetic properties for Ca(2+) transport and different abilities to bind to CASK, our study suggests a mechanism for combining the functional attributes of both PMCA4 variants, leading to heightened efficiency of the pump in the maintenance of Ca(2+) homeostasis, which is crucial for normal motility and male fertility.

  6. TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

    Science.gov (United States)

    Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

    2018-02-28

    Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

  7. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    International Nuclear Information System (INIS)

    Chiu Yali; Ouyang Pin

    2006-01-01

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function

  8. Performance comparison of two androgen receptor splice variant 7 (AR-V7) detection methods.

    Science.gov (United States)

    Bernemann, Christof; Steinestel, Julie; Humberg, Verena; Bögemann, Martin; Schrader, Andres Jan; Lennerz, Jochen K

    2018-01-23

    To compare the performance of two established androgen receptor splice variant 7 (AR-V7) mRNA detection systems, as paradoxical responses to next-generation androgen-deprivation therapy in AR-V7 mRNA-positive circulating tumour cells (CTC) of patients with castration-resistant prostate cancer (CRPC) could be related to false-positive classification using detection systems with different sensitivities. We compared the performance of two established mRNA-based AR-V7 detection technologies using either SYBR Green or TaqMan chemistries. We assessed in vitro performance using eight genitourinary cancer cell lines and serial dilutions in three AR-V7-positive prostate cancer cell lines, as well as in 32 blood samples from patients with CRPC. Both assays performed identically in the cell lines and serial dilutions showed identical diagnostic thresholds. Performance comparison in 32 clinical patient samples showed perfect concordance between the assays. In particular, both assays determined AR-V7 mRNA-positive CTCs in three patients with unexpected responses to next-generation anti-androgen therapy. Thus, technical differences between the assays can be excluded as the underlying reason for the unexpected responses to next-generation anti-androgen therapy in a subset of AR-V7 patients. Irrespective of the method used, patients with AR-V7 mRNA-positive CRPC should not be systematically precluded from an otherwise safe treatment option. © 2018 The Authors BJU International © 2018 BJU International Published by John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    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

  10. 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

  11. Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

    Science.gov (United States)

    Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

    2015-01-01

    The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Differentially regulated splice variants and systems biology analysis of Kaposi's sarcoma-associated herpesvirus-infected lymphatic endothelial cells.

    Science.gov (United States)

    Chang, Ting-Yu; Wu, Yu-Hsuan; Cheng, Cheng-Chung; Wang, Hsei-Wei

    2011-09-01

    Alternative RNA splicing greatly increases proteome diversity, and the possibility of studying genome-wide alternative splicing (AS) events becomes available with the advent of high-throughput genomics tools devoted to this issue. Kaposi's sarcoma associated herpesvirus (KSHV) is the etiological agent of KS, a tumor of lymphatic endothelial cell (LEC) lineage, but little is known about the AS variations induced by KSHV. We analyzed KSHV-controlled AS using high-density microarrays capable of detecting all exons in the human genome. Splicing variants and altered exon-intron usage in infected LEC were found, and these correlated with protein domain modification. The different 3'-UTR used in new transcripts also help isoforms to escape microRNA-mediated surveillance. Exome-level analysis further revealed information that cannot be disclosed using classical gene-level profiling: a significant exon usage difference existed between LEC and CD34(+) precursor cells, and KSHV infection resulted in LEC-to-precursor, dedifferentiation-like exon level reprogramming. Our results demonstrate the application of exon arrays in systems biology research, and suggest the regulatory effects of AS in endothelial cells are far more complex than previously observed. This extra layer of molecular diversity helps to account for various aspects of endothelial biology, KSHV life cycle and disease pathogenesis that until now have been unexplored.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. High-resolution temporal and regional mapping of MAPT expression and splicing in human brain development.

    Science.gov (United States)

    Hefti, Marco M; Farrell, Kurt; Kim, SoongHo; Bowles, Kathryn R; Fowkes, Mary E; Raj, Towfique; Crary, John F

    2018-01-01

    The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. Identification of a novel splice acceptor in the HIV-1 genome: independent expression of the cytoplasmic tail of the envelope protein

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J. L.

    1996-01-01

    Multiple splicing sites exist in the RNA genome of the human immunodeficiency virus type 1 (HIV-1). In a screen for subgenomic forms of the HIV-1 genome that could be transferred to fresh cells by virus infection, we identified a novel spliced variant of HIV-1 RNA that uses a hitherto unknown splice

  18. 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

    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...

  19. Kinetic and structural characterization of an alternatively spliced variant of human mitochondrial 5'(3')-deoxyribonucleotidase

    Czech Academy of Sciences Publication Activity Database

    Pachl, Petr; Fábry, Milan; Veverka, Václav; Brynda, Jiří; Řezáčová, Pavlína

    2015-01-01

    Roč. 30, č. 1 (2015), 63-68 ISSN 1475-6366 R&D Projects: GA ČR GA203/09/0820; GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : 5'(3')-deoxyribonucleotidase * alternative splicing * crystal structure * hydrolase * mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.428, year: 2015

  20. Androgen receptor and its splice variant, AR-V7, differentially regulate FOXA1 sensitive genes in LNCaP prostate cancer cells.

    Science.gov (United States)

    Krause, William C; Shafi, Ayesha A; Nakka, Manjula; Weigel, Nancy L

    2014-09-01

    Prostate cancer (PCa) is an androgen-dependent disease, and tumors that are resistant to androgen ablation therapy often remain androgen receptor (AR) dependent. Among the contributors to castration-resistant PCa are AR splice variants that lack the ligand-binding domain (LBD). Instead, they have small amounts of unique sequence derived from cryptic exons or from out of frame translation. The AR-V7 (or AR3) variant is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequence in AR-V7, or simply to loss of the LBD. To examine transcriptional regulation by AR-V7, we have used lentiviruses encoding AR-V7 (amino acids 1-627 of AR with the 16 amino acids unique to the variant) to prepare a derivative of the androgen-dependent LNCaP cells with inducible expression of AR-V7. An additional cell line was generated with regulated expression of AR-NTD (amino acids 1-660 of AR); this mutant lacks the LBD but does not have the AR-V7 specific sequence. We find that AR and AR-V7 have distinct activities on target genes that are co-regulated by FOXA1. Transcripts regulated by AR-V7 were similarly regulated by AR-NTD, indicating that loss of the LBD is sufficient for the observed differences. Differential regulation of target genes correlates with preferential recruitment of AR or AR-V7 to specific cis-regulatory DNA sequences providing an explanation for some of the observed differences in target gene regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

    Science.gov (United States)

    Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

    2017-10-30

    Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

  2. Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia

    Directory of Open Access Journals (Sweden)

    Colby A. Souders

    2015-06-01

    Full Text Available Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1, are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

  3. Complement Factor H-Related Protein 4A Is the Dominant Circulating Splice Variant of CFHR4

    Directory of Open Access Journals (Sweden)

    Richard B. Pouw

    2018-04-01

    Full Text Available Recent research has elucidated circulating levels of almost all factor H-related (FHR proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH, fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

  4. Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling.

    Science.gov (United States)

    Wang, Qiong; Sun, Rui; Wu, Leyan; Huang, Junfeng; Wang, Ping; Yuan, Hailong; Qiu, Feifei; Xu, Xiaohong; Wu, Di; Yu, Ying; Liu, Xin; Zhang, Qing

    2013-12-01

    The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. 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

    DEFF Research Database (Denmark)

    de la Hoya, Miguel; Soukarieh, Omar; López-perolio, Irene

    2016-01-01

    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...

  6. 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.

  7. Insulin-like growth factor I (IGF-1 Ec/Mechano Growth factor--a splice variant of IGF-1 within the growth plate.

    Directory of Open Access Journals (Sweden)

    Werner Schlegel

    Full Text Available Human insulin-like growth factor 1 Ec (IGF-1Ec, also called mechano growth factor (MGF, is a splice variant of insulin-like growth factor 1 (IGF-1, which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

  8. Insulin-like growth factor I (IGF-1) Ec/Mechano Growth factor--a splice variant of IGF-1 within the growth plate.

    Science.gov (United States)

    Schlegel, Werner; Raimann, Adalbert; Halbauer, Daniel; Scharmer, Daniela; Sagmeister, Susanne; Wessner, Barbara; Helmreich, Magdalena; Haeusler, Gabriele; Egerbacher, Monika

    2013-01-01

    Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

  9. Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits.

    Science.gov (United States)

    Zhang, Yanghai; Cui, Yang; Zhang, Xuelian; Wang, Yimin; Gao, Jiayang; Yu, Ting; Lv, Xiaoyan; Pan, Chuanying

    2018-05-31

    Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P StAR mRNA expression level was significantly higher in LCs than others (P StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    International Nuclear Information System (INIS)

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-01-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten -/- fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten -/- cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten -/- cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  11. Biologico-clinical significance of DNMT3A variants expression in acute myeloid leukemia.

    Science.gov (United States)

    Lin, Na; Fu, Wei; Zhao, Chen; Li, Bixin; Yan, Xiaojing; Li, Yan

    2017-12-09

    DNA methyltransferase 3A (DNMT3A) catalyzes de novo DNA methylation and plays important roles in the pathogenesis of acute myeloid leukemia. However, the expression status of DNMT3A variants in acute myeloid leukemia remains obscure. This study aimed to assess the expression levels of alternative splicing of DNMT3A variants and explore their roles in acute myeloid leukemia (AML). DNMT3A variants gene expression were assessed, measuring their effects on cell proliferation. In addition, the expression of DNMT3A variants were evaluated in acute myeloid leukemia patients. Four DNMT3A variants were identified, with DNMT3A1 and DNMT3A2V found to be dominant in acute myeloid leukemia cell lines. Moreover, DNMT3A2V overexpression delayed cell proliferation; while, DNMT3A2V R882H mutation promoted cell proliferation. Further, DNMT3A1 and DNMT3A2V were detected in newly diagnosed acute myeloid leukemia (AML) patients and controls with non-malignant hematological disease, with DNMT3A2V significantly up-regulated in AML patients. The main transcript switched from DNMT3A1 to DNMT3A2V in some patients, especially the low risk group based on the NCCN 2016 guidelines. These findings suggest that DNMT3A1 and DNMT3A2V are the main variants in acute myeloid leukemia with different clinical association, and might play important roles in the pathophysiology of acute myeloid leukemia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. First Trimester Pregnancy Loss and the Expression of alternatively spliced NKp30 isoforms in Maternal Blood and Placental Tissue

    Directory of Open Access Journals (Sweden)

    Avishai eShemesh

    2015-06-01

    Full Text Available In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group was comprised of women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expression was mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms -a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. In contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10 and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss.

  13. Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Ryder, L R; Woetmann, A; Madsen, H O

    2010-01-01

    OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure...... the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full......-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls...

  14. Alternative splice variant of the thiazide-sensitive NaCl cotransporter

    DEFF Research Database (Denmark)

    Tutakhel, Omar A Z; Jeleń, Sabina; Valdez-Flores, Marco

    2016-01-01

    in comparison to NCC3. Mimicking a constitutively active phosphorylation site at residue 811 (S811D) in NCC1 further augmented Na(+) transport, while a non-phosphorylatable variant (S811A) of NCC1 prevented this enhanced response. Analysis of human urinary exosomes demonstrated that water loading in human...

  15. Targeting Splicing in Prostate Cancer

    OpenAIRE

    Effrosyni Antonopoulou; Michael Ladomery

    2018-01-01

    Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform speci...

  16. High expression of PTBP1 promote invasion of colorectal cancer by alternative splicing of cortactin.

    Science.gov (United States)

    Wang, Zhi-Na; Liu, Dan; Yin, Bin; Ju, Wen-Yi; Qiu, Hui-Zhong; Xiao, Yi; Chen, Yuan-Jia; Peng, Xiao-Zhong; Lu, Chong-Mei

    2017-05-30

    Polypyrimidine tract-binding protein 1 (PTBP1) involving in almost all steps of mRNA regulation including alternative splicing metabolism during tumorigenesis due to its RNA-binding activity. Initially, we found that high expressed PTBP1 and poor prognosis was interrelated in colorectal cancer (CRC) patients with stages II and III CRC, which widely different in prognosis and treatment, by immunohistochemistry. PTBP1 was also upregulated in colon cancer cell lines. In our study, knockdown of PTBP1 by siRNA transfection decreased cell proliferation and invasion in vitro. Denovirus shRNA knockdown of PTBP1 inhibited colorectal cancer growth in vivo. Furthermore, PTBP1 regulates alternative splicing of many target genes involving in tumorgenesis in colon cancer cells. We confirmed that the splicing of cortactin exon 11 which was only contained in cortactin isoform-a, as a PTBP1 target. Knockdown of PTBP1 decreased the expression of cortactin isoform-a by exclusion of exon 11. Also the mRNA levels of PTBP1 and cortactin isoform-a were cooperatively expressed in colorectal cancer tissues. Knocking down cortactin isoform-a significantly decreased cell migration and invasion in colorectal cancer cells. Overexpression of cortactin isoform-a could rescue PTBP1-knockdown effect of cell motility. In summary the study revealed that PTBP1 facilitates colorectal cancer migration and invasion activities by inclusion of cortactin exon 11.

  17. Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse

    OpenAIRE

    Ohtsuki, Takashi; Otsuki, Mariko; Murakami, Yousuke; Maekawa, Tetsuya; Yamamoto, Takashi; Akasaka, Koji; Takeuchi, Sakae; Takahashi, Sumio

    2005-01-01

    Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse ...

  18. Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

    Directory of Open Access Journals (Sweden)

    Rosner William

    2009-05-01

    Full Text Available Abstract Background Human sex hormone-binding globulin (SHBG regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (PL. The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (PT, and two minor transcripts. Results We report that transcriptional expression of the human SHBG gene is far more complex than previously described. PL and PT direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of PL and PT, and present evidence for a third SHBG gene promoter (PN within the neighboring FXR2 gene; PN regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N. Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. PL- PT- and PN-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells. Conclusion These results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Splice form variant and amino acid changes in MDR49 confers DDT resistance in transgenic Drosophila.

    Science.gov (United States)

    Seong, Keon Mook; Sun, Weilin; Clark, John M; Pittendrigh, Barry R

    2016-03-22

    The ATP-binding cassette (ABC) transporters represent a superfamily of proteins that have important physiological roles in both prokaryotes and eukaryotes. In insects, ABC transporters have previously been implicated in insecticide resistance. The 91-R strain of Drosophila melanogaster has been intensely selected with DDT over six decades. A recent selective sweeps analysis of 91-R implicated the potential role of MDR49, an ABC transporter, in DDT resistance, however, to date the details of how MDR49 may play a role in resistance have not been elucidated. In this study, we investigated the impact of structural changes and an alternative splicing event in MDR49 on DDT-resistance in 91-R, as compared to the DDT susceptible strain 91-C. We observed three amino acid differences in MDR49 when 91-R was compared with 91-C, and only one isoform (MDR49B) was implicated in DDT resistance. A transgenic Drosophila strain containing the 91-R-MDR49B isoform had a significantly higher LD50 value as compared to the 91-C-MDR49B isoform at the early time points (6 h to 12 h) during DDT exposure. Our data support the hypothesis that the MDR49B isoform, with three amino acid mutations, plays a role in the early aspects of DDT resistance in 91-R.

  1. Activity, splice variants, conserved peptide motifs, and phylogeny of two new alpha1,3-fucosyltransferase families (FUT10 and FUT11).

    Science.gov (United States)

    Mollicone, Rosella; Moore, Stuart E H; Bovin, Nicolai; Garcia-Rosasco, Marcela; Candelier, Jean-Jacques; Martinez-Duncker, Iván; Oriol, Rafael

    2009-02-13

    We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    Ando, Hitoshi; Natsume, Atsushi; Iwami, Kenichiro; Ohka, Fumiharu; Kuchimaru, Takahiro; Kizaka-Kondoh, Shinae; Ito, Kengo; Saito, Kiyoshi; Sugita, Sachi; Hoshino, Tsuneyoshi; Wakabayashi, Toshihiko

    2013-01-01

    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

  4. Differential control of ageing and lifespan by isoforms and splice variants across the mTOR network.

    Science.gov (United States)

    Razquin Navas, Patricia; Thedieck, Kathrin

    2017-07-15

    Ageing can be defined as the gradual deterioration of physiological functions, increasing the incidence of age-related disorders and the probability of death. Therefore, the term ageing not only reflects the lifespan of an organism but also refers to progressive functional impairment and disease. The nutrient-sensing kinase mTOR (mammalian target of rapamycin) is a major determinant of ageing. mTOR promotes cell growth and controls central metabolic pathways including protein biosynthesis, autophagy and glucose and lipid homoeostasis. The concept that mTOR has a crucial role in ageing is supported by numerous reports on the lifespan-prolonging effects of the mTOR inhibitor rapamycin in invertebrate and vertebrate model organisms. Dietary restriction increases lifespan and delays ageing phenotypes as well and mTOR has been assigned a major role in this process. This may suggest a causal relationship between the lifespan of an organism and its metabolic phenotype. More than 25 years after mTOR's discovery, a wealth of metabolic and ageing-related effects have been reported. In this review, we cover the current view on the contribution of the different elements of the mTOR signalling network to lifespan and age-related metabolic impairment. We specifically focus on distinct roles of isoforms and splice variants across the mTOR network. The comprehensive analysis of mouse knockout studies targeting these variants does not support a tight correlation between lifespan prolongation and improved metabolic phenotypes and questions the strict causal relationship between them. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  5. MAPT expression and splicing is differentially regulated by brain region: relation to genotype and implication for tauopathies

    Science.gov (United States)

    Trabzuni, Daniah; Wray, Selina; Vandrovcova, Jana; Ramasamy, Adaikalavan; Walker, Robert; Smith, Colin; Luk, Connie; Gibbs, J. Raphael; Dillman, Allissa; Hernandez, Dena G.; Arepalli, Sampath; Singleton, Andrew B.; Cookson, Mark R.; Pittman, Alan M.; de Silva, Rohan; Weale, Michael E.; Hardy, John; Ryten, Mina

    2012-01-01

    The MAPT (microtubule-associated protein tau) locus is one of the most remarkable in neurogenetics due not only to its involvement in multiple neurodegenerative disorders, including progressive supranuclear palsy, corticobasal degeneration, Parksinson's disease and possibly Alzheimer's disease, but also due its genetic evolution and complex alternative splicing features which are, to some extent, linked and so all the more intriguing. Therefore, obtaining robust information regarding the expression, splicing and genetic regulation of this gene within the human brain is of immense importance. In this study, we used 2011 brain samples originating from 439 individuals to provide the most reliable and coherent information on the regional expression, splicing and regulation of MAPT available to date. We found significant regional variation in mRNA expression and splicing of MAPT within the human brain. Furthermore, at the gene level, the regional distribution of mRNA expression and total tau protein expression levels were largely in agreement, appearing to be highly correlated. Finally and most importantly, we show that while the reported H1/H2 association with gene level expression is likely to be due to a technical artefact, this polymorphism is associated with the expression of exon 3-containing isoforms in human brain. These findings would suggest that contrary to the prevailing view, genetic risk factors for neurodegenerative diseases at the MAPT locus are likely to operate by changing mRNA splicing in different brain regions, as opposed to the overall expression of the MAPT gene. PMID:22723018

  6. 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

  7. 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

  8. Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

    Science.gov (United States)

    Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

    2017-10-01

    Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  10. The Role of Platelet-Derived Growth Factor C and Its Splice Variant in Breast Cancer

    Science.gov (United States)

    2012-02-01

    epithelial-mesenchymal transition leading instead to apoptosis (8). Furthermore, inhibition of PDGFR signaling by the PDGFR inhibitor STI571...PDGFRs are expressed in many different cell types including endothelial, epithelial and neural cells and are necessary for embryological development (as

  11. Cre-loxP–mediated Inactivation of the α6A Integrin Splice Variant In Vivo: Evidence for a Specific Functional Role of α6A in Lymphocyte Migration but Not in Heart Development

    Science.gov (United States)

    Gimond, Clotilde; Baudoin, Christian; van der Neut, Ronald; Kramer, Duco; Calafat, Jero; Sonnenberg, Arnoud

    1998-01-01

    Two splice variants of the α6 integrin subunit, α6A and α6B, with different cytoplasmic domains, have previously been described. While α6B is expressed throughout the development of the mouse, the expression of α6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, α6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of α6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of α6A in embryonic stem cells, and the deletion resulted in the expression of α6B in all tissues that normally express α6A. We show that α6A−/− mice develop normally and are fertile. The substitution of α6A by α6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in α6A−/− mice. However, the substitution of α6A by α6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin–ligand interactions, was not affected in the α6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of α6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions. PMID:9763436

  12. Expressed Centromere Specific Histone 3 (CENH3 Variants in Cultivated Triploid and Wild Diploid Bananas (Musa spp.

    Directory of Open Access Journals (Sweden)

    Kariuki S. Muiruri

    2017-06-01

    Full Text Available Centromeres are specified by a centromere specific histone 3 (CENH3 protein, which exists in a complex environment, interacting with conserved proteins and rapidly evolving satellite DNA sequences. The interactions may become more challenging if multiple CENH3 versions are introduced into the zygote as this can affect post-zygotic mitosis and ultimately sexual reproduction. Here, we characterize CENH3 variant transcripts expressed in cultivated triploid and wild diploid progenitor bananas. We describe both splice- and allelic-[Single Nucleotide Polymorphisms (SNP] variants and their effects on the predicted secondary structures of protein. Expressed CENH3 transcripts from six banana genotypes were characterized and clustered into three groups (MusaCENH-1A, MusaCENH-1B, and MusaCENH-2 based on similarity. The CENH3 groups differed with SNPs as well as presence of indels resulting from retained and/or skipped exons. The CENH3 transcripts from different banana genotypes were spliced in either 7/6, 5/4 or 6/5 exons/introns. The 7/6 and the 5/4 exon/intron structures were found in both diploids and triploids, however, 7/6 was most predominant. The 6/5 exon/introns structure was a result of failure of the 7/6 to splice correctly. The various transcripts obtained were predicted to encode highly variable N-terminal tails and a relatively conserved C-terminal histone fold domain (HFD. The SNPs were predicted in some cases to affect the secondary structure of protein by lengthening or shorting the affected domains. Sequencing of banana CENH3 transcripts predicts SNP variations that affect amino acid sequences and alternatively spliced transcripts. Most of these changes affect the N-terminal tail of CENH3.

  13. Investigation of tissue-specific human orthologous alternative splice events in pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Salicio, Susanna Cirera

    2010-01-01

    Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have ...... in preservation of open reading frame are indicative of a functional significance of the splice variants of the gene....

  14. Expression of the Hippocampal NMDA Receptor GluN1 Subunit and Its Splicing Isoforms in Schizophrenia: Postmortem Study

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Šťastný, František; Horáček, J.; Lochman, J.; Šerý, O.; Peková, S.; Klaschka, Jan; Höschl, C.

    2010-01-01

    Roč. 35, č. 7 (2010), s. 994-1002 ISSN 0364-3190 Grant - others:GA MZd(CZ) NR9324 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10300504 Keywords : schizophrenia * hippocampus * GluN1 subunit of NMDA receptor * splice variants * laterality Subject RIV: FL - Psychiatry, Sexuology Impact factor: 2.608, year: 2010

  15. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

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    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  16. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

    Science.gov (United States)

    Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

    2016-11-07

    The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  17. 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.

  18. Acute hypoxia stress induced abundant differential expression genes and alternative splicing events in heart of tilapia.

    Science.gov (United States)

    Xia, Jun Hong; Li, Hong Lian; Li, Bi Jun; Gu, Xiao Hui; Lin, Hao Ran

    2018-01-10

    Hypoxia is one of the critical environmental stressors for fish in aquatic environments. Although accumulating evidences indicate that gene expression is regulated by hypoxia stress in fish, how genes undergoing differential gene expression and/or alternative splicing (AS) in response to hypoxia stress in heart are not well understood. Using RNA-seq, we surveyed and detected 289 differential expressed genes (DEG) and 103 genes that undergo differential usage of exons and splice junctions events (DUES) in heart of a hypoxia tolerant fish, Nile tilapia, Oreochromis niloticus following 12h hypoxic treatment. The spatio-temporal expression analysis validated the significant association of differential exon usages in two randomly selected DUES genes (fam162a and ndrg2) in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under acute hypoxia treatment. Functional analysis significantly associated the differential expressed genes with the categories related to energy conservation, protein synthesis and immune response. Different enrichment categories were found between the DEG and DUES dataset. The Isomerase activity, Oxidoreductase activity, Glycolysis and Oxidative stress process were significantly enriched for the DEG gene dataset, but the Structural constituent of ribosome and Structural molecule activity, Ribosomal protein and RNA binding protein were significantly enriched only for the DUES genes. Our comparative transcriptomic analysis reveals abundant stress responsive genes and their differential regulation function in the heart tissues of Nile tilapia under acute hypoxia stress. Our findings will facilitate future investigation on transcriptome complexity and AS regulation during hypoxia stress in fish. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

    Science.gov (United States)

    Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

    2006-01-01

    Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice

  20. Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

    Science.gov (United States)

    Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

    2006-03-06

    Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely

  1. Alternative RNA splicing and gastric cancer.

    Science.gov (United States)

    Li, Ying; Yuan, Yuan

    2017-07-01

    Alternative splicing (AS) linked to diseases, especially to tumors. Recently, more and more studies focused on the relationship between AS and gastric cancer (GC). This review surveyed the hot topic from four aspects: First, the common types of AS in cancer, including exon skipping, intron retention, mutually exclusive exon, alternative 5 ' or 3' splice site, alternative first or last exon and alternative 3' untranslated regions. Second, basic mechanisms of AS and its relationship with cancer. RNA splicing in eukaryotes follows the GT-AG rule by both cis-elements and trans-acting factors regulatory. Through RNA splicing, different proteins with different forms and functions can be produced and may be associated with carcinogenesis. Third, AS types of GC-related genes and their splicing variants. In this paper, we listed 10 common genes with AS and illustrated its possible molecular mechanisms owing to genetic variation (mutation and /or polymorphism). Fourth, the splicing variants of GC-associated genes and gastric carcinogenesis, invasion and metastasis. Many studies have found that the different splicing variants of the same gene are differentially expressed in GC and its precancerous diseases, suggesting AS has important implications in GC development. Taking together, this review highlighted the role of AS and splicing variants in the process of GC. We hope that this is not only beneficial to advances in the study field of GC, but also can provide valuable information to other similar tumor research.Although we already know some gene splicing and splicing variants play an important role in the development of GC, but many phenomena and mechanisms are still unknown. For example, how the tumor microenvironment and signal transduction pathway effect the forming and function of AS? Unfortunately, this review did not cover the contents because the current study is limited. It is no doubt that clarifying the phenomena and mechanisms of these unknown may help to reveal

  2. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

    Directory of Open Access Journals (Sweden)

    Purcell Damian FJ

    2008-02-01

    Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

  3. A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung D. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Seo, Kwang W. [Department of Internal Medicines, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Lee, Eun A.; Quang, Nguyen N. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Cho, Hong R. [Department of Surgery, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Kwon, Byungsuk, E-mail: bskwon@mail.ulsan.as.kr [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of)

    2011-07-01

    Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

  4. The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

    Directory of Open Access Journals (Sweden)

    Guo Yajun

    2006-01-01

    Full Text Available Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested and for metastatic melanoma of 96% (146/151 tested in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

  5. Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

    Science.gov (United States)

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A; Vajja, Venkata G; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K; Levi, Amnon; Wehner, Todd; Reddy, Umesh K

    2015-03-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. A study of alternative splicing in the pig

    Directory of Open Access Journals (Sweden)

    Jørgensen Claus B

    2010-05-01

    Full Text Available Abstract Background Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. Results The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR. Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. Conclusions In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue

  7. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

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    El Andaloussi Samir

    2011-05-01

    Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

  8. Multiple Functional Variants in cis Modulate PDYN Expression.

    Science.gov (United States)

    Babbitt, Courtney C; Silverman, Jesse S; Haygood, Ralph; Reininga, Jennifer M; Rockman, Matthew V; Wray, Gregory A

    2010-02-01

    Understanding genetic variation and its functional consequences within cis-regulatory regions remains an important challenge in human genetics and evolution. Here, we present a fine-scale functional analysis of segregating variation within the cis-regulatory region of prodynorphin, a gene that encodes an endogenous opioid precursor with roles in cognition and disease. In order to characterize the functional consequences of segregating variation in cis in a region under balancing selection in different human populations, we examined associations between specific polymorphisms and gene expression in vivo and in vitro. We identified five polymorphisms within the 5' flanking region that affect transcript abundance: a 68-bp repeat recognized in prior studies, as well as two microsatellites and two single nucleotide polymorphisms not previously implicated as functional variants. The impact of these variants on transcription differs by brain region, sex, and cell type, implying interactions between cis genotype and the differentiated state of cells. The effects of individual variants on expression level are not additive in some combinations, implying epistatic interactions between nearby variants. These data reveal an unexpectedly complex relationship between segregating genetic variation and its expression-trait consequences and highlights the importance of close functional scrutiny of natural genetic variation within even relatively well-studied cis-regulatory regions.

  9. SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2016-02-05

    Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

  10. Genetic Variants Contribute to Gene Expression Variability in Humans

    Science.gov (United States)

    Hulse, Amanda M.; Cai, James J.

    2013-01-01

    Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed. PMID:23150607

  11. Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1 in normal and transformed cerebellar cells

    Directory of Open Access Journals (Sweden)

    Baader Stephan L

    2007-10-01

    Full Text Available Abstract Background Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. Results During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. Conclusion Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

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

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    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  13. rSeqNP: a non-parametric approach for detecting differential expression and splicing from RNA-Seq data.

    Science.gov (United States)

    Shi, Yang; Chinnaiyan, Arul M; Jiang, Hui

    2015-07-01

    High-throughput sequencing of transcriptomes (RNA-Seq) has become a powerful tool to study gene expression. Here we present an R package, rSeqNP, which implements a non-parametric approach to test for differential expression and splicing from RNA-Seq data. rSeqNP uses permutation tests to access statistical significance and can be applied to a variety of experimental designs. By combining information across isoforms, rSeqNP is able to detect more differentially expressed or spliced genes from RNA-Seq data. The R package with its source code and documentation are freely available at http://www-personal.umich.edu/∼jianghui/rseqnp/. jianghui@umich.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

    Science.gov (United States)

    Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

    2018-02-20

    Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  15. Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

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    Marcin Szczot

    2017-12-01

    Full Text Available Summary: Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. : Szczot et al. find that the mechanoreceptor Piezo2 is extensively alternatively spliced, generating multiple distinct isoforms. Their findings indicate that these splice products have specific tissue and cell type expression patterns and exhibit differences in receptor properties. Keywords: Piezo, touch, sensation, ion-channel, splicing

  16. [The impact of the androgen receptor splice variant AR-V7 on the prognosis and treatment of advanced prostate cancer].

    Science.gov (United States)

    Thelen, P; Taubert, H; Duensing, S; Kristiansen, G; Merseburger, A S; Cronauer, M V

    2018-01-25

    A recently discovered mechanism enabling prostate cancer cells to escape the effects of endocrine therapies consists in the synthesis of C-terminally truncated, constitutively active androgen receptor (AR) splice variants (AR-V). Devoid of a functional C-terminal hormone/ligand binding domain, various AR-Vs are insensitive to therapies targeting the androgen/AR signalling axis. Preliminary studies suggest that AR-V7, the most common AR-V, is a promising predictive tumour marker and a relevant selection marker for the treatment of advanced prostate cancer. This review critically outlines recent advances in AR-V7 diagnostics and presents an overview of current AR-V7 targeted therapies. © Georg Thieme Verlag KG Stuttgart · New York.

  17. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Wulf, Helle; Hay-Schmidt, Anders

    2009-01-01

    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expresse...

  18. Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner.

    Science.gov (United States)

    Rezania, S; Kammerer, S; Li, C; Steinecker-Frohnwieser, B; Gorischek, A; DeVaney, T T J; Verheyen, S; Passegger, C A; Tabrizi-Wizsy, N Ghaffari; Hackl, H; Platzer, D; Zarnani, A H; Malle, E; Jahn, S W; Bauernhofer, T; Schreibmayer, W

    2016-08-12

    Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

  19. Identification of a truncated splice variant of IL-18 receptor alpha in the human and rat, with evidence of wider evolutionary conservation

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    Chris S. Booker

    2014-09-01

    Full Text Available Interleukin-18 (IL-18 is a pro-inflammatory cytokine which stimulates activation of the nuclear factor kappa beta (NF-κB pathway via interaction with the IL-18 receptor. The receptor itself is formed from a dimer of two subunits, with the ligand-binding IL-18Rα subunit being encoded by the IL18R1 gene. A splice variant of murine IL18r1, which has been previously described, is formed by transcription of an unspliced intron (forming a ‘type II’ IL18r1 transcript and is predicted to encode a receptor with a truncated intracellular domain lacking the capacity to generate downstream signalling. In order to examine the relevance of this finding to human IL-18 function, we assessed the presence of a homologous transcript by reverse transcription-polymerase chain reaction (RT-PCR in the human and rat as another common laboratory animal. We present evidence for type II IL18R1 transcripts in both species. While the mouse and rat transcripts are predicted to encode a truncated receptor with a novel 5 amino acid C-terminal domain, the human sequence is predicted to encode a truncated protein with a novel 22 amino acid sequence bearing resemblance to the ‘Box 1’ motif of the Toll/interleukin-1 receptor (TIR domain, in a similar fashion to the inhibitory interleukin-1 receptor 2. Given that transcripts from these three species are all formed by inclusion of homologous unspliced intronic regions, an analysis of homologous introns across a wider array of 33 species with available IL18R1 gene records was performed, which suggests similar transcripts may encode truncated type II IL-18Rα subunits in other species. This splice variant may represent a conserved evolutionary mechanism for regulating IL-18 activity.

  20. Cellulase variants with improved expression, activity and stability, and use thereof

    Science.gov (United States)

    Aehle, Wolfgang; Bott, Richard R; Bower, Benjamin; Caspi, Jonathan; Estell, David A; Goedegebuur, Frits; Hommes, Ronaldus W.J.; Kaper, Thijs; Kelemen, Bradley; Kralj, Slavko; Van Lieshout, Johan; Nikolaev, Igor; Van Stigt Thans, Sander; Wallace, Louise; Vogtentanz, Gudrun; Sandgren, Mats

    2014-03-25

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

  1. Cellulase variants with improved expression, activity and stability, and use thereof

    Energy Technology Data Exchange (ETDEWEB)

    Aehle, Wolfgang; Bott, Richard R.; Bower, Benjamin S.; Caspi, Jonathan; Goedegebuur, Frits; Hommes, Ronaldus Wilhelmus Joannes; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Van Lieshout, Johannes Franciscus Thomas; Nikolaev, Igor; Wallace, Louise; Van Stigt Thans, Sander; Vogtentanz, Gudrun; Sandgren, Mats

    2016-12-20

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

  2. Antitumorigenic potential of STAT3 alternative splicing modulation.

    Science.gov (United States)

    Zammarchi, Francesca; de Stanchina, Elisa; Bournazou, Eirini; Supakorndej, Teerawit; Martires, Kathryn; Riedel, Elyn; Corben, Adriana D; Bromberg, Jacqueline F; Cartegni, Luca

    2011-10-25

    Signal transducer and activator of transcription 3 (STAT3) plays a central role in the activation of multiple oncogenic pathways. Splicing variant STAT3β uses an alternative acceptor site within exon 23 that leads to a truncated isoform lacking the C-terminal transactivation domain. Depending on the context, STAT3β can act as a dominant-negative regulator of transcription and promote apoptosis. We show that modified antisense oligonucleotides targeted to a splicing enhancer that regulates STAT3 exon 23 alternative splicing specifically promote a shift of expression from STAT3α to STAT3β. Induction of endogenous STAT3β leads to apoptosis and cell-cycle arrest in cell lines with persistent STAT3 tyrosine phosphorylation compared with total STAT3 knockdown obtained by forced splicing-dependent nonsense-mediated decay (FSD-NMD). Comparison of the molecular effects of splicing redirection to STAT3 knockdown reveals a unique STAT3β signature, with a down-regulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1β) distinct from canonical STAT3 targets typically associated with total STAT3 knockdown. Furthermore, similar in vivo redirection of STAT3 alternative splicing leads to tumor regression in a xenograft cancer model, demonstrating how pharmacological manipulation of a single key splicing event can manifest powerful antitumorigenic properties and validating endogenous splicing reprogramming as an effective cancer therapeutic approach.

  3. Corneal epithelium expresses a variant of P2X(7 receptor in health and disease.

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    Courtney Mankus

    Full Text Available Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X(7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X(7 form (defined as the canonical receptor and its truncated forms. When Ca(2+ mobilization is induced by BzATP, a P2X(7 agonist, it is attenuated in the presence of extracellular Mg(2+ or Zn(2+, negligible in the absence of extracellular Ca(2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X(7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X(7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X(7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X(7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X(7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X(7, which ultimately allows P2X(7 to function as a multifaceted receptor that can mediate cell proliferation and

  4. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

    Science.gov (United States)

    Kwon, Young-Ju; Park, Mi-Jeong; Kim, Sang-Gyu; Baldwin, Ian T; Park, Chung-Mo

    2014-05-19

    The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5' splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress

  5. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

    Science.gov (United States)

    2014-01-01

    Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock

  6. The 0.3-kb fragment containing the R-U5-5'leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA.

    Science.gov (United States)

    Choo, Yeng Cheng; Seki, Yohei; Machinaga, Akihito; Ogita, Nobuo; Takase-Yoden, Sayaka

    2013-04-19

    A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5'leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood. Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5'splice site (5'ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5'ss. The 0.3-kb fragment containing the R-U5-5'leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing

  7. Expression analysis of an evolutionarily conserved alternative splicing factor, Sfrs10, in age-related macular degeneration.

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    Devi Krishna Priya Karunakaran

    Full Text Available Age-related macular degeneration (AMD is the most common cause of blindness in the elderly population. Hypoxic stress created in the micro-environment of the photoreceptors is thought to be the underlying cause that results in the pathophysiology of AMD. However, association of AMD with alternative splicing mediated gene regulation is not well explored. Alternative Splicing is one of the primary mechanisms in humans by which fewer protein coding genes are able to generate a vast proteome. Here, we investigated the expression of a known stress response gene and an alternative splicing factor called Serine-Arginine rich splicing factor 10 (Sfrs10. Sfrs10 is a member of the serine-arginine (SR rich protein family and is 100% identical at the amino acid level in most mammals. Immunoblot analysis on retinal extracts from mouse, rat, and chicken showed a single immunoreactive band. Further, immunohistochemistry on adult mouse, rat and chicken retinae showed pan-retinal expression. However, SFRS10 was not detected in normal human retina but was observed as distinct nuclear speckles in AMD retinae. This is in agreement with previous reports that show Sfrs10 to be a stress response gene, which is upregulated under hypoxia. The difference in the expression of Sfrs10 between humans and lower mammals and the upregulation of SFRS10 in AMD is further reflected in the divergence of the promoter sequence between these species. Finally, SFRS10+ speckles were independent of the SC35+ SR protein speckles or the HSF1+ stress granules. In all, our data suggests that SFRS10 is upregulated and forms distinct stress-induced speckles and might be involved in AS of stress response genes in AMD.

  8. Transforming growth factor-β1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

    International Nuclear Information System (INIS)

    Han Fei; Gilbert, James R.; Harrison, Gerald; Adams, Christopher S.; Freeman, Theresa; Tao Zhuliang; Zaka, Raihana; Liang Hongyan; Williams, Charlene; Tuan, Rocky S.; Norton, Pamela A.; Hickok, Noreen J.

    2007-01-01

    Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-β1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-β1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-β1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-β1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression

  9. Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β gene with differential expression patterns and regulatory functions.

    Directory of Open Access Journals (Sweden)

    Linjie Wang

    Full Text Available Glycogen synthase kinase 3 (GSK3α and GSK3β are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer's disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.

  10. 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.

  11. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    in the anion exchange, indicating that the protein still exists in different oligomer forms. This was also observed in the gel filtration. Variants of both hTPH1 and hTPH2 containing the regulatory domain or parts of it were constructed and tested for expression in Escherichia coli as well as solubility....... It was observed that changes in the amino acid sequence of the regulatory domain by point mutations or truncations in the N-terminal had a huge impact on the solubility of the protein and caused the protein to be insoluble. The regulatory domain of human TPH1 (rhTPH1), and two fusion proteins of rhTPH1 fused...... to the green fluorescent protein (GFP) in the C-terminal and the glutathione S-transferase (GST) in the N-terminal, respectively, were expressed in a soluble form. The purification trials of the variants containing the regulatory domain showed that a high salt concentration was necessary to stabilize...

  12. Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells

    Directory of Open Access Journals (Sweden)

    Carin K. Ingemarsdotter

    2017-06-01

    Full Text Available Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

  13. The neurogenetics of alternative splicing.

    Science.gov (United States)

    Vuong, Celine K; Black, Douglas L; Zheng, Sika

    2016-05-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

    Science.gov (United States)

    Baert, Annelot; Machackova, Eva; Coene, Ilse; Cremin, Carol; Turner, Kristin; Portigal-Todd, Cheryl; Asrat, Marie Jill; Nuk, Jennifer; Mindlin, Allison; Young, Sean; MacMillan, Andree; Van Maerken, Tom; Trbusek, Martin; McKinnon, Wendy; Wood, Marie E; Foulkes, William D; Santamariña, Marta; de la Hoya, Miguel; Foretova, Lenka; Poppe, Bruce; Vral, Anne; Rosseel, Toon; De Leeneer, Kim; Vega, Ana; Claes, Kathleen B M

    2018-04-01

    For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies. © 2017 Wiley Periodicals, Inc.

  16. Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner

    International Nuclear Information System (INIS)

    Rezania, S.; Kammerer, S.; Li, C.; Steinecker-Frohnwieser, B.; Gorischek, A.; DeVaney, T. T. J.; Verheyen, S.; Passegger, C. A.; Tabrizi-Wizsy, N. Ghaffari; Hackl, H.; Platzer, D.; Zarnani, A. H.; Malle, E.; Jahn, S. W.; Bauernhofer, T.; Schreibmayer, W.

    2016-01-01

    Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K + channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235–402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer. The online

  17. Expression and distribution of spastin and spliced variants in glioblastomas and human glioblastoma cell lines

    Czech Academy of Sciences Publication Activity Database

    Katsetos, C.; Dráberová, Eduarda; Liu, S.P.; Sládková, Vladimíra; Solowska, J.; De Chaderevian, J.P.; Legido, A.; Mork, S.; Baas, P.; Dráber, Pavel

    2010-01-01

    Roč. 69, č. 5 (2010), s. 536-537 ISSN 0022-3069. [Annual Meeting of the American-Association-of-Neuropathologists /86./. 10.06.10-13.06.10, Phidadelphia] Institutional research plan: CEZ:AV0Z50520514

  18. PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators

    International Nuclear Information System (INIS)

    Lamba, Vishal; Yasuda, Kazuto; Lamba, Jatinder K.; Assem, Mahfoud; Davila, Julio; Strom, Stephen; Schuetz, Erin G.

    2004-01-01

    To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues

  19. PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators.

    Science.gov (United States)

    Lamba, Vishal; Yasuda, Kazuto; Lamba, Jatinder K; Assem, Mahfoud; Davila, Julio; Strom, Stephen; Schuetz, Erin G

    2004-09-15

    To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues.

  20. A novel splice variant of the stem cell marker LGR5/GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients

    International Nuclear Information System (INIS)

    Rot, Swetlana; Taubert, Helge; Bache, Matthias; Greither, Thomas; Würl, Peter; Eckert, Alexander W; Schubert, Johannes; Vordermark, Dirk; Kappler, Matthias

    2011-01-01

    The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet. Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified. A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA. An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested

  1. Identification and characterization of novel ERC-55 interacting proteins: evidence for the existence of several ERC-55 splicing variants; including the cytosolic ERC-55-C.

    Science.gov (United States)

    Ludvigsen, Maja; Jacobsen, Christian; Maunsbach, Arvid B; Honoré, Bent

    2009-12-01

    ERC-55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC-55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC-55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC-55 splicing variants including ERC-55-C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub-cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin-6, kininogen and lysozyme with ERC-55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca(2+)] of approximately 10(-7) M or greater, while calcyclin interaction requires [Ca(2+)] of >10(-5) M. Interaction with peroxiredoxin-6 is independent of Ca(2+). Co-localization of lactoferrin, S100P and calcyclin with ERC-55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC-55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.

  2. Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

    Science.gov (United States)

    2012-09-01

    expression in prostate cancer (4). Lower or no ERG expression in a subset of aggressive tumors with TMPRSS2-ERG fusion may reflect attenuation of androgen...Services University of the Health Sciences, Bethesda MD 20814, USA 2. Cancer Vaccine Development Laboratory, Department of Surgery, United...negative for ERG. The ERG MAb did not show cross reactivity to FLI-1 in LNCaP cells infected with a FLI-1 adenovirus expression vector (Fig 1A

  3. Validation and Interrogation of Differentially Expressed and Alternately Spliced Genes in African American Prostate Cancer

    Science.gov (United States)

    2017-10-01

    receptor (AR) and epidermal growth factor receptor (EGFR) pathways in AA versus white prostate cancer . Thus, there is an urgent need to develop a novel...androgen receptor signaling and aggressive prostate cancer . AACR Conference on The Science of Cancer Health Disparities in Racial/Ethnic Minorities and...Freedman and S. R. Patierno. Race-related differential splicing of the insulin receptor : A novel target underlying prostate cancer disparities. AACR

  4. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

    International Nuclear Information System (INIS)

    Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

    2003-01-01

    The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

  5. Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein

    Science.gov (United States)

    Puizdar, Vida; Zajc, Tajana; Žerovnik, Eva; Renko, Miha; Pieper, Ursula; Eswar, Narayanan; Šali, Andrej; Dolenc, Iztok; Turk, Vito

    2014-01-01

    Cathepsin E splice variant 2 appears in a number of gastric carcinoma. Here, we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variant 1 and variant 2 of cathepsins E, with propeptide and without it, in Echerichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by Atomic Force Microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS), and used to rationalize its conformational properties and loss of activity. PMID:22718633

  6. Aberrant alternative splicing is another hallmark of cancer.

    Science.gov (United States)

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  7. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    OpenAIRE

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  8. Identification of new TSGA10 transcript variants in human testis with conserved regulatory RNA elements in 5'untranslated region and distinct expression in breast cancer.

    Science.gov (United States)

    Salehipour, Pouya; Nematzadeh, Mahsa; Mobasheri, Maryam Beigom; Afsharpad, Mandana; Mansouri, Kamran; Modarressi, Mohammad Hossein

    2017-09-01

    Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia. Copyright © 2017. Published by Elsevier B.V.

  9. IL-7 splicing variant IL-7δ5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

    International Nuclear Information System (INIS)

    Pan, Deshun; Liu, Bing; Jin, Xiaobao; Zhu, Jiayong

    2012-01-01

    Highlights: ► This study confirms the role of IL-7δ5 in breast cancer cell proliferation. ► IL-7δ5 promotes breast cancer cell proliferation and cell cycle progression. ► IL-7δ5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7δ5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7δ5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27 kip1 expression. Mechanistically, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7δ5. In conclusion, our findings demonstrate that IL-7δ5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target for human breast cancer therapeutics intervention.

  10. 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

    was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown...

  11. Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

    Science.gov (United States)

    Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

    2015-03-01

    Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

  12. Molecular characterization, expression profile of the FSHR gene and ...

    Indian Academy of Sciences (India)

    2016-10-24

    Oct 24, 2016 ... Muscovy duckswere lived under the same normal conditions of management and feeding ..... The authors declare that they have no conflict of interest. .... acid: functional expression of alternatively spliced variants and receptor.

  13. Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

    International Nuclear Information System (INIS)

    Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana; Rozieres, Sohela de; Elder, John H.

    2008-01-01

    Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication

  14. Clinical Expression and New SPINK5 Splicing Defects in Netherton Syndrome: Unmasking a Frequent Founder Synonymous Mutation and Unconventional Intronic Mutations

    DEFF Research Database (Denmark)

    Lacroix, Matthieu; Lacaze-Buzy, Laetitia; Furio, Laetitia

    2012-01-01

    a clinical triad suggestive of NS with variations in inter- and intra-familial disease expression. We identified a new and frequent synonymous mutation c.891C>T (p.Cys297Cys) in exon 11 of the 12 NS patients. This mutation disrupts an exonic splicing enhancer sequence and causes out-of-frame skipping of exon...

  15. Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome

    Directory of Open Access Journals (Sweden)

    Olatz Villate

    2018-01-01

    Full Text Available Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

  16. Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome.

    Science.gov (United States)

    Villate, Olatz; Ibarluzea, Nekane; Fraile-Bethencourt, Eugenia; Valenzuela, Alberto; Velasco, Eladio A; Grozeva, Detelina; Raymond, F L; Botella, María P; Tejada, María-Isabel

    2018-01-01

    Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD ® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

  17. Region-specific expression and hormonal regulation of the first exon variants of rat prolactin receptor mRNA in rat brain and anterior pituitary gland.

    Science.gov (United States)

    Nogami, H; Hoshino, R; Ogasawara, K; Miyamoto, S; Hisano, S

    2007-08-01

    Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

  18. Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals.

    Science.gov (United States)

    Zhang, Yong; Liu, Yun; Huang, Xigui; Liu, Xiaochun; Jiao, Baowei; Meng, Zining; Zhu, Pei; Li, Shuisheng; Lin, Haoran; Cheng, Christopher H K

    2008-12-01

    Two GPR39 transcripts, designated as sbGPR39-1a and sbGPR39-1b, were identified in black seabream (Acanthopagrus schlegeli). The deduced amino acid (aa) sequence of sbGPR39-1a contains 423 residues with seven putative transmembrane (TM) domains. On the other hand, sbGPR39-1b contains 284 aa residues with only five putative TM domains. Northern blot analysis confirmed the presence of two GPR39 transcripts in the seabream intestine, stomach, and liver. Apart from seabream, the presence of two GPR39 transcripts was also found to exist in a number of teleosts (zebrafish and pufferfish) and mammals (human and mouse). Analysis of the GPR39 gene structure in different species suggests that the two GPR39 transcripts are generated by alternative splicing. When the seabream receptors were expressed in cultured HEK293 cells, Zn(2)(+) could trigger sbGPR39-1a signaling through the serum response element pathway, but no such functionality could be detected for the sbGPR39-1b receptor. The two receptors were found to be differentially expressed in seabream tissues. sbGPR39-1a is predominantly expressed in the gastrointestinal tract. On the other hand, sbGPR39-1b is widely expressed in most central and peripheral tissues except muscle and ovary. The expression of sbGPR39-1a in the intestine and the expression of sbGPR39-1b in the hypothalamus were decreased significantly during food deprivation in seabream. On the contrary, the expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream. The reciprocal regulatory patterns of expression of these two genes suggest that both of them are involved in controlling the physiological response of the organism during starvation.

  19. Human Splicing Finder: an online bioinformatics tool to predict splicing signals

    OpenAIRE

    Desmet, Francois-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Beroud, Gwenaelle; Claustres, Mireille; Beroud, Christophe

    2009-01-01

    International audience; Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effec...

  20. Single molecule analysis of c-myb alternative splicing reveals novel classifiers for precursor B-ALL.

    Directory of Open Access Journals (Sweden)

    Ye E Zhou

    Full Text Available The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.

  1. 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...

  2. Allelic variation, alternative splicing and expression analysis of Psy1 gene in Hordeum chilense Roem. et Schult.

    Directory of Open Access Journals (Sweden)

    Cristina Rodríguez-Suárez

    Full Text Available BACKGROUND: The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1 is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. METHODOLOGY/PRINCIPAL FINDINGS: We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16 representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5' UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids, are functional in the bacterial system. CONCLUSIONS/SIGNIFICANCE: The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.

  3. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

    International Nuclear Information System (INIS)

    Grant, Nicola J.; Coates, Philip J.; Woods, Yvonne L.; Bray, Susan E.; Morrice, Nicholas A.; Hastie, C. James; Lamont, Douglas J.; Carey, Francis A.; Sutherland, Calum

    2015-01-01

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  4. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

    Science.gov (United States)

    Grant, Nicola J; Coates, Philip J; Woods, Yvonne L; Bray, Susan E; Morrice, Nicholas A; Hastie, C James; Lamont, Douglas J; Carey, Francis A; Sutherland, Calum

    2015-11-10

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  5. The connection between splicing and cancer

    OpenAIRE

    Srebrow, Anabella; Kornblihtt, Alberto Rodolfo

    2017-01-01

    Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cisacting splicing elements or changes in the activity of regulatory proteins that compromise the accuracy of either constitutive or alternativ...

  6. Association of breast cancer risk with genetic variants showing differential allelic expression

    DEFF Research Database (Denmark)

    Hamdi, Yosr; Soucy, Penny; Adoue, Véronique

    2016-01-01

    There are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are functional...

  7. Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility.

    Directory of Open Access Journals (Sweden)

    Zhihua Ju

    Full Text Available Neutrophil cytosolic factor 4 (NCF4 is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice variants and functional single-nucleotide polymorphism (SNP of NCF4 were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as NCF4-TV and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the NCF4-reference main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether NCF4-TV could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant NCF4 g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of NCF4 was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced NCF4-TV with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows. NCF4 expression was also regulated by alternative splicing. This study proposes that NCF4 splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows.

  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. Alternative splicing and expression of the insulin-like growth factor (IGF-1) gene in osteoblasts under mechanical stretch

    Institute of Scientific and Technical Information of China (English)

    XIAN Chengyu; WANG Yuanliang; ZHANG Bingbing; TANG Liling; PAN Jun; LUO Yanfeng; JIANG Peng; LI Dajun

    2006-01-01

    Insulin-like growth factor 1 (IGF-1) promotes osteoblasts differentiation and bone formation,and its expression is induced by mechanical stretch,thus IGF-1 has been considered an effector molecule that links mechanical stimulation and local tissue responses. In this study, a mechanical stretching device was designed to apply physiological level static or cyclic stretching stimulation to osteoblasts.Different isoforms of IGF-1 mRNA were amplified by RT-PCR from the cells using respective primers and these amplified products were sequenced. An isoform of IGF-1 splicing product was found to be selectively produced by osteoblasts under stretching stimulation. This IGF-1 isoform had identical sequence with the mechano growth factor (MGF) which was originally identified in muscle cells. Regulations of the expression of the liver-type IGF (L.IGF-1) and MGF in osteoblasts under stretch stimulation were further studied using semi-quantitative RT-PCR.Stretch stimulation was found to promot the expression of IGF-1 (L.IGF-1 and MGF), and for both isoforms expression was more effectively stimulated by cyclic stretch than static stretch. MGF was detected only in osteoblasts subjected to mechanical stretch,suggesting MGF was a stretch sensitive growth factor.Expression of MGF peaked earlier than that of L.IGF-1, which was similar to their regulation in muscie and suggested similar roles of MGF and L.IGF-1in bone as in muscle cells. The functions of MGF and L.IGF-1 in osteoblasts shall be established by further experimental studies.

  10. Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice

    International Nuclear Information System (INIS)

    Selden, R.F.; Wagner, T.E.; Blethen, S.; Yun, J.S.; Rowe, M.E.; Goodman, H.M.

    1988-01-01

    The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, the authors have expressed a mouse methallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties

  11. Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression.

    Science.gov (United States)

    Hesson, Luke B; Packham, Deborah; Kwok, Chau-To; Nunez, Andrea C; Ng, Benedict; Schmidt, Christa; Fields, Michael; Wong, Jason W H; Sloane, Mathew A; Ward, Robyn L

    2015-06-01

    Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5'untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5'UTR in the pathogenesis of Lynch syndrome. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  12. Tissue-specific alternative splicing and expression of ATP1B2 gene ...

    African Journals Online (AJOL)

    After heat-stress, the expression levels of the different transcripts were lower in different tissues; however, the expression of the ATP1B2-complete transcript increased in heart and lung tissues. The results of this research provide some useful information for further studies into the function of the bovine ATP1B2 gene.

  13. The Expression and Regulation of the Cell Adhesion Molecule CD44 in Human Breast Cancer

    National Research Council Canada - National Science Library

    Ge, Lisheng

    1997-01-01

    ... alternative splicing signals of CD44 variant exons as control elements in CEPT. In our colon cancer metastasis to liver model, we developed dual modulation vectors to increase tissue-specific expression of cytosine deaminase (CD...

  14. Spliced leader-based analyses reveal the effects of polycyclic aromatic hydrocarbons on gene expression in the copepod Pseudodiaptomus poplesia.

    Science.gov (United States)

    Zhuang, Yunyun; Yang, Feifei; Xu, Donghui; Chen, Hongju; Zhang, Huan; Liu, Guangxing

    2017-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are a group of toxic and carcinogenic pollutants that can adversely affect the development, growth and reproduction of marine organisms including copepods. However, knowledge on the molecular mechanisms regulating the response to PAH exposure in marine planktonic copepods is limited. In this study, we investigated the survival and gene expression of the calanoid copepod Pseudodiaptomus poplesia upon exposure to two PAHs, 1, 2-dimethylnaphthalene (1, 2-NAPH) and pyrene. Acute toxicity responses resulted in 96-h LC 50 of 788.98μgL -1 and 54.68μgL -1 for 1, 2-NAPH and pyrene, respectively. Using the recently discovered copepod spliced leader as a primer, we constructed full-length cDNA libraries from copepods exposed to sublethal concentrations and revealed 289 unique genes of diverse functions, including stress response genes and novel genes previously undocumented for this species. Eighty-three gene families were specifically expressed in PAH exposure libraries. We further analyzed the expression of seven target genes by reverse transcription-quantitative PCR in a time-course test with three sublethal concentrations. These target genes have primary roles in detoxification, oxidative defense, and signal transduction, and include different forms of glutathione S-transferase (GST), glutathione peroxidases (GPX), peroxiredoxin (PRDX), methylmalonate-semialdehyde dehydrogenase (MSDH) and ras-related C3 botulinum toxin substrate (RAC1). Expression stability of seven candidate reference genes were evaluated and the two most stable ones (RPL15 and RPS20 for 1, 2-NAPH exposure, RPL15 and EF1D for pyrene exposure) were used to normalize the expression levels of the target genes. Significant upregulation was detected in GST-T, GST-DE, GPX4, PRDX6 and RAC1 upon 1, 2-NAPH exposure, and GST-DE and MSDH upon pyrene exposure. These results indicated that the oxidative stress was induced and that signal transduction might be affected by PAH

  15. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

    Science.gov (United States)

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-01-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  16. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  17. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  18. Tissue-specific alternative splicing and expression of ATP1B2 gene

    African Journals Online (AJOL)

    user6

    2012-05-15

    May 15, 2012 ... 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 ...

  19. Enhanced hepatocarcinogenesis in mouse models and human hepatocellular carcinoma by coordinate KLF6 depletion and increased messenger RNA splicing

    NARCIS (Netherlands)

    Vetter, Diana; Cohen-Naftaly, Michal; Villanueva, Augusto; Lee, Youngmin A.; Kocabayoglu, Peri; Hannivoort, Rebekka; Narla, Goutham; M. Llovet, Josep; Thung, Swan N.; Friedman, Scott L.

    2012-01-01

    KLF6-SV1 (SV1), the major splice variant of KLF6, antagonizes the KLF6 tumor suppressor by an unknown mechanism. Decreased KLF6 expression in human hepatocellular carcinoma (HCC) correlates with increased mortality, but the contribution of increased SV1 is unknown. We sought to define the impact of

  20. 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

  1. AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

    KAUST Repository

    Zhang, Runxuan

    2016-05-06

    Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

  2. Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations

    International Nuclear Information System (INIS)

    Kalari, Krishna R.; Rossell, David; Necela, Brian M.; Asmann, Yan W.; Nair, Asha

    2012-01-01

    KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

  3. Pre- and Post-Conditions Expressed in Variants of the Modal µ-Calculus

    Science.gov (United States)

    Tanabe, Yoshinori; Sekizawa, Toshifusa; Yuasa, Yoshifumi; Takahashi, Koichi

    Properties of Kripke structures can be expressed by formulas of the modal µ-calculus. Despite its strong expressive power, the validity problem of the modal µ-calculus is decidable, and so are some of its variants enriched by inverse programs, graded modalities, and nominals. In this study, we show that the pre- and post-conditions of transformations of Kripke structures, such as addition/deletion of states and edges, can be expressed using variants of the modal µ-calculus. Combined with decision procedures we have developed for those variants, the properties of sequences of transformations on Kripke structures can be deduced. We show that these techniques can be used to verify the properties of pointer-manipulating programs.

  4. 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.

  5. Li-Fraumeni syndrome with simultaneous osteosarcoma and liver cancer: Increased expression of a CD44 variant isoform after chemotherapy

    International Nuclear Information System (INIS)

    Yoshida, Go J; Kuroda, Tatsuo; Fuchimoto, Yasushi; Osumi, Tomoo; Shimada, Hiroyuki; Hosaka, Seiichi; Morioka, Hideo; Mukai, Makio; Masugi, Yohei; Sakamoto, Michiie

    2012-01-01

    Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome that is commonly associated with a germline mutation in the tumor suppressor gene p53. Loss of p53 results in increased expression of CD44, a cancer stem cell (CSC) marker, which is involved in the scavenging of reactive oxygen species (ROS). Here, we report a change in the expression of a CD44 variant isoform (CD44v8-10) in an 8-year-old female LFS patient with osteosarcoma and atypical liver cancer after chemotherapy. The patient visited a clinic with a chief complaint of chronic pain in a bruise on her right knee. Magnetic resonance imaging (MRI) raised the possibility of a bone malignancy. Biochemical testing also revealed significantly elevated levels of AFP, which strongly suggested the existence of a primary malignancy in the liver. MRI imaging showed the simultaneous development of osteosarcoma and liver cancer, both of which were confirmed upon biopsy. Combined therapy with surgical resection after chemotherapy was successful in this patient. Regardless of the absence of a familial history of hereditary cancer, a germline mutation in p53 was identified (a missense mutation defined as c.722 C>T, p.Ser241Phe). To better understand the cancer progression and response to treatment, immunohistochemical (IHC) analysis of biopsy specimens obtained before and after chemotherapy was performed using a specific antibody against CD44v8-10. This case demonstrates the ectopic up-regulation of CD44v8-10 in a biopsy sample obtained after cytotoxic chemotherapy, which confers high levels of oxidative stress on cancer cells. Because the alternative splicing of CD44 is tightly regulated epigenetically, it is possible that micro-environmental stress resulting from chemotherapy caused the ectopic induction of CD44v8-10 in vivo

  6. Unique features of Myf-5 in turtles: nucleotide deletion, alternative splicing, and unusual expression pattern.

    Science.gov (United States)

    Ohya, Yoshie Kawashima; Usuda, Ryo; Kuraku, Shigehiro; Nagashima, Hiroshi; Kuratani, Shigeru

    2006-01-01

    Turtles characteristically possess a bony shell and show an extensive reduction of the trunk muscles. To gain insight into the evolution of this animal group, we focused on the underlying mechanism of the turtle-specific developmental pattern associated with the somitic mesoderm, which differentiates into both skeleton and muscle. We isolated Myf-5, a member of the myogenic-transcription-factor-encoding gene family expressed in the myotome, from the Chinese soft-shelled turtle Pelodiscus sinensis. We detected a deletion of 12 sequential nucleotides in P. sinensis Myf-5 (PsMyf-5), which appears to be shared by the turtle group. The expression pattern of PsMyf-5 in P. sinensis embryos differed from those of its orthologs in other amniotes, especially in the hypaxial region of the flank. We also identified two isoforms of the PsMyf-5 protein, a normal form similar to those of other vertebrates, and a short form produced by a translational frameshift. The short PsMyf-5 showed weaker myogenic activity in cultured cells than that of the normal protein, although the tissue distribution of the two isoforms overlapped perfectly. We propose that the unusual features of PsMyf-5 may be related to the unique developmental patterns of this animal group, and constitute one of the molecular bases for their evolutionary origin.

  7. Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

    Science.gov (United States)

    Martínez-Montiel, Nancy; Anaya-Ruiz, Maricruz; Pérez-Santos, Martín; Martínez-Contreras, Rebeca D

    2017-10-05

    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.

  8. Impact of gene variants on sex-specific regulation of human Scavenger receptor class B type 1 (SR-BI expression in liver and association with lipid levels in a population-based study

    Directory of Open Access Journals (Sweden)

    Barrett-Connor Elizabeth

    2010-01-01

    Full Text Available Abstract Background Several studies have noted that genetic variants of SCARB1, a lipoprotein receptor involved in reverse cholesterol transport, are associated with serum lipid levels in a sex-dependent fashion. However, the mechanism underlying this gene by sex interaction has not been explored. Methods We utilized both epidemiological and molecular methods to study how estrogen and gene variants interact to influence SCARB1 expression and lipid levels. Interaction between 35 SCARB1 haplotype-tagged polymorphisms and endogenous estradiol levels was assessed in 498 postmenopausal Caucasian women from the population-based Rancho Bernardo Study. We further examined associated variants with overall and SCARB1 splice variant (SR-BI and SR-BII expression in 91 human liver tissues using quantitative real-time PCR. Results Several variants on a haplotype block spanning intron 11 to intron 12 of SCARB1 showed significant gene by estradiol interaction affecting serum lipid levels, the strongest for rs838895 with HDL-cholesterol (p = 9.2 × 10-4 and triglycerides (p = 1.3 × 10-3 and the triglyceride:HDL cholesterol ratio (p = 2.7 × 10-4. These same variants were associated with expression of the SR-BI isoform in a sex-specific fashion, with the strongest association found among liver tissue from 52 young women Conclusions Estrogen and SCARB1 genotype may act synergistically to regulate expression of SCARB1 isoforms and impact serum levels of HDL cholesterol and triglycerides. This work highlights the importance of considering sex-dependent effects of gene variants on serum lipid levels.

  9. Cloning and expression of a novel human profilin variant, profilin II

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Andersen, A H

    1993-01-01

    We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kid...

  10. Establishment of Sf9 transformants constitutively expressing PBAN receptor variants: application to functional evaluation

    Science.gov (United States)

    To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines stably expressing a number of fluorescent Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants incl...

  11. Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers

    DEFF Research Database (Denmark)

    Rennig, Maja; Daley, Daniel O.; Nørholm, Morten H. H.

    2018-01-01

    Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can...

  12. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  13. An XPA gene splicing mutation resulting in trace protein expression in an elderly patient with xeroderma pigmentosum group A without neurological abnormalities.

    Science.gov (United States)

    Takahashi, Y; Endo, Y; Kusaka-Kikushima, A; Nakamaura, S; Nakazawa, Y; Ogi, T; Uryu, M; Tsuji, G; Furue, M; Moriwaki, S

    2017-07-01

    A certain relationship between XPA gene mutations and the severity of symptoms has been observed in patients with xeroderma pigmentosum group A (XP-A). Patients with mutations within the DNA-binding domain usually exhibit severe symptoms, whereas splicing mutations in the same domain sometimes cause very mild symptoms. This inconsistency can be explained by a small amount of functional XPA protein produced from normally spliced transcripts. We herein report the case of an adult Japanese patient with XP-A with unusually mild symptoms. We identified a homozygous c.529G>A mutation in exon 4 of the XPA gene, which resulted in aberrant splicing with a 29-bp deletion in exon 4 causing a frameshift. Intact mRNA was observable, but a Western blot analysis failed to detect any normal XPA protein. We therefore evaluated the DNA repair capacity in normal cells in which the XPA expression was artificially diminished. The repair capacity was still present in cells with trace levels of the XPA protein. The repair capacity of the cells derived from our patient with mild symptoms was poor by comparison, but still significant compared with that of the cells derived from a patient with XP-A with severe symptoms. These results provide strong evidence that a trace level of XPA protein can still exert a relatively strong repair capacity, resulting in only a mild phenotype. © 2016 British Association of Dermatologists.

  14. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

    Energy Technology Data Exchange (ETDEWEB)

    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T., E-mail: a.t.das@amc.uva.nl

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

  15. Evaluation of Parkinson disease risk variants as expression-QTLs.

    Directory of Open Access Journals (Sweden)

    Jeanne C Latourelle

    Full Text Available The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci SNCA, MAPT, GAK/DGKQ, and HLA and identified a novel risk locus at RIT2. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26 and controls (N = 24 by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for cis-acting effects on all probes within 250 kb of each locus. Trans-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of cis-acting SNP effects, several SNPs in the MAPT region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene LOC644246 and the duplicated genes LRRC37A and LRRC37A2, and a third uncorrelated probe targeting the gene DCAKD. Significant cis-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine trans effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8 × 10(-8 including SNPs from the SNCA, MAPT and RIT2 regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.

  16. Evaluation of Parkinson disease risk variants as expression-QTLs.

    Science.gov (United States)

    Latourelle, Jeanne C; Dumitriu, Alexandra; Hadzi, Tiffany C; Beach, Thomas G; Myers, Richard H

    2012-01-01

    The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci SNCA, MAPT, GAK/DGKQ, and HLA and identified a novel risk locus at RIT2. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26) and controls (N = 24) by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for cis-acting effects on all probes within 250 kb of each locus. Trans-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of cis-acting SNP effects, several SNPs in the MAPT region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene LOC644246 and the duplicated genes LRRC37A and LRRC37A2, and a third uncorrelated probe targeting the gene DCAKD. Significant cis-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine trans effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8 × 10(-8)) including SNPs from the SNCA, MAPT and RIT2 regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.

  17. Over-expression of SR-cyclophilin, an interaction partner of nuclear pinin, releases SR family splicing factors from nuclear speckles

    International Nuclear Information System (INIS)

    Lin, C.-L.; Leu, Steve; Lu, M.-C.; Ouyang Pin

    2004-01-01

    Pre-mRNA splicing takes place within a dynamic ribonucleoprotein particle called the spliceosome and occurs in an ordered pathway. Although it is known that spliceosome consists of five small nuclear RNAs and at least 50 proteins, little is known about how the interaction among the proteins changes during splicing. Here we identify that SR-cyp, a Moca family of nuclear cyclophilin, interacts and colocalizes with nuclear pinin (pnn), a SR-related protein involving in pre-mRNA splicing. Nuclear pnn interacts with SR-cyp via its C-terminal RS domain. Upon SR-cyp over-expression, however, the subnuclear distribution of nuclear pnn is altered, resulting in its redistribution from nuclear speckles to a diffuse nucleoplasmic form. The diffuse subnuclear distribution of nuclear pnn is not due to epitope masking, accelerated protein turnover or post-translational modification. Furthermore, we find that SR-cyp regulates the subnuclear distribution of other SR family proteins, including SC35 and SRm300, in a similar manner as it does on nuclear pnn. This result is significant because it suggests that SR-cyp plays a general role in modulating the distribution pattern of SR-like and SR proteins, similar to that of Clk (cdc2-like kinase)/STY on SR family splicing factors. SR-cyp might direct its effect via either alteration of protein folding/conformation or of protein-protein interaction and thus may add another control level of regulation of SR family proteins and modification of their functions

  18. Temporal expression profiling identifies pathways mediating effect of causal variant on phenotype.

    Directory of Open Access Journals (Sweden)

    Saumya Gupta

    2015-06-01

    Full Text Available Even with identification of multiple causal genetic variants for common human diseases, understanding the molecular processes mediating the causal variants' effect on the disease remains a challenge. This understanding is crucial for the development of therapeutic strategies to prevent and treat disease. While static profiling of gene expression is primarily used to get insights into the biological bases of diseases, it makes differentiating the causative from the correlative effects difficult, as the dynamics of the underlying biological processes are not monitored. Using yeast as a model, we studied genome-wide gene expression dynamics in the presence of a causal variant as the sole genetic determinant, and performed allele-specific functional validation to delineate the causal effects of the genetic variant on the phenotype. Here, we characterized the precise genetic effects of a functional MKT1 allelic variant in sporulation efficiency variation. A mathematical model describing meiotic landmark events and conditional activation of MKT1 expression during sporulation specified an early meiotic role of this variant. By analyzing the early meiotic genome-wide transcriptional response, we demonstrate an MKT1-dependent role of novel modulators, namely, RTG1/3, regulators of mitochondrial retrograde signaling, and DAL82, regulator of nitrogen starvation, in additively effecting sporulation efficiency. In the presence of functional MKT1 allele, better respiration during early sporulation was observed, which was dependent on the mitochondrial retrograde regulator, RTG3. Furthermore, our approach showed that MKT1 contributes to sporulation independent of Puf3, an RNA-binding protein that steady-state transcription profiling studies have suggested to mediate MKT1-pleiotropic effects during mitotic growth. These results uncover interesting regulatory links between meiosis and mitochondrial retrograde signaling. In this study, we highlight the advantage

  19. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...... constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli...

  20. 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.

  1. Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression.

    Science.gov (United States)

    Fairfax, Benjamin P; Humburg, Peter; Makino, Seiko; Naranbhai, Vivek; Wong, Daniel; Lau, Evelyn; Jostins, Luke; Plant, Katharine; Andrews, Robert; McGee, Chris; Knight, Julian C

    2014-03-07

    To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor-modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants.

  2. HOLLYWOOD: a comparative relational database of alternative splicing.

    Science.gov (United States)

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  3. 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.

  4. 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.

  5. Identification of new alternative splice events in the TCIRG1 gene in different human tissues

    International Nuclear Information System (INIS)

    Smirnova, Anna S.; Morgun, Andrey; Shulzhenko, Natalia; Silva, Ismael D.C.G.; Gerbase-DeLima, Maria

    2005-01-01

    Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption

  6. Sequence, 'subtle' alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors

    International Nuclear Information System (INIS)

    Vitale, Lorenza; Coppola, Domenico; Strippoli, Pierluigi; Frabetti, Flavia; Huntsman, Shane A; Canaider, Silvia; Casadei, Raffaella; Lenzi, Luca; Facchin, Federica; Carinci, Paolo; Zannotti, Maria

    2007-01-01

    CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG - isoform, the first described mRNA for CYYR1 locus) or the presence (CAG + isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG - or CAG + isoform expression compared to control tissues. CYYR1 CAG - isoform was significantly more expressed than CAG + isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. A new 'subtle' splicing isoform (CAG + ) of CYYR1 mRNA, the sequence and

  7. Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

    Science.gov (United States)

    Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

    2016-11-01

    The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Primate-specific spliced PMCHL RNAs are non-protein coding in human and macaque tissues

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    Delerue-Audegond Audrey

    2008-12-01

    Full Text Available Abstract Background Brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in Homo sapiens. PMCHL1 arose from retroposition of a pro-melanin-concentrating hormone (PMCH antisense mRNA on the ancestral human chromosome 5p14 when platyrrhines and catarrhines diverged. Mutations before divergence of hylobatidae led to creation of new exons and finally PMCHL1 duplicated in an ancestor of hominids to generate PMCHL2 at the human chromosome 5q13. A complex pattern of spliced and unspliced PMCHL RNAs were found in human brain and testis. Results Several novel spliced PMCHL transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. Sequencing of PMCHL genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin (CDH12 gene, soon after platyrrhine/catarrhine divergence, i.e. 30–35 Mya, and was concomitant with the insertion of an AluSg element. Sequence analysis of the spliced PMCHL transcripts identified only short ORFs of less than 300 bp, with low (VMCH-p8 and protein variants or no evolutionary conservation. Western blot analyses of human and macaque tissues expressing PMCHL RNA failed to reveal any protein corresponding to VMCH-p8 and protein variants encoded by spliced transcripts. Conclusion Our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric PMCHL genes. These genes produce multiple spliced transcripts, bearing short, non-conserved and apparently non-translated ORFs that may function as mRNA-like non-coding RNAs.

  9. 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.

  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

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    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. Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

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    Katrina Soderquest

    2017-02-01

    Full Text Available The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21 specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn's disease, ulcerative colitis (UC and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

  12. The group A streptococcal collagen-like protein 1, Scl1, mediates biofilm formation by targeting the EDA-containing variant of cellular fibronectin expressed in wounded tissue

    Science.gov (United States)

    Oliver-Kozup, Heaven; Martin, Karen H.; Schwegler-Berry, Diane; Green, Brett J.; Betts, Courtney; Shinde, Arti V.; Van De Water, Livingston; Lukomski, Slawomir

    2012-01-01

    Summary Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C′ loop region recognized by the α9β1 integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization. PMID:23217101

  13. Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

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    Marta A Ślimak

    2014-01-01

    Full Text Available The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3 and β4 nicotinic acetylcholine receptor (nAChR subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs with allele frequencies > 0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS, showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine

  14. Integrated analysis of genetic variation and gene expression reveals novel variant for increased warfarin dose requirement in African Americans

    NARCIS (Netherlands)

    Hernandez, W.; Gamazon, E. R.; Aquino-Michaels, K.; Smithberger, E.; O'Brien, T. J.; Harralson, A. F.; Tuck, M.; Barbour, A.; Cavallari, L. H.; Perera, M. A.

    2017-01-01

    Essentials Genetic variants controlling gene regulation have not been explored in pharmacogenomics. We tested liver expression quantitative trait loci for association with warfarin dose response. A novel predictor for increased warfarin dose response in African Americans was identified. Precision

  15. Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

    Science.gov (United States)

    Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

    2011-01-01

    Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

  16. CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants

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    Roseli da Silva

    2013-01-01

    Full Text Available OBJECTIVES: We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor. METHODS: Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1 mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information. RESULTS: CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1 were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants. CONCLUSIONS: A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.

  17. Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity

    OpenAIRE

    Larrimore, Katherine E.; Kazan, I. Can; Kannan, Latha; Kendle, R. Player; Jamal, Tameem; Barcus, Matthew; Bolia, Ashini; Brimijoin, Stephen; Zhan, Chang-Guo; Ozkan, S. Banu; Mor, Tsafrir S.

    2017-01-01

    Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme?s ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in p...

  18. Single nucleotide polymorphisms at erythropoietin, superoxide dismutase 1, splicing factor, arginine/serin-rich 15 and plasmacytoma variant translocation genes association with diabetic nephropathy

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    Maisaa Alwohhaib

    2014-01-01

    Full Text Available A number of genes have been identified in diabetic nephropathy. Association between diabetes-associated nephropathy and polymorphisms in the erythropoietin (EPO gene, variants in the superoxide dismutase 1 (SOD1 gene and plasmacytoma variant translocation 1 (PVT1 gene have been identified. The EPO, SOD1:SFRS15 and PVT1 genes were genotyped using the single nucleotide polymorphism (SNP technique in 38 diabetic nephropathy patients (Group 1 compared with 64 diabetic type 2 subjects without nephropathy (Group 2 at the Mubarak Alkabeer Hospital, Kuwait. The frequency of the risk allele T of the EPO (rs1617640 gene was high in both groups (0.96 in Group 1 and 0.92 in Group 2. Similarly, SNPs of the PVT1 (rs2720709 gene showed a higher frequency of the risk allele G in both groups (0.70 in the Group 1 and 0.68 in Group 2. Although the frequency of the risk allele A was higher than the frequency of the non-risk allele C of the SOD1:SFRS15 gene in both groups, the lowest probability value was observed in those gene SNPs (P = 0.05. We observed that the A allele of the SOD1:SFRS15 gene (rs17880135 was more frequently present in Group 1 (0.75 compared with Group 2 (0.62. Susceptibility to diabetes-associated nephropathy is partially mediated by genetic predisposition, and screening tests may open the gate for new therapeutic approaches.

  19. Calling genotypes from public RNA-sequencing data enables identification of genetic variants that affect gene-expression levels

    NARCIS (Netherlands)

    Deelen, Patrick; Zhernakova, Daria V.; de Haan, Mark; van der Sijde, Marijke; Bonder, Marc Jan; Karjalainen, Juha; van der Velde, K. Joeri; Abbott, Kristin M.; Fu, Jingyuan; Wijmenga, Cisca; Sinke, Richard J.; Swertz, Morris A.; Franke, Lude

    2015-01-01

    Background: RNA-sequencing (RNA-seq) is a powerful technique for the identification of genetic variants that affect gene-expression levels, either through expression quantitative trait locus (eQTL) mapping or through allele-specific expression (ASE) analysis. Given increasing numbers of RNA-seq

  20. Alternative Splicing in Neurogenesis and Brain Development.

    Science.gov (United States)

    Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

    2018-01-01

    Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  1. Alternative Splicing in Neurogenesis and Brain Development

    Directory of Open Access Journals (Sweden)

    Chun-Hao Su

    2018-02-01

    Full Text Available Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  2. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

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    Penny David

    2007-10-01

    Full Text Available Abstract Background Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, 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, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. Conclusion Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

  3. NF-κB and androgen receptor variant expression correlate with human BPH progression.

    Science.gov (United States)

    Austin, David C; Strand, Douglas W; Love, Harold L; Franco, Omar E; Jang, Alex; Grabowska, Magdalena M; Miller, Nicole L; Hameed, Omar; Clark, Peter E; Fowke, Jay H; Matusik, Robert J; Jin, Ren J; Hayward, Simon W

    2016-04-01

    Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.

  4. Serine/arginine rich splicing factor 2 expression and clinic pathological features indicating a prognostic factor in human hepatocellular carcinoma patients.

    Science.gov (United States)

    Wang, Pingan; Guo, Lingyu; Li, Kaipeng; Ning, Shanglei; Shi, Weichen; Liu, Zhaochen; Chen, Yuxin

    2018-02-14

    This research was aimed to study the expression of Serine/arginine rich splicing factor 2 (SRSF2) in tissues of hepatocellular carcinoma, and explore the relationship between the expression and the clinic pathological and prognosis of human hepatocellular carcinoma (HCC). One hundred and fifty-three pairs HCC tissues and adjacent normal tissue were collected from January 2010 to March 2013. The expression of SRSF2 gene was detected by immunohistochemistry, western blotting and real-time quantitative polymerase chain reaction (PCR), and the relationship between the expression and the clinic pathological and prognosis of HCC being analyzed. In 153 cases of hepatocellular carcinoma, SRSF2 was highly expressed in 93 cases, low expression of 60 cases, immunohistochemistry score (6.50 ± 2.82), which was significantly higher than that in adjacent normal tissues (2.94 ± 1.23) (Phepatocellular carcinoma was positively correlated (r = 0.704, Phepatocellular carcinoma were 74.19%, 44.09%, 26.88%, 24.73% and 21.51% at 1 year, 2 years, 3 years, 4 years and 5 years respectively, which were lower than those of SRSF2 low expression group (93.33%, 71.67%, 56.67%, 51.67% and 50.00%). SRSF2 is highly expressed in hepatocellular carcinoma and its expression increases with the degree of tumor differentiation and TNM staging. It is related to lymph node metastasis and metastasis of tumor cells, and is positively related to serum alpha fetoprotein content, and affects the postoperative survival time of HCC patients.

  5. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

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    Lee, Seung Heon; Kang, Sukmo [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of); Dong, Mi Sook [School of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Park, Jung-Duck [College of Medicine, Chung-Ang University, Seoul (Korea, Republic of); Park, Jinseo; Rhee, Sangkee [College of Agriculture of Life Science, Seoul National University, Seoul (Korea, Republic of); Ryu, Doug-Young, E-mail: dyryu@snu.ac.kr [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of)

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.

  6. Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

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    Petter Michaela

    2008-07-01

    Full Text Available Abstract Background Plasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation. Methods Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process. Results Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown. Conclusion The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with

  7. Genome-wide association between DNA methylation and alternative splicing in an invertebrate

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    Flores Kevin

    2012-09-01

    Full Text Available Abstract Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee and Nasonia vitripennis (jewel wasp analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice

  8. 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.

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    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.

  9. Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy.

    Science.gov (United States)

    Rafael, Julianny Freitas; Cruz, Fernando Eugênio Dos Santos; Carvalho, Antônio Carlos Campos de; Gottlieb, Ilan; Cazelli, José Guilherme; Siciliano, Ana Paula; Dias, Glauber Monteiro

    2017-04-01

    Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease. This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression. A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed. The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H) in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM. We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband. Resumo A cardiomiopatia hipertrófica (CMH) é uma doença autossômica dominante causada por mutações em genes que codificam as proteínas dos sarcômeros. É a principal causa de morte súbita cardíaca em atletas jovens de alto nível. Estudos têm demonstrado um pior prognóstico associado a mutações específicas. A associação entre genótipo e fenótipo em CMH tem sido objeto de diversos estudos desde a descoberta da origem genética dessa doença. Este trabalho apresenta o efeito de uma mutação composta em MYBPC3 na expressão fenotípica da CMH. Uma família na qual um jovem tem o diagnóstico clínico de CMH foi

  10. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

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    Pohl Martina

    2010-10-01

    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  11. Alternative Splicing Control of Abiotic Stress Responses.

    Science.gov (United States)

    Laloum, Tom; Martín, Guiomar; Duque, Paula

    2018-02-01

    Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai.

    Science.gov (United States)

    Kim, Mi Ae; Rhee, Jae-Sung; Kim, Tae Ha; Lee, Jung Sick; Choi, Ah-Young; Choi, Beom-Soon; Choi, Ik-Young; Sohn, Young Chang

    2017-03-09

    In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA) libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

  13. Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai

    Directory of Open Access Journals (Sweden)

    Mi Ae Kim

    2017-03-01

    Full Text Available In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

  14. TransAtlasDB: an integrated database connecting expression data, metadata and variants

    Science.gov (United States)

    Adetunji, Modupeore O; Lamont, Susan J; Schmidt, Carl J

    2018-01-01

    Abstract High-throughput transcriptome sequencing (RNAseq) is the universally applied method for target-free transcript identification and gene expression quantification, generating huge amounts of data. The constraint of accessing such data and interpreting results can be a major impediment in postulating suitable hypothesis, thus an innovative storage solution that addresses these limitations, such as hard disk storage requirements, efficiency and reproducibility are paramount. By offering a uniform data storage and retrieval mechanism, various data can be compared and easily investigated. We present a sophisticated system, TransAtlasDB, which incorporates a hybrid architecture of both relational and NoSQL databases for fast and efficient data storage, processing and querying of large datasets from transcript expression analysis with corresponding metadata, as well as gene-associated variants (such as SNPs) and their predicted gene effects. TransAtlasDB provides the data model of accurate storage of the large amount of data derived from RNAseq analysis and also methods of interacting with the database, either via the command-line data management workflows, written in Perl, with useful functionalities that simplifies the complexity of data storage and possibly manipulation of the massive amounts of data generated from RNAseq analysis or through the web interface. The database application is currently modeled to handle analyses data from agricultural species, and will be expanded to include more species groups. Overall TransAtlasDB aims to serve as an accessible repository for the large complex results data files derived from RNAseq gene expression profiling and variant analysis. Database URL: https://modupeore.github.io/TransAtlasDB/ PMID:29688361

  15. Expression of human placental lactogen and variant growth hormone genes in placentas.

    Science.gov (United States)

    Martinez-Rodriguez, H G; Guerra-Rodriguez, N E; Iturbe-Cantu, M A; Martinez-Torres, A; Barrera-Saldaña, H A

    1997-01-01

    Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.

  16. Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line

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    Radosław Januchowski

    2014-01-01

    Full Text Available Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  17. SpliceSeq: a resource for analysis and visualization of RNA-Seq data on alternative splicing and its functional impacts.

    Science.gov (United States)

    Ryan, Michael C; Cleland, James; Kim, RyangGuk; Wong, Wing Chung; Weinstein, John N

    2012-09-15

    SpliceSeq is a resource for RNA-Seq data that provides a clear view of alternative splicing and identifies potential functional changes that result from splice variation. It displays intuitive visualizations and prioritized lists of results that highlight splicing events and their biological consequences. SpliceSeq unambiguously aligns reads to gene splice graphs, facilitating accurate analysis of large, complex transcript variants that cannot be adequately represented in other formats. SpliceSeq is freely available at http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview. The application is a Java program that can be launched via a browser or installed locally. Local installation requires MySQL and Bowtie. mryan@insilico.us.com Supplementary data are available at Bioinformatics online.

  18. Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer

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    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

  19. 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.

  20. Human Splicing Finder: an online bioinformatics tool to predict splicing signals.

    Science.gov (United States)

    Desmet, François-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Béroud, Gwenaëlle; Claustres, Mireille; Béroud, Christophe

    2009-05-01

    Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.

  1. CDKL5 variants

    Science.gov (United States)

    Kalscheuer, Vera M.; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A.; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E.S.; Cobb, Stuart R.

    2017-01-01

    Objective: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. Methods: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. Results: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. Conclusions: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain. PMID:29264392

  2. Depolarization-mediated regulation of alternative splicing

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    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.

  3. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  4. The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

    Science.gov (United States)

    Schuster-Gossler, K; Bilinski, P; Sado, T; Ferguson-Smith, A; Gossler, A

    1998-06-01

    We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

  5. Altered expression of estrogen receptor-α variant messenger RNAs between adjacent normal breast and breast tumor tissues

    International Nuclear Information System (INIS)

    Leygue, Etienne; Dotzlaw, Helmut; Watson, Peter H; Murphy, Leigh C

    2000-01-01

    Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-α messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-α (ERC4) messenger RNA and a lower exon 3 deleted ER-α variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-α varient (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-α variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast tumorigenesis. Estrogen receptor (ER)-α and ER-β are believed to mediate the action of estradiol in target tissues. Several ER-α and ER-β variant messenger RNAs have been identified in both normal and neoplastic human tissues. Most of these variants contain a deletion of one or more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that are encoded by these variant messenger RNAs would therefore be missing some functional domains of the WT receptors, and might interfere with WT-ER signaling pathways. The detection of ER-α variants in both normal and neoplastic human breast tissues raised the question of their possible role in breast tumorigenesis. We have previously reported an increased relative expression of exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α variant truncated of all sequences following the exon 2 of the WT ER-α (ERC4) messenger RNA in breast tumor samples versus independent normal breast tissues. In contrast, a decreased relative expression of exon 3 deleted ER

  6. Variation in alternative splicing across human tissues

    OpenAIRE

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most p...

  7. U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

    Science.gov (United States)

    Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

    2014-04-25

    The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

    2012-01-01

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  9. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

    2012-07-25

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  10. Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

    Directory of Open Access Journals (Sweden)

    Orth Angela

    2009-04-01

    Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

  11. Digital PCR (dPCR) analysis reveals that the homozygous c.315-48T>C variant in the FECH gene might cause erythropoietic protoporphyria (EPP).

    Science.gov (United States)

    Brancaleoni, Valentina; Granata, Francesca; Missineo, Pasquale; Fustinoni, Silvia; Graziadei, Giovanna; Di Pierro, Elena

    2018-06-13

    Alterations in the ferrochelatase gene (FECH) are the basis of the phenotypic expressions in erythropoietic protoporphyria. The phenotype is due to the presence of a mutation in the FECH gene associated in trans to the c.315-48 T > C variant in the intron 3. The latter is able to increase the physiological quota of alternative splicing events in the intron 3. Other two variants in the FECH gene (c.1-252A > G and c.68-23C > T) have been found to be associated to the intron 3 variant in some populations and together, they constitute a haplotype (ACT/GTC), but eventually, their role in the alternative splicing event has never been elucidated. The absolute number of the aberrantly spliced FECH mRNA molecules and the absolute expression of the FECH gene were evaluated by digital PCR technique in a comprehensive cohort. The number of splicing events that rose in the presence of the c.315-48 T > C variant, both in the heterozygous and homozygous condition was reported for the first time. Also, the percentage of the inserted FECH mRNA increased, even doubled in the T/C cases, compared to T/T cases. The constant presence of variants in the promoter and intron 2 did not influence or modulate the aberrant splicing. The results of FECH gene expression suggested that the homozygosity for the c.315-48 T > C variant could be considered pathological. Thus, this study identified the homozygotes for the c.315-48 T > C variant as pathological. By extension, when the samples were categorised according to the haplotypes, the GTC haplotype in homozygosis was pathological. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. 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.

  13. Expression of a LINE-1 endonuclease variant in gastric cancer: its association with clinicopathological parameters

    International Nuclear Information System (INIS)

    Wang, Gangshi; Wu, Benyan; Wang, Mengwei; Gao, Jie; Huang, Haili; Tian, Yu; Xue, Liyan; Wang, Weihua; You, Weidi; Lian, Hongwei; Duan, Xiaojian

    2013-01-01

    -specific PCR. BLASTP program analysis revealed that GCRG213p peptide shared 83.0% alignment with the C-terminal region of L1 endonuclease (L1-EN). GCRG213p sequence possesses the important residues that compose the conserved features of L1-EN. GCRG213p could be a variant of L1-EN, a functional member of L1-EN family. Overexpression of GCRG213p is common in both primary gastric cancer and lymph node metastasis. These findings provide evidence of somatic L1 expression in gastric cancer, and its potential consequences in the form of tumor

  14. Alternative Splicing of AMPA subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys following Chronic Ethanol Self-Administration

    Directory of Open Access Journals (Sweden)

    Glen eAcosta

    2012-01-01

    Full Text Available Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA subunit variant and kainate (GRIK subunit mRNA expression were studied in the orbitofrontal cortex (OFC, dorsolateral prefrontal cortex (DLPFC and anterior cingulate cortex (ACC of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted.

  15. 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.

  16. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Vogelsang, Matjaz; Comino, Aleksandra; Zupanec, Neja [Department for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Hudler, Petra [Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana (Slovenia); Komel, Radovan [Department for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana (Slovenia)

    2009-10-28

    Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

  17. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    Directory of Open Access Journals (Sweden)

    Hudler Petra

    2009-10-01

    Full Text Available Abstract Background Loss of DNA mismatch repair (MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Methods Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. Results The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Conclusion Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

  18. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    International Nuclear Information System (INIS)

    Vogelsang, Matjaz; Comino, Aleksandra; Zupanec, Neja; Hudler, Petra; Komel, Radovan

    2009-01-01

    Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene

  19. Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis

    Science.gov (United States)

    Srivastava, Jyoti; Premi, Sanjay; Kumar, Sudhir; Parwez, Iqbal; Ali, Sher

    2007-01-01

    Background Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. Results We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual

  20. Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells.

    Science.gov (United States)

    Zhou, Rui; Park, Juw Won; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Zavala, Kathryn; Sea, Jessica L; Lu, Zhi-Xiang; Xu, Jianzhong; Adams, John S; Xing, Yi; Hewison, Martin

    2017-01-25

    Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) 2 D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH) 2 D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH) 2 D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH) 2 D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH) 2 D-mediated induction of transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Expression pattern of the AHP gene family from Arabidopsis thaliana and organ specific alternative splicing in the AHP5 gene

    Czech Academy of Sciences Publication Activity Database

    Hradilová, Jana; Brzobohatý, Břetislav

    2007-01-01

    Roč. 51, č. 2 (2007), s. 257-267 ISSN 0006-3134 Grant - others:GA MŠk(CZ) LN00A081; GA AV ČR(CZ) IAA600040612 Program:LN; IA Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : Arabidopsis two component systems * gene expression analysis * real time RT-PCR Subject RIV: BO - Biophysics Impact factor: 1.259, year: 2007

  2. Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

    Science.gov (United States)

    2010-01-01

    Background Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts

  3. 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.

    Science.gov (United States)

    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

    2016-04-01

    Previously, we reported a rare X-linked disorder, Uruguay syndrome in a single family. The main features are pugilistic facies, skeletal deformities, and muscular hypertrophy despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death. An ≈19 Mb critical region on X chromosome was identified through identity-by-descent analysis of 3 affected males. Exome sequencing was conducted on one affected male to identify the disease-causing gene and variant. A splice site variant (c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants. 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. Targeted analysis showed that this splice site variant cosegregated with disease in the family. Western blot and immunohistochemical analysis of muscle from the proband showed a significant decrease in protein expression of FHL1A. Real-time polymerase chain reaction analysis of different isoforms of FHL1 demonstrated that the FHL1C is markedly increased. Mutations in the FHL1 gene have been reported in disorders with skeletal and cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of FHL1A and the abundance of FHL1C, which may contribute to the complex and severe phenotype. Mutation screening of the FHL1 gene should be considered for patients with uncharacterized myopathies and cardiomyopathies. © 2016 American Heart Association, Inc.

  4. A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction.

    Science.gov (United States)

    Nisar, Shaista P; Lordkipanidzé, Marie; Jones, Matthew L; Dawood, Ban; Murden, Sherina; Cunningham, Margaret R; Mumford, Andrew D; Wilde, Jonathan T; Watson, Steve P; Mundell, Stuart J; Lowe, Gillian C

    2014-05-05

    A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

  5. Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis

    DEFF Research Database (Denmark)

    Hinrichsen, Inga; Brieger, Angela; Trojan, Jörg

    2013-01-01

    Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results...

  6. Salt-Sensitive Hypertension and Cardiac Hypertrophy in Transgenic Mice Expressing a Corin Variant Identified in African Americans

    Science.gov (United States)

    Wang, Wei; Cui, Yujie; Shen, Jianzhong; Jiang, Jingjing; Chen, Shenghan; Peng, Jianhao; Wu, Qingyu

    2012-01-01

    African Americans represent a high risk population for salt-sensitive hypertension and heart disease but the underlying mechanism remains unclear. Corin is a cardiac protease that regulates blood pressure by activating natriuretic peptides. A corin gene variant (T555I/Q568P) was identified in African Americans with hypertension and cardiac hypertrophy. In this study, we test the hypothesis that the corin variant contributes to the hypertensive and cardiac hypertrophic phenotype in vivo. Transgenic mice were generated to express wild-type or T555I/Q568P variant corin in the heart under the control of α-myosin heavy chain promoter. The mice were crossed into a corin knockout background to create KO/TgWT and KO/TgV mice that expressed WT or variant corin, respectively, in the heart. Functional studies showed that KO/TgV mice had significantly higher levels of pro-atrial natriuretic peptide in the heart compared with that in control KO/TgWT mice, indicating that the corin variant was defective in processing natriuretic peptides in vivo. By radiotelemetry, corin KO/TgV mice were found to have hypertension that was sensitive to dietary salt loading. The mice also developed cardiac hypertrophy at 12–14 months of age when fed a normal salt diet or at a younger age when fed a high salt diet. The phenotype of salt-sensitive hypertension and cardiac hypertrophy in KO/TgV mice closely resembles the pathological findings in African Americans who carry the corin variant. The results indicate that corin defects may represent an important mechanism in salt-sensitive hypertension and cardiac hypertrophy in African Americans. PMID:22987923

  7. Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

    Science.gov (United States)

    Boroni, Mariana; Sammeth, Michael; Gava, Sandra Grossi; Jorge, Natasha Andressa Nogueira; Macedo, Andréa Mara; Machado, Carlos Renato; Mourão, Marina Moraes; Franco, Glória Regina

    2018-03-01

    Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.

  8. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-01-01

    Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125 I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125 I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen

  9. Mechanical rebar splicing

    Directory of Open Access Journals (Sweden)

    Milosavljević Branko

    2014-01-01

    Full Text Available Different mechanical rebar splicing systems are presented, and design situations where mechanical splicing has advantage over reinforcement splicing by overlapping and welding are defined in this paper. New international standards for testing and proof of systems for mechanical rebar splicing quality are considered. Mechanical splicing system for rebar and bolt connection, usable in steel and reinforced concrete structural elements connections, is presented in this paper. There are only few examples of mechanical rebar splicing in our country. The most significant one - the pylon and beam connection at Ada Bridge in Belgrade is presented in the paper. Intensive development of production and use of mechanical rebar splicing systems, research in this area, as well as the publication of international standards prescribing requirements for quality and procedures for proof of quality, represent very good base for development of the corresponding technical norms in Serbia. The legislation in this area would quicken proof of quality procedures, attest and approval issuing for individual products, leading to wider use of this system in all situations where it is in advantage over the classical reinforcement splicing.

  10. 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...

  11. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

    Directory of Open Access Journals (Sweden)

    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.

  12. Expression Profile of Three Splicing Factors in Pleural Cells Based on the Underlying Etiology and Its Clinical Values in Patients with Pleural Effusion

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    A-Lum Han

    2018-02-01

    Full Text Available Splicing factors (SFs are involved in oncogenesis or immune modulation, the common underlying processes giving rise to pleural effusion (PE. The expression profiles of three SFs (HNRNPA1, SRSF1, and SRSF3 and their clinical values have never been assessed in PE. The three SFs (in pellets of PE and conventional tumor markers were analyzed using PE samples in patients with PE (N = 336. The sum of higher–molecular weight (Mw forms of HNRNPA1 (Sum-HMws-HNRNPA1 and SRSF1 (Sum-HMws-SRSF1 and SRSF3 levels were upregulated in malignant PE (MPE compared to benign PE (BPE; they were highest in cytology-positive MPE, followed by tuberculous PE and parapneumonic PE. Meanwhile, the lowest-Mw HNRNPA1 (LMw-HNRNPA1 and SRSF1 (LMw-SRSF1 levels were not upregulated in MPE. Sum-HMws-HNRNPA1, Sum-HMws-SRSF1, and SRSF3, but neither LMw-HNRNPA1 nor LMw-SRSF1, showed positive correlations with cancer cell percentages in MPE. The detection accuracy for MPE was high in the order of carcinoembryonic antigen (CEA, 85%, Sum-HMws-HNRNPA1 (76%, Sum-HMws-SRSF1 (68%, SRSF3, cytokeratin-19 fragments (CYFRA 21-1, LMw-HNRNPA1, and LMw-SRSF1. Sum-HMws-HNRNPA1 detected more than half of the MPE cases that were undetected by cytology and CEA. Sum-HMws-HNRNPA1, but not other SFs or conventional tumor markers, showed an association with longer overall survival among patients with MPE receiving chemotherapy. Our results demonstrated different levels of the three SFs with their Mw-specific profiles depending on the etiology of PE. We suggest that Sum-HMws-HNRNPA1 is a supplementary diagnostic marker for MPE and a favorable prognostic indicator for patients with MPE receiving chemotherapy.

  13. Novel Variants in ZNF34 and Other Brain-Expressed Transcription Factors are Shared Among Early-Onset MDD Relatives

    Science.gov (United States)

    Subaran, Ryan L.; Odgerel, Zagaa; Swaminathan, Rajeswari; Glatt, Charles E.; Weissman, Myrna M.

    2018-01-01

    There are no known genetic variants with large effects on susceptibility to major depressive disorder (MDD). Although one proposed study approach is to increase sensitivity by increasing sample sizes, another is to focus on families with multiple affected individuals to identify genes with rare or novel variants with strong effects. Choosing the family-based approach, we performed whole-exome analysis on affected individuals (n = 12) across five MDD families, each with at least five affected individuals, early onset, and prepubertal diagnoses. We identified 67 genes where novel deleterious variants were shared among affected relatives. Gene ontology analysis shows that of these 67 genes, 18 encode transcriptional regulators, eight of which are expressed in the human brain, including four KRAB-A box-containing Zn2+ finger repressors. One of these, ZNF34, has been reported as being associated with bipolar disorder and as differentially expressed in bipolar disorder patients compared to healthy controls. We found a novel variant—encoding a non-conservative P17R substitution in the conserved repressor domain of ZNF34 protein—segregating completely with MDD in all available individuals in the family in which it was discovered. Further analysis showed a common ZNF34 coding indel segregating with MDD in a separate family, possibly indicating the presence of an unobserved, linked, rare variant in that particular family. Our results indicate that genes encoding transcription factors expressed in the brain might be an important group of MDD candidate genes and that rare variants in ZNF34 might contribute to susceptibility to MDD and perhaps other affective disorders. PMID:26823146

  14. A robust approach to identifying tissue-specific gene expression regulatory variants using personalized human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Je-Hyuk Lee

    2009-11-01

    Full Text Available Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.

  15. Genome-wide data-mining of candidate human splice translational efficiency polymorphisms (STEPs and an online database.

    Directory of Open Access Journals (Sweden)

    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/.

  16. Metabotropic glutamate receptor 1 splice variants mGluR1a and mGluR1b combine in mGluR1a/b dimers in vivo

    Czech Academy of Sciences Publication Activity Database

    Techlovská, Šárka; Chambers, Jayne Nicole; Dvořáková, Michaela; Petralia, R.S.; Wang, Y.X.; Hájková, Alena; Franková, Daniela; Prezeau, L.; Blahoš, Jaroslav

    2014-01-01

    Roč. 86, November (2014), s. 329-326 ISSN 0028-3908 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : Glutamate receptors * GPCR * alternative splicing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.106, year: 2014

  17. 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.

  18. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    International Nuclear Information System (INIS)

    Zacapala-Gómez, Ana Elvira; Del Moral-Hernández, Oscar; Villegas-Sepúlveda, Nicolás; Hidalgo-Miranda, Alfredo; Romero-Córdoba, Sandra Lorena

    2016-01-01

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  19. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    Energy Technology Data Exchange (ETDEWEB)

    Zacapala-Gómez, Ana Elvira, E-mail: zak_ana@yahoo.com.mx [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Del Moral-Hernández, Oscar, E-mail: odelmoralh@gmail.com [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Villegas-Sepúlveda, Nicolás, E-mail: nvillega@cinvestav.mx [Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México, D.F., México (Mexico); Hidalgo-Miranda, Alfredo, E-mail: ahidalgo@inmegen.gob.mx [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); Romero-Córdoba, Sandra Lorena, E-mail: sromero_cordoba@hotmail.com [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); and others

    2016-01-15

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  20. Alternative Splicing as a Target for Cancer Treatment.

    Science.gov (United States)

    Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

    2018-02-11

    Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

  1. Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning.

    Science.gov (United States)

    Wasser, Catherine R; Masiulis, Irene; Durakoglugil, Murat S; Lane-Donovan, Courtney; Xian, Xunde; Beffert, Uwe; Agarwala, Anandita; Hammer, Robert E; Herz, Joachim

    2014-11-25

    Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory. Copyright © 2014, American Association for the Advancement of Science.

  2. Dyslexia risk variant rs600753 is linked with dyslexia-specific differential allelic expression of DYX1C1

    Directory of Open Access Journals (Sweden)

    Bent Müller

    2018-02-01

    Full Text Available Abstract An increasing number of genetic variants involved in dyslexia development were discovered during the last years, yet little is known about the molecular functional mechanisms of these SNPs. In this study we investigated whether dyslexia candidate SNPs have a direct, disease-specific effect on local expression levels of the assumed target gene by using a differential allelic expression assay. In total, 12 SNPs previously associated with dyslexia and related phenotypes were suitable for analysis. Transcripts corresponding to four SNPs were sufficiently expressed in 28 cell lines originating from controls and a family affected by dyslexia. We observed a significant effect of rs600753 on expression levels of DYX1C1 in forward and reverse sequencing approaches. The expression level of the rs600753 risk allele was increased in the respective seven cell lines from members of the dyslexia family which might be due to a disturbed transcription factor binding sites. When considering our results in the context of neuroanatomical dyslexia-specific findings, we speculate that this mechanism may be part of the pathomechanisms underlying the dyslexia-specific brain phenotype. Our results suggest that allele-specific DYX1C1 expression levels depend on genetic variants of rs600753 and contribute to dyslexia. However, these results are preliminary and need replication.

  3. Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

    Science.gov (United States)

    Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

    2012-01-01

    Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDRschizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

  4. Local binary pattern variants-based adaptive texture features analysis for posed and nonposed facial expression recognition

    Science.gov (United States)

    Sultana, Maryam; Bhatti, Naeem; Javed, Sajid; Jung, Soon Ki

    2017-09-01

    Facial expression recognition (FER) is an important task for various computer vision applications. The task becomes challenging when it requires the detection and encoding of macro- and micropatterns of facial expressions. We present a two-stage texture feature extraction framework based on the local binary pattern (LBP) variants and evaluate its significance in recognizing posed and nonposed facial expressions. We focus on the parametric limitations of the LBP variants and investigate their effects for optimal FER. The size of the local neighborhood is an important parameter of the LBP technique for its extraction in images. To make the LBP adaptive, we exploit the granulometric information of the facial images to find the local neighborhood size for the extraction of center-symmetric LBP (CS-LBP) features. Our two-stage texture representations consist of an LBP variant and the adaptive CS-LBP features. Among the presented two-stage texture feature extractions, the binarized statistical image features and adaptive CS-LBP features were found showing high FER rates. Evaluation of the adaptive texture features shows competitive and higher performance than the nonadaptive features and other state-of-the-art approaches, respectively.

  5. Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

    International Nuclear Information System (INIS)

    Manzotti, G; Mariani, S A; Corradini, F; Bussolari, R; Cesi, V; Vergalli, J; Ferrari-Amorotti, G; Fragliasso, V; Soliera, A R; Cattelani, S; Raschellà, G; Holyoake, T L; Calabretta, B

    2012-01-01

    The c-Myb gene encodes the p75 c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p c-Mybex9b , which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of p c-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p c-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75 c-Myb , p c-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p c-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p c-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34 + cells, without affecting the levels of p75 c-Myb . Together, these studies indicate that expression of the low-abundance p c-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  6. Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2010-11-01

    Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

  7. Vitamin D and alternative splicing of RNA.

    Science.gov (United States)

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  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. Characterization of a rare variant (c.2635-2A>G) of the MSH2 gene in a family with Lynch syndrome.

    Science.gov (United States)

    Cariola, Filomena; Disciglio, Vittoria; Valentini, Anna M; Lotesoriere, Claudio; Fasano, Candida; Forte, Giovanna; Russo, Luciana; Di Carlo, Antonio; Guglielmi, Floranna; Manghisi, Andrea; Lolli, Ivan; Caruso, Maria L; Simone, Cristiano

    2018-04-01

    Lynch syndrome is caused by germline mutations in one of the mismatch repair genes ( MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2).

  11. A SImplified method for Segregation Analysis (SISA) to determine penetrance and expression of a genetic variant in a family.

    Science.gov (United States)

    Møller, Pål; Clark, Neal; Mæhle, Lovise

    2011-05-01

    A method for SImplified rapid Segregation Analysis (SISA) to assess penetrance and expression of genetic variants in pedigrees of any complexity is presented. For this purpose the probability for recombination between the variant and the gene is zero. An assumption is that the variant of undetermined significance (VUS) is introduced into the family once only. If so, all family members in between two members demonstrated to carry a VUS, are obligate carriers. Probabilities for cosegregation of disease and VUS by chance, penetrance, and expression, may be calculated. SISA return values do not include person identifiers and need no explicit informed consent. There will be no ethical complications in submitting SISA return values to central databases. Values for several families may be combined. Values for a family may be updated by the contributor. SISA is used to consider penetrance whenever sequencing demonstrates a VUS in the known cancer-predisposing genes. Any family structure at hand in a genetic clinic may be used. One may include an extended lineage in a family through demonstrating the same VUS in a distant relative, and thereby identifying all obligate carriers in between. Such extension is a way to escape the selection biases through expanding the families outside the clusters used to select the families. © 2011 Wiley-Liss, Inc.

  12. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

    Directory of Open Access Journals (Sweden)

    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  13. Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

    Science.gov (United States)

    Wu, Liang; Zhang, Xiaolong; Zhao, Zhikun; Wang, Ling; Li, Bo; Li, Guibo; Dean, Michael; Yu, Qichao; Wang, Yanhui; Lin, Xinxin; Rao, Weijian; Mei, Zhanlong; Li, Yang; Jiang, Runze; Yang, Huan; Li, Fuqiang; Xie, Guoyun; Xu, Liqin; Wu, Kui; Zhang, Jie; Chen, Jianghao; Wang, Ting; Kristiansen, Karsten; Zhang, Xiuqing; Li, Yingrui; Yang, Huanming; Wang, Jian; Hou, Yong; Xu, Xun

    2015-01-01

    Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

  14. Behavioral and Neural Manifestations of Reward Memory in Carriers of Low-Expressing versus High-Expressing Genetic Variants of the Dopamine D2 Receptor

    Science.gov (United States)

    Richter, Anni; Barman, Adriana; Wüstenberg, Torsten; Soch, Joram; Schanze, Denny; Deibele, Anna; Behnisch, Gusalija; Assmann, Anne; Klein, Marieke; Zenker, Martin; Seidenbecher, Constanze; Schott, Björn H.

    2017-01-01

    Dopamine is critically important in the neural manifestation of motivated behavior, and alterations in the human dopaminergic system have been implicated in the etiology of motivation-related psychiatric disorders, most prominently addiction. Patients with chronic addiction exhibit reduced dopamine D2 receptor (DRD2) availability in the striatum, and the DRD2 TaqIA (rs1800497) and C957T (rs6277) genetic polymorphisms have previously been linked to individual differences in striatal dopamine metabolism and clinical risk for alcohol and nicotine dependence. Here, we investigated the hypothesis that the variants of these polymorphisms would show increased reward-related memory formation, which has previously been shown to jointly engage the mesolimbic dopaminergic system and the hippocampus, as a potential intermediate phenotype for addiction memory. To this end, we performed functional magnetic resonance imaging (fMRI) in 62 young, healthy individuals genotyped for DRD2 TaqIA and C957T variants. Participants performed an incentive delay task, followed by a recognition memory task 24 h later. We observed effects of both genotypes on the overall recognition performance with carriers of low-expressing variants, namely TaqIA A1 carriers and C957T C homozygotes, showing better performance than the other genotype groups. In addition to the better memory performance, C957T C homozygotes also exhibited a response bias for cues predicting monetary reward. At the neural level, the C957T polymorphism was associated with a genotype-related modulation of right hippocampal and striatal fMRI responses predictive of subsequent recognition confidence for reward-predicting items. Our results indicate that genetic variations associated with DRD2 expression affect explicit memory, specifically for rewarded stimuli. We suggest that the relatively better memory for rewarded stimuli in carriers of low-expressing DRD2 variants may reflect an intermediate phenotype of addiction memory. PMID

  15. Behavioral and Neural Manifestations of Reward Memory in Carriers of Low-Expressing versus High-Expressing Genetic Variants of the Dopamine D2 Receptor

    Directory of Open Access Journals (Sweden)

    Anni Richter

    2017-05-01

    Full Text Available Dopamine is critically important in the neural manifestation of motivated behavior, and alterations in the human dopaminergic system have been implicated in the etiology of motivation-related psychiatric disorders, most prominently addiction. Patients with chronic addiction exhibit reduced dopamine D2 receptor (DRD2 availability in the striatum, and the DRD2 TaqIA (rs1800497 and C957T (rs6277 genetic polymorphisms have previously been linked to individual differences in striatal dopamine metabolism and clinical risk for alcohol and nicotine dependence. Here, we investigated the hypothesis that the variants of these polymorphisms would show increased reward-related memory formation, which has previously been shown to jointly engage the mesolimbic dopaminergic system and the hippocampus, as a potential intermediate phenotype for addiction memory. To this end, we performed functional magnetic resonance imaging (fMRI in 62 young, healthy individuals genotyped for DRD2 TaqIA and C957T variants. Participants performed an incentive delay task, followed by a recognition memory task 24 h later. We observed effects of both genotypes on the overall recognition performance with carriers of low-expressing variants, namely TaqIA A1 carriers and C957T C homozygotes, showing better performance than the other genotype groups. In addition to the better memory performance, C957T C homozygotes also exhibited a response bias for cues predicting monetary reward. At the neural level, the C957T polymorphism was associated with a genotype-related modulation of right hippocampal and striatal fMRI responses predictive of subsequent recognition confidence for reward-predicting items. Our results indicate that genetic variations associated with DRD2 expression affect explicit memory, specifically for rewarded stimuli. We suggest that the relatively better memory for rewarded stimuli in carriers of low-expressing DRD2 variants may reflect an intermediate phenotype of

  16. SPA: a probabilistic algorithm for spliced alignment.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non

  17. Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

    Science.gov (United States)

    Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. Transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese supero...

  18. Aberrant splicing in transgenes containing introns, exons, and V5 epitopes: lessons from developing an FSHD mouse model expressing a D4Z4 repeat with flanking genomic sequences.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD. This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV and strong viral control elements (CMV promoter, SV40 poly A to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.

  19. Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

    DEFF Research Database (Denmark)

    Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

    2006-01-01

    Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

  20. Association of breast cancer risk with genetic variants showing differential allelic expression: Identification of a novel breast cancer susceptibility locus at 4q21

    NARCIS (Netherlands)

    Y. Hamdi (Yosr); Soucy, P. (Penny); Adoue, V. (Véronique); K. Michailidou (Kyriaki); S. Canisius (Sander); Lemaçon, A. (Audrey); A. Droit (Arnaud); I.L. Andrulis (Irene); H. Anton-Culver (Hoda); Arndt, V. (Volker); Baynes, C. (Caroline); C. Blomqvist (Carl); N.V. Bogdanova (Natalia); S.E. Bojesen (Stig); M.K. Bolla (Manjeet K.); B. Bonnani (Bernardo); A.-L. Borresen-Dale (Anne-Lise); J.S. Brand (Judith S.); H. Brauch (Hiltrud); Brenner, H. (Hermann); A. Broeks (Annegien); B. Burwinkel (Barbara); J. Chang-Claude (Jenny); Couch, F.J. (Fergus J.); A. Cox (Angela); S.S. Cross (Simon); K. Czene (Kamila); H. Darabi (Hatef); J. Dennis (Joe); P. Devilee (Peter); T. Dörk (Thilo); I. dos Santos Silva (Isabel); M. Eriksson (Mats); P.A. Fasching (Peter); J.D. Figueroa (Jonine); H. Flyger (Henrik); M. García-Closas (Montserrat); Giles, G.G. (Graham G.); M.S. Goldberg (Mark); A. González-Neira (Anna); G. Grenaker Alnæs (Grethe); P. Guénel (Pascal); L. Haeberle (Lothar); C.A. Haiman (Christopher); U. Hamann (Ute); Hallberg, E. (Emily); M.J. Hooning (Maartje); J.L. Hopper (John); A. Jakubowska (Anna); M. Jones (Michael); M. Kabisch (Maria); V. Kataja (Vesa); Lambrechts, D. (Diether); L. Le Marchand (Loic); A. Lindblom (Annika); J. Lubinski (Jan); A. Mannermaa (Arto); M. Maranian (Melanie); S. Margolin (Sara); Marme, F. (Frederik); R.L. Milne (Roger); S.L. Neuhausen (Susan); H. Nevanlinna (Heli); P. Neven (Patrick); C. Olswold (Curtis); J. Peto (Julian); Plaseska-Karanfilska, D. (Dijana); K. Pykäs (Katri); P. Radice (Paolo); A. Rudolph (Anja); E.J. Sawyer (Elinor); M.K. Schmidt (Marjanka); X.-O. Shu (Xiao-Ou); M.C. Southey (Melissa); A.J. Swerdlow (Anthony ); R.A.E.M. Tollenaar (Rob); I.P. Tomlinson (Ian); D. Torres (Diana); T. Truong (Thérèse); C. Vachon (Celine); A.M.W. van den Ouweland (Ans); Q. Wang (Qin); R. Winqvist (Robert); W. Zheng (Wei); J. Benítez (Javier); G. Chenevix-Trench (Georgia); A.M. Dunning (Alison); P.D.P. Pharoah (Paul); Kristensen, V. (Vessela); P. Hall (Per); D.F. Easton (Douglas); T. Pastinen (Tomi); S. Nord (Silje); J. Simard (Jacques)

    2016-01-01

    textabstractThere are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are

  1. The function and developmental expression of alternatively spliced isoforms of amphioxus and Xenopus laevis Pax2/5/8 genes: revealing divergence at the invertebrate to vertebrate transition

    Czech Academy of Sciences Publication Activity Database

    Short, S.; Kozmik, Zbyněk; Holland, L. Z.

    2012-01-01

    Roč. 318, č. 7 (2012), s. 555-571 ISSN 1552-5007 R&D Projects: GA ČR GAP305/10/2141; GA MŠk LH12047 Grant - others:NSF(US) MCB 06-20019 Institutional research plan: CEZ:AV0Z50520514 Keywords : Pax2/5/8 * alternative splicing * eye development * amphioxus * Xenopus laevis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.123, year: 2012

  2. Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase.

    Science.gov (United States)

    Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

    2009-04-01

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

  3. Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

    Science.gov (United States)

    Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

    2009-01-01

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

  4. Reduced Penetrance and Variable Expression of SCN5A Mutations and the Importance of Co-inherited Genetic Variants: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    T. Robyns, MD.

    2014-05-01

    Full Text Available Mutations in the SCN5A gene are responsible for multiple phenotypical presentations including Brugada syndrome, long QT syndrome, progressive familial heart block, sick sinus syndrome, dilated cardiomyopathy, lone atrial fibrillation and multiple overlap syndromes. These different phenotypic expressions of a mutation in a single gene can be explained by variable expression and reduced penetrance. One of the possible explanations of these phenomena is the co-inheritance of genetic variants. We describe a family where the individuals exhibit a compound heterozygosity in the SCN5A gene including a mutation (R1632H and a new variant (M858L. Individuals with both the mutation and new variant present with a more severe phenotype including spontaneous atrial tachyarrhythmia at young age. We give an overview of the different phenotypes of "SCN5A disease" and discuss the importance of co-inherited genetic variants in the expression of SCN5A disease.

  5. GCPII Variants, Paralogs and Orthologs

    Czech Academy of Sciences Publication Activity Database

    Hlouchová, Klára; Navrátil, Václav; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

    2012-01-01

    Roč. 19, č. 9 (2012), s. 1316-1322 ISSN 0929-8673 R&D Projects: GA ČR GAP304/12/0847 Institutional research plan: CEZ:AV0Z40550506 Keywords : PSMA * GCPIII * NAALADase L * splice variants * homologs * PSMAL Subject RIV: CE - Biochemistry Impact factor: 4.070, year: 2012

  6. 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

    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 of w...

  7. Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus.

    Science.gov (United States)

    Rivera-Casas, Ciro; González-Romero, Rodrigo; Vizoso-Vazquez, Ángel; Cheema, Manjinder S; Cerdán, M Esperanza; Méndez, Josefina; Ausió, Juan; Eirin-Lopez, Jose M

    2016-10-01

    Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.

  8. Differential expression of upstream stimulatory factor (USF 2 variants in eutopic endometria from women with endometriosis: estradiol regulation

    Directory of Open Access Journals (Sweden)

    Jazmin Castro

    2015-01-01

    Full Text Available BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1. Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b, a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control endometriosis and the involvement of nuclear estrogen receptors (ER and G-coupled protein estrogen receptor (GPER-1 RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b and late (USF2a and USF2b secretory phase in eutopic endometria from endometriosis than control (p < 0.05. In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the