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Sample records for splicing inhibition splicing-sensitive

  1. Microbial and Natural Metabolites That Inhibit Splicing: A Powerful Alternative for Cancer Treatment

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    Nancy Martínez-Montiel

    2016-01-01

    Full Text Available In eukaryotes, genes are frequently interrupted with noncoding sequences named introns. Alternative splicing is a nuclear mechanism by which these introns are removed and flanking coding regions named exons are joined together to generate a message that will be translated in the cytoplasm. This mechanism is catalyzed by a complex machinery known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression when assembled. It has been proposed that several genetic diseases are related to defects in the splicing process, including cancer. For this reason, natural products that show the ability to regulate splicing have attracted enormous attention due to its potential use for cancer treatment. Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanism responsible for this inhibition is being studied for future applications. Here, we summarize the main types of natural products that have been characterized as splicing inhibitors, the recent advances regarding molecular and cellular effects related to these molecules, and the applications reported so far in cancer therapeutics.

  2. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...... mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208-735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell...... extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors....

  3. Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

    Science.gov (United States)

    Waldsich, C; Semrad, K; Schroeder, R

    1998-12-01

    The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

  4. Baicalin hydrate inhibits cancer progression in nasopharyngeal carcinoma by affecting genome instability and splicing.

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    Lai, Weiwei; Jia, Jiantao; Yan, Bin; Jiang, Yiqun; Shi, Ying; Chen, Ling; Mao, Chao; Liu, Xiaoli; Tang, Haosheng; Gao, Menghui; Cao, Ya; Liu, Shuang; Tao, Yongguang

    2018-01-02

    Baicalin hydrate (BH), a natural compound, has been investigated for many years because of its traditional medicinal properties. However, the anti-tumor activities of BH and its epigenetic role in NPC have not been elucidated. In this study, we identified that BH inhibits NPC cell growth in vivo and in vitro by inducing apoptosis and cell cycle arrest. BH epigenetically regulated genome instability by up-regulating the expression of satellite 2 (Sat2), alpha satellite (α-Sat), and major satellite (Major-Sat). BH also increased the level of IKKα, Suv39H1, and H3K9me3 and decreased LSH expression. Interestingly, BH promoted the splicing of Suv39H1 via the enhancement of m6A RNA methylation, rather than DNA methylation. Taken together, our results demonstrated that BH has an anti-tumor role in NPC and revealed a unique role of BH in genome instability and splicing in response to DNA damage.

  5. WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

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    Wen Jiang

    Full Text Available Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.

  6. Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

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    Nadia Bakkour

    2007-10-01

    Full Text Available The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16 that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

  7. A five' splice-region G → C mutation in exon 1 of the human β-globin gene inhibits pre-mRNA splicing: A mechanism for β+-thalassemia

    International Nuclear Information System (INIS)

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M.; Gattoni, R.; Stevenin, J.; Chibani, J.

    1989-01-01

    The authors have characterized a Mediterranean β-thalassemia allele containing a sequence change at codon 30 that alters both β-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G → C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg → Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5

  8. Splice variants of the forkhead box protein AFX exhibit dominant negative activity and inhibit AFXalpha-mediated tumor cell apoptosis.

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    Eun Jig Lee

    2008-07-01

    Full Text Available Loss-of-function in the apoptosis-inducing genes is known to facilitate tumorigenesis. AFX (FOXO4, a member of forkhead transcription factors functions as a tumor suppressor and has 2 isoforms, AFXalpha (505 a.a. and AFXzeta (450 a.a.. In human cancer cells, we identified an N-terminally deleted form of AFXalpha (alpha198-505, translated from a downstream start and 2 short N-terminal AFX proteins (90, and 101 a.a. produced by aberrant splicing.We investigated the expression and role of these AFX variants. Cell transduction study revealed that short N-terminal AFX proteins were not stable. Though alpha(198-505 protein expression was detected in the cytoplasm and nucleus, alpha(198-505 expressing cells did not show a nucleocytoplasmic shuttling mediated by PI3 kinase signaling. Whereas, we observed this shuttling in cells expressing either AFXalpha or AFXzeta protein. AFXzeta and alpha(198-505 lost the ability to transactivate BCL6 or suppress cyclin D2 gene expression. These variants did not induce cancer cell death whereas AFXalpha resulted in apoptosis. We found that AFXzeta and alpha(198-505 suppress the AFXalpha stimulation of BCL6 promoter in a dose dependent manner, indicating dominant negative activity. These variants also inhibited AFXalpha induction of apoptosis.Loss of function by aberrant splicing and the dominant negative activity of AFX variants may provide a mechanism for enhanced survival of neoplastic cells.

  9. The soluble form of the EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection of target cells.

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    Lin, Yue-Zhi; Yang, Fei; Zhang, Shu-Qin; Sun, Liu-Ke; Wang, Xue-Feng; Du, Cheng; Zhou, Jian-Hua

    2013-01-01

    Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. Because ELR1-DE presumably encodes a peptide of a mere 23 residues, only ELR1-IN was further analyzed. The expression of a soluble form of ELR1 (sELR1) by ELR1-IN was confirmed by Western blot and immunofluorescence analyses. Similar to ELR1, the transcription level of ELR1-IN varied among individual horses and at different time points in the same individuals. The ratio of ELR1-IN mRNA species to ELR1 mRNA was approximately 1∶2.5. Pre-incubation of the recombinant sELR1 with EIAV significantly inhibited EIAV infection in equine macrophages, the primary in vivo target cell of the virus. Fetal equine dermal (FED) cells are susceptible to EIAV in vitro, and the replication of EIAV in FED cells transiently transfected with ELR1-IN was markedly reduced when compared with replication in cells transfected with the empty vector. Finally, the expression levels of both forms of the EIAV receptor were significantly regulated by infection with this virus. Taken together, our data indicate that sELR1 acts as a secreted cellular factor that inhibits EIAV infection in host cells.

  10. The soluble form of the EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection of target cells.

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    Yue-Zhi Lin

    Full Text Available Equine lentivirus receptor 1 (ELR1 has been identified as the sole receptor for equine infectious anemia virus (EIAV and is a member of the tumor necrosis factor receptor (TNFR superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs. One major spliced species (ELR1-IN contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. Because ELR1-DE presumably encodes a peptide of a mere 23 residues, only ELR1-IN was further analyzed. The expression of a soluble form of ELR1 (sELR1 by ELR1-IN was confirmed by Western blot and immunofluorescence analyses. Similar to ELR1, the transcription level of ELR1-IN varied among individual horses and at different time points in the same individuals. The ratio of ELR1-IN mRNA species to ELR1 mRNA was approximately 1∶2.5. Pre-incubation of the recombinant sELR1 with EIAV significantly inhibited EIAV infection in equine macrophages, the primary in vivo target cell of the virus. Fetal equine dermal (FED cells are susceptible to EIAV in vitro, and the replication of EIAV in FED cells transiently transfected with ELR1-IN was markedly reduced when compared with replication in cells transfected with the empty vector. Finally, the expression levels of both forms of the EIAV receptor were significantly regulated by infection with this virus. Taken together, our data indicate that sELR1 acts as a secreted cellular factor that inhibits EIAV infection in host cells.

  11. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells

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    Ge Changhui

    2010-04-01

    Full Text Available Abstract Background PCBP1 (or alpha CP1 or hnRNP E1, a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. Results We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. Conclusions We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

  12. Morpholino antisense oligo inhibits trans-splicing of pre-inositol 1,4,5-trisphosphate receptor mRNA of Trypanosoma cruzi and suppresses parasite growth and infectivity.

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    Hashimoto, Muneaki; Nara, Takeshi; Mita, Toshihiro; Mikoshiba, Katsuhiko

    2016-06-01

    Morpholino antisense oligos (MAOs) are used to investigate physiological gene function by inhibiting gene translation or construction of specific alternative splicing variants by blocking cis-splicing. MAOs are attractive drug candidates for viral- and bacterial-infectious disease therapy because of properties such as in vivo stability and specificity to target genes. Recently, we showed that phosphorothioate antisense oligos against Trypanosoma cruzi inositol 1,4,5-trisphosphate receptor (TcIP(3)R) mRNA inhibit the parasite host cell infection. In the present study, we identified the spliced leader (SL) acceptor of pre-TcIP(3)R mRNA and synthesized MAO, which inhibited trans-splicing of the transcript (MAO-1). MAO-1 was found to inhibit the addition of SL-RNA to pre-TcIP(3)R mRNA by real-time RT-PCR analysis. Treatment of the parasites with MAO-1 significantly impaired the growth and infectivity into host cells. These results indicate that MAO-1 is a potential novel drug for Chagas disease and that MAOs inhibiting trans-splicing can be used to investigate the physiology of trypanosomal genes leading to the development of novel drugs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

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    Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

    2009-10-06

    The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

  14. Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

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    Yuanyuan Xiao

    2005-09-01

    Full Text Available Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

  15. Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

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    Xiaoze Li

    Full Text Available The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16 genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

  16. Fine-scale variation and genetic determinants of alternative splicing across individuals.

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    Jasmin Coulombe-Huntington

    2009-12-01

    Full Text Available Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre-mRNA splicing (AS in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72% candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and

  17. Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.

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    Kasper Daniel Hansen

    2009-06-01

    Full Text Available Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA-binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD-affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD-target mRNAs had significantly longer 3' untranslated regions (UTRs than the nontarget isoforms of the same genes, supporting a role for 3' UTR length in the recognition of NMD targets in fly.

  18. Targeting the splicing of mRNA in autoimmune diseases: BAFF inhibition in Sjögren's syndrome as a proof of concept

    NARCIS (Netherlands)

    Roescher, N.; Vosters, J. L.; Alsaleh, G.; Dreyfus, P.; Jacques, S.; Chiocchia, G.; Sibilia, J.; Tak, P. P.; Chiorini, J. A.; Mariette, X.; Gottenberg, Jacques-Eric

    2014-01-01

    BAFF (B-cell-activating factor of the tumor necrosis factor family), a pivotal cytokine for B-cell activation, is overexpressed by salivary gland (SG) epithelial cells in primary Sjogren's syndrome (pSS). ΔBAFF, a physiological inhibitor of BAFF, is a minor alternative splice variant of BAFF. A U7

  19. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

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    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  20. tRNA splicing

    OpenAIRE

    Abelson, John; Trotta, Christopher R.; Li, Hong

    1998-01-01

    Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an iden...

  1. 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...

  2. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    Science.gov (United States)

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  3. Multiset splicing systems.

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    Dassow, Jürgen; Vaszil, György

    2004-01-01

    We consider splicing systems reflecting two important aspects of the behaviour of DNA molecules in nature or in laboratory experiments which so far have not been studied in the literature. We examine the effect of splicing rules applied to finite multisets of words using sequential and different types of parallel derivation strategies and compare the sets of words or sets of multisets which can be obtained.

  4. The neurogenetics of alternative splicing.

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    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.

  5. Alternative Splicing in Lung Cancer

    OpenAIRE

    Pio, Ruben; Montuenga, Luis M.

    2009-01-01

    Abstract: Alterations in alternative splicing affect essential biologic processes and are the basis for a number of pathologic conditions, including cancer. In this review we will summarize the evidence supporting the relevance of alternative splicing in lung cancer. An example that illustrates this relevance is the altered balance between Bcl-xL and Bcl-xS, two splice variants of the apoptosis regulator Bcl-x. Splice modifications in cancer-related genes can be associated ...

  6. Regulation of HIV-1 splicing

    NARCIS (Netherlands)

    Müller, N.

    2016-01-01

    Human immunodeficiency virus type-1 (HIV-1) produces a single primary RNA transcript. The full-length transcript functions as RNA genome that is packaged into virions and as mRNA for translation of the Gag and Pol proteins. HIV-1 RNA contains several splice donor (5’splice site; 5’ss) and splice

  7. Where splicing joins chromatin

    Czech Academy of Sciences Publication Activity Database

    Hnilicová, Jarmila; Staněk, David

    2011-01-01

    Roč. 2, č. 3 (2011), s. 182-188 ISSN 1949-1034 R&D Projects: GA ČR GAP305/10/0424; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z50520514 Keywords : chromatin * exon * alternative splicing * transcription * snRNP Subject RIV: EB - Genetics ; Molecular Biology

  8. Expressiveness of basic Splice

    NARCIS (Netherlands)

    J.C. van de Pol (Jaco)

    2000-01-01

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

  9. Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-03-27

    Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  10. Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jason Gabunilas

    2016-04-01

    Full Text Available In Saccharomyces cerevisiae, splicing is critical for expression of ribosomal protein genes (RPGs, which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the RPL22B pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p in vivo and splicing inhibition in vivo and in vitro and can rescue splicing inhibition in vitro when added in trans to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the RPL22B locus. We further demonstrate that the inhibition of RPL22B pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis.

  11. Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gabunilas, Jason; Chanfreau, Guillaume

    2016-04-01

    In Saccharomyces cerevisiae, splicing is critical for expression of ribosomal protein genes (RPGs), which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the RPL22B pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p in vivo and splicing inhibition in vivo and in vitro and can rescue splicing inhibition in vitro when added in trans to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the RPL22B locus. We further demonstrate that the inhibition of RPL22B pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis.

  12. Alternative REST Splicing Underappreciated

    OpenAIRE

    Chen, Guo-Lin; Miller, Gregory

    2017-01-01

    As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription, an N-terminal REST4 isoform caused by alternative splicing - inclusion of an extra exon (N3c) which introduces a pre-mature stop codon - has been implicated in...

  13. The neurogenetics of alternative splicing

    OpenAIRE

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

    2016-01-01

    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 r...

  14. Work organization for splice consolidation

    CERN Document Server

    Bertinelli, F

    2011-01-01

    The Splices Task Force has worked in 2010 to prepare the necessary interventions for 7 TeV operation. The design solution for consolidating the main interconnection splices is well advanced. The required activities to implement it are described, highlighting working assumptions, missing resources and schedule considerations. Progress has also been made in assessing other splices, 6 kA praying hands and corrector circuits: results and ongoing work are presented, highlighting priorities for the remaining work.

  15. Stabilized cyclopropane analogs of the splicing inhibitor FD-895.

    Science.gov (United States)

    Villa, Reymundo; Kashyap, Manoj Kumar; Kumar, Deepak; Kipps, Thomas J; Castro, Januario E; La Clair, James J; Burkart, Michael D

    2013-09-12

    Targeting the spliceosome with small molecule inhibitors provides a new avenue to target cancer by intercepting alternate splicing pathways. Although our understanding of alternate mRNA splicing remains poorly understood, it provides an escape pathway for many cancers resistant to current therapeutics. These findings have encouraged recent academic and industrial efforts to develop natural product spliceosome inhibitors, including FD-895 (1a), pladienolide B (1b), and pladienolide D (1c), into next-generation anticancer drugs. The present study describes the application of semisynthesis and total synthesis to reveal key structure-activity relationships for the spliceosome inhibition by 1a. This information is applied to deliver new analogs with improved stability and potent activity at inhibiting splicing in patient derived cell lines.

  16. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  17. 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.

  18. 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.

  19. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2008-09-01

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

  20. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  1. Identification of a novel function of CX-4945 as a splicing regulator.

    Directory of Open Access Journals (Sweden)

    Hyeongki Kim

    Full Text Available Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2 and a molecule currently in clinical trials (Phase II for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3-90 nM was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.

  2. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

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

  3. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    Energy Technology Data Exchange (ETDEWEB)

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  4. 0-6652 : spliced Texas girder bridges.

    Science.gov (United States)

    2015-02-01

    Spliced girder technology continues to attract : attention due to its versatility over traditional : prestressed concrete highway bridge construction. : By joining multiple precast concrete girders using : post-tensioning, spliced girder technology :...

  5. Detained introns are a novel, widespread class of post-transcriptionally spliced introns.

    Science.gov (United States)

    Boutz, Paul L; Bhutkar, Arjun; Sharp, Phillip A

    2015-01-01

    Deep sequencing of embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within polyadenylated transcripts; we classified these as "detained" introns (DIs). We identified thousands of DIs, many of which are evolutionarily conserved, in human and mouse cell lines as well as the adult mouse liver. DIs can have half-lives of over an hour yet remain in the nucleus and are not subject to nonsense-mediated decay (NMD). Drug inhibition of Clk, a stress-responsive kinase, triggered rapid splicing changes for a specific subset of DIs; half showed increased splicing, and half showed increased intron detention, altering transcript pools of >300 genes. Srsf4, which undergoes a dramatic phosphorylation shift in response to Clk kinase inhibition, regulates the splicing of some DIs, particularly in genes encoding RNA processing and splicing factors. The splicing of some DIs-including those in Mdm4, a negative regulator of p53-was also altered following DNA damage. After 4 h of Clk inhibition, the expression of >400 genes changed significantly, and almost one-third of these are p53 transcriptional targets. These data suggest a widespread mechanism by which the rate of splicing of DIs contributes to the level of gene expression. © 2015 Boutz et al.; Published by Cold Spring Harbor Laboratory Press.

  6. cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing

    International Nuclear Information System (INIS)

    Liao, Huey-Jane; Baker, Carl C.; Princler, Gerald L.; Derse, David

    2004-01-01

    Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping

  7. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  8. Titin Diversity—Alternative Splicing Gone Wild

    Directory of Open Access Journals (Sweden)

    Wei Guo

    2010-01-01

    Full Text Available Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

  9. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar P

    2014-03-01

    Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  10. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Science.gov (United States)

    Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

    2014-03-01

    TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  11. Supplementary Material for: Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-01-01

    Abstract Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

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

    Energy Technology Data Exchange (ETDEWEB)

    White, Eric S., E-mail: docew@umich.edu [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI (United States); Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B. [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI (United States); Ritzenthaler, Jeffrey D.; Roman, Jesse [Department of Medicine, University of Louisville School of Medicine, Louisville, KY (United States); Muro, Andres F. [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy)

    2010-10-01

    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-{beta}, 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{sup -/-} 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{sup -/-} 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{sup -/-} 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.

  13. HIV-1 splicing at the major splice donor site is restricted by RNA structure.

    Science.gov (United States)

    Mueller, Nancy; van Bel, Nikki; Berkhout, Ben; Das, Atze T

    2014-11-01

    The 5' leader region of the HIV-1 RNA contains the major 5' splice site (ss) that is used in the production of all spliced viral RNAs. This splice-donor (SD) region can fold a stem-loop structure. We demonstrate that whereas stabilization of this SD hairpin reduces splicing efficiency, destabilization increases splicing. Both stabilization and destabilization reduce viral fitness. These results demonstrate that the stability of the SD hairpin can modulate the level of splicing, most likely by controlling the accessibility of the 5'ss for the splicing machinery. The natural stability of the SD hairpin restricts splicing and this stability seems to be fine-tuned to reach the optimal balance between unspliced and spliced RNAs for efficient virus replication. The 5'ss region of different HIV-1 isolates and the related SIVmac239 can fold a similar structure. This evolutionary conservation supports the importance of this structure in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection

    Directory of Open Access Journals (Sweden)

    Mark A. Chua

    2013-01-01

    Full Text Available Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm, and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic nonstructural segment that encodes the spliced virus product responsible for genomic nuclear export. We find that utilization of an erroneous splice site ensures the slow accumulation of the viral nuclear export protein (NEP while generating excessive levels of an antagonist that inhibits the cellular response to infection. Modulation of this simple transcriptional event results in improperly timed export and loss of virus infection. Together, these data demonstrate that coordination of the influenza A virus life cycle is set by a “molecular timer” that operates on the inefficient splicing of a virus transcript.

  15. 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.

  16. Alternative Splicing in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Shengming Yang

    2014-06-01

    Full Text Available Alternative splicing (AS occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel.

  17. Capacity of columns with splice imperfections

    International Nuclear Information System (INIS)

    Popov, E.P.; Stephen, R.M.

    1977-01-01

    To study the behavior of spliced columns subjected to tensile forces simulating situations which may develop in an earthquake, all of the spliced specimens were tested to failure in tension after first having been subjected to large compressive loads. The results of these tests indicate that the lack of perfect contact at compression splices of columns may not be important, provided that the gaps are shimmed and welding is used to maintain the sections in alignment

  18. 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...

  19. Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein

    OpenAIRE

    Bar, Aileen; Marchand, Virginie; Khoury, Georges; Dreumont, Natacha; Mougin, Annie; Robas, Nathalie; Stévenin, James; Visvikis, Athanase; Branlant, Christiane

    2010-01-01

    Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5′-part is considered as an ESE binding SR proteins. Its 3′-part contains a decoy 5′-splice site (ss), which inhibits splicing at the bona fide 5′-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entir...

  20. 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.

  1. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid conjugates targeting intron-exon junctions

    Directory of Open Access Journals (Sweden)

    Nielsen Peter E

    2010-06-01

    Full Text Available Abstract Background Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. Methods We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512 targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT. Results We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406 targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512 targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. Conclusion We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.

  2. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    Science.gov (United States)

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Pre-mRNA splicing is a determinant of histone H3K36 methylation.

    Science.gov (United States)

    Kim, Soojin; Kim, Hyunmin; Fong, Nova; Erickson, Benjamin; Bentley, David L

    2011-08-16

    A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3' splice site in intron 2 or in both introns 1 and 2 of an integrated β-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5' ends and toward 3' ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5' ends in favor of 3' ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification.

  4. Alcoholism and Alternative Splicing of Candidate Genes

    Directory of Open Access Journals (Sweden)

    Toshikazu Sasabe

    2010-03-01

    Full Text Available Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  5. 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.

  6. 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.

  7. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    Human gene therapy (HGT) aims to cure disease by inserting or editing the DNA of patients with genetic conditions. Since foundational genetic techniques came into use in the 1970s, the field has developed to the point that now three therapies have market approval, and over 1800 clinical trials have...... been initiated. In this article I present a brief history of HGT, showing how the ethical and practical viability of the field was achieved by key scientific and regulatory actors. These parties carefully articulated gene therapy’s scope, limiting it to therapeutic interventions on somatic cells......, and cultivated alliances and divisions that bolstered the field’s legitimacy. At times these measures faltered, and then practitioners and sometimes patients would invoke an ethical imperative, posing gene therapy as the best solution to life and death problems. I suggest that we consider how boundary...

  8. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  9. Pre-mRNA splicing repression triggers abiotic stress signaling in plants

    KAUST Repository

    Ling, Yu

    2016-09-24

    Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A

  10. RAGE splicing variants in mammals.

    Science.gov (United States)

    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.

  11. Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis

    KAUST Repository

    Cui, Peng

    2014-01-07

    Background: Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing.Results: Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes.Conclusions: We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote. 2014 Cui et al.; licensee BioMed Central Ltd.

  12. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_patte...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  13. Regular languages, regular grammars and automata in splicing systems

    Science.gov (United States)

    Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

    2013-04-01

    Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

  14. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function

    Science.gov (United States)

    Gallagher, Thomas L.; Arribere, Joshua A.; Geurts, Paul A.; Exner, Cameron R. T.; McDonald, Kent L.; Dill, Kariena K.; Marr, Henry L.; Adkar, Shaunak S.; Garnett, Aaron T.; Amacher, Sharon L.; Conboy, John G.

    2012-01-01

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function. PMID:21925157

  15. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

    Science.gov (United States)

    Gallagher, Thomas L; Arribere, Joshua A; Geurts, Paul A; Exner, Cameron R T; McDonald, Kent L; Dill, Kariena K; Marr, Henry L; Adkar, Shaunak S; Garnett, Aaron T; Amacher, Sharon L; Conboy, John G

    2011-11-15

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions. Published by Elsevier Inc.

  16. DNA damage regulates alternative splicing through changes in POL II elongation

    International Nuclear Information System (INIS)

    Munoz, M.J.; Perez Santangelo, M.S.; De la Mata, M.; Kornblihtt, A.R.

    2008-01-01

    Many apoptotic genes are regulated via alternative splicing (AS) but little is known about the mechanisms controlling AS in stress situations derived from DNA damage. Here we show that ultraviolet (UV) radiation affects co-transcriptional, but not post transcriptional, AS through a systemic mechanism involving a CDK-9-dependent hyper phosphorylation of RNA polymerase II carboxy terminal domain (CTD) and a subsequent and unprecedented inhibition of transcriptional elongation, estimated in vivo and in real time by FRAP. To mimic this hyper phosphorylation we used CTD mutants with serines 2 or 5 substituted by glutamic acids and found that they not only display lower elongation rates but duplicate the effects of UV light on AS in the absence of irradiation. Consistently, substitution of the serines with alanines prevents the UV effect on splicing. These results represent the first in vivo proof of modulation of elongation in response to an environmental signal, affecting in turn the kinetic coupling between transcription and splicing. (authors)

  17. The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers

    DEFF Research Database (Denmark)

    Cohen-Eliav, Michal; Golan-Gerstl, Regina; Siegfried, Zahava

    2013-01-01

    to be tumorigenic in mice. In contrast, knockdown of SRSF6 in lung and colon cancer cell lines inhibited their tumorigenic abilities. SRSF6 up- or down regulation altered the splicing of several tumor suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumor suppressive isoforms. Our data...... and lung tumors and found that the gene encoding for the splicing factor SRSF6 is amplified and overexpressed in these cancers. Moreover, overexpression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them...... suggests that the splicing factor SRSF6 is an oncoprotein which regulates proliferation and survival of lung and colon cancer cells....

  18. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  19. 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.

  20. Mechanism of alternative splicing and its regulation.

    Science.gov (United States)

    Wang, Yan; Liu, Jing; Huang, B O; Xu, Yan-Mei; Li, Jing; Huang, Lin-Feng; Lin, Jin; Zhang, Jing; Min, Qing-Hua; Yang, Wei-Ming; Wang, Xiao-Zhong

    2015-03-01

    Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases.

  1. 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.

  2. BRCA1 Exon 11, a CERES (Composite Regulatory Element of Splicing Element Involved in Splice Regulation

    Directory of Open Access Journals (Sweden)

    Claudia Tammaro

    2014-07-01

    Full Text Available Unclassified variants (UV of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES.

  3. 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.

  4. Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

    Directory of Open Access Journals (Sweden)

    Sushmita Poddar

    2014-06-01

    Full Text Available To date the Simian Virus 40 (SV40 is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

  5. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern in Cae...

  6. Identification of Common Genetic Variation That Modulates Alternative Splicing

    OpenAIRE

    Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

    2007-01-01

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by nat...

  7. Universal Alternative Splicing of Noncoding Exons

    DEFF Research Database (Denmark)

    Deveson, Ira W; Brunck, Marion E; Blackburn, James

    2018-01-01

    The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed......, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution....

  8. 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

  9. 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.

  10. The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

    DEFF Research Database (Denmark)

    Honoré, B

    2000-01-01

    The hnRNP 2H9 gene products are involved in the splicing process and participate in early heat shock-induced splicing arrest. By combining low/high stringency hybridisation, database search, Northern and Western blotting it is shown that the gene is alternatively spliced into at least six transcr...

  11. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor

  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. Survey of gene splicing algorithms based on reads.

    Science.gov (United States)

    Si, Xiuhua; Wang, Qian; Zhang, Lei; Wu, Ruo; Ma, Jiquan

    2017-11-02

    Gene splicing is the process of assembling a large number of unordered short sequence fragments to the original genome sequence as accurately as possible. Several popular splicing algorithms based on reads are reviewed in this article, including reference genome algorithms and de novo splicing algorithms (Greedy-extension, Overlap-Layout-Consensus graph, De Bruijn graph). We also discuss a new splicing method based on the MapReduce strategy and Hadoop. By comparing these algorithms, some conclusions are drawn and some suggestions on gene splicing research are made.

  14. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    by facilitating or hindering the interaction with factors and small nuclear ribonucleoproteins (snRNPs) that regulate splicing. Moreover, the secondary structure could play a fundamental role in the splicing of yeast species, which lack many of the regulatory splicing factors present in metazoans. This chapter......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

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

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    Erik van der Wal

    2017-06-01

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

  16. Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein.

    Science.gov (United States)

    Bar, Aileen; Marchand, Virginie; Khoury, Georges; Dreumont, Natacha; Mougin, Annie; Robas, Nathalie; Stévenin, James; Visvikis, Athanase; Branlant, Christiane

    2011-04-01

    Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5'-part is considered as an ESE binding SR proteins. Its 3'-part contains a decoy 5'-splice site (ss), which inhibits splicing at the bona fide 5'-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entire RSV NRS. Structural analysis of other avian NRSs and comparison with all sequenced avian NRSs is in favour of a phylogenetic conservation of the NRS 2D structure. By combination of approaches: (i) in vitro and in cellulo splicing assays, (ii) footprinting assays and (iii) purification and analysis of reconstituted RNP complex, we define a small NRS element retaining splicing inhibitory property. We also demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo. Altogether these data bring new insights on how NRS fine tune splicing activity.

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

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2007-01-01

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...

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

    Directory of Open Access Journals (Sweden)

    Gomez-Roman Javier

    2010-06-01

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

  19. Alteration of RNA splicing by small molecule inhibitors of the interaction between NHP2L1 and U4

    Science.gov (United States)

    Diouf, Barthelemy; Lin, Wenwei; Goktug, Asli; Grace, Christy R. R.; Waddell, Michael Brett; Bao, Ju; Shao, Youming; Heath, Richard J.; Zheng, Jie J.; Shelat, Anang A.; Relling, Mary V.; Chen, Taosheng; Evans, William E.

    2018-01-01

    Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remains a challenge. We developed an assay based on time-resolved FRET (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs which could facilitate the development of therapeutic strategies to modify splicing and alter gene function. PMID:28985478

  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. RNA G-quadruplex secondary structure promotes alternative splicing via the RNA-binding protein hnRNPF.

    Science.gov (United States)

    Huang, Huilin; Zhang, Jing; Harvey, Samuel E; Hu, Xiaohui; Cheng, Chonghui

    2017-11-15

    It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Destroying G-quadruplex-forming capacity while keeping G tracts intact abrogates exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in heterogeneous nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA (The Cancer Genome Atlas) data sets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression. © 2017 Huang et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

    Science.gov (United States)

    Erkelenz, Steffen; Theiss, Stephan; Otte, Marianne; Widera, Marek; Peter, Jan Otto; Schaal, Heiner

    2014-01-01

    Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Evolution of Nova-dependent splicing regulation in the brain.

    Directory of Open Access Journals (Sweden)

    Nejc Jelen

    2007-10-01

    Full Text Available A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.

  4. Alternative mRNA Splicing in the Pathogenesis of Obesity

    Directory of Open Access Journals (Sweden)

    Chi-Ming Wong

    2018-02-01

    Full Text Available Alternative mRNA splicing is an important mechanism in expansion of proteome diversity by production of multiple protein isoforms. However, emerging evidence indicates that only a limited number of annotated protein isoforms by alternative splicing are detected, and the coding sequence of alternative splice variants usually is only slightly different from that of the canonical sequence. Nevertheless, mis-splicing is associated with a large array of human diseases. Previous reviews mainly focused on hereditary and somatic mutations in cis-acting RNA sequence elements and trans-acting splicing factors. The importance of environmental perturbations contributed to mis-splicing is not assessed. As significant changes in exon skipping and splicing factors expression levels are observed with diet-induced obesity, this review focuses on several well-known alternatively spliced metabolic factors and discusses recent advances in the regulation of the expressions of splice variants under the pathophysiological conditions of obesity. The potential of targeting the alternative mRNA mis-splicing for obesity-associated diseases therapies will also be discussed.

  5. Intronic alternative splicing regulators identified by comparative genomics in nematodes.

    Directory of Open Access Journals (Sweden)

    Jennifer L Kabat

    2006-07-01

    Full Text Available Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (TGCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis

  6. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

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

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....... with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA...

  7. Alternative Splicing Regulated by Butyrate in Bovine Epithelial Cells

    Science.gov (United States)

    Wu, Sitao; Li, Congjun; Huang, Wen; Li, Weizhong; Li, Robert W.

    2012-01-01

    As a signaling molecule and an inhibitor of histone deacetylases (HDACs), butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control (CT) groups. 201,326 potential splicing junctions detected were supported by ≥3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ∼3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC inhibitors. PMID:22720068

  8. 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.

  9. DNA computing based on splicing: universality results.

    Science.gov (United States)

    Csuhaj-Varjú, E; Freund, R; Kari, L; Păun, G

    1996-01-01

    The paper extends some of the most recently obtained results on the computational universality of specific variants of H systems (e.g. with regular sets of rules) and proves that we can construct universal computers based on various types of H systems with a finite set of splicing rules as well as a finite set of axioms, i.e. we show the theoretical possibility to design programmable universal DNA computers based on the splicing operation. For H systems working in the multiset style (where the numbers of copies of all available strings are counted) we elaborate how a Turing machine computing a partial recursive function can be simulated by an equivalent H system computing the same function; in that way, from a universal Turning machine we obtain a universal H system. Considering H systems as language generating devices we have to add various simple control mechanisms (checking the presence/absence of certain symbols in the spliced strings) to systems with a finite set of splicing rules as well as with a finite set of axioms in order to obtain the full computational power, i.e. to get a characterization of the family of recursively enumerable languages. We also introduce test tube systems, where several H systems work in parallel in their tubes and from time to time the contents of each tube are redistributed to all tubes according to certain separation conditions. By the construction of universal test tube systems we show that also such systems could serve as the theoretical basis for the development of biological (DNA) computers.

  10. Stochastic principles governing alternative splicing of RNA.

    OpenAIRE

    Jianfei Hu; Eli Boritz; William Wylie; Daniel C Douek

    2017-01-01

    Author summary Alternative RNA splicing within eukaryotic cells enables each gene to generate multiple different mature transcripts which further encode proteins with distinct or even opposing functions. The relative frequencies of the transcript isoforms generated by a particular gene are essential to the maintenance of normal cellular physiology; however, the underlying mechanisms and principles that govern these frequencies are unknown. We analyzed the frequency distribution of all transcr...

  11. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

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

    Science.gov (United States)

    Deschênes, Mathieu; Chabot, Benoit

    2017-10-01

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

  13. Accumulation of GC donor splice signals in mammals

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  14. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Jose E. Kroll

    2015-11-01

    Full Text Available Motivation. Alternative splicing events (ASEs are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background.Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events.Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress.

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

    Directory of Open Access Journals (Sweden)

    Jeremy Hull

    2007-06-01

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

  16. Alternative Splicing of FOXP3-Virtue and Vice.

    Science.gov (United States)

    Mailer, Reiner K W

    2018-01-01

    FOXP3 is the lineage-defining transcription factor of CD4+ CD25+ regulatory T cells. While many aspects of its regulation, interaction, and function are conserved among species, alternatively spliced FOXP3 isoforms are expressed only in human cells. This review summarizes current knowledge about alternative splicing of FOXP3 and the specific functions of FOXP3 isoforms in health and disease. Future perspectives in research and the therapeutic potential of manipulating alternative splicing of FOXP3 are discussed.

  17. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

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    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

  18. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

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    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  19. Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast

    OpenAIRE

    Aslanzadeh, Vahid; Huang, Yuanhua; Sanguinetti, Guido; Beggs, Jean D.

    2018-01-01

    The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggest...

  20. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...... cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5;- and the 3;-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3;-splice site of intron3 with a complementarity of 4 bases to intron......3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). RESULTS: We show that several of these PNAs effectively...

  1. Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD.

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    Mariaelena Pistoni

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1 in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6- is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

  2. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs...

  3. Alternative Splicing of the RAGE Cytoplasmic Domain Regulates Cell Signaling and Function

    Science.gov (United States)

    Jules, Joel; Maiguel, Dony; Hudson, Barry I.

    2013-01-01

    The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling. PMID:24260107

  4. 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.

  5. RNA Splicing: Regulation and Dysregulation in the Heart

    NARCIS (Netherlands)

    van den Hoogenhof, Maarten M. G.; Pinto, Yigal M.; Creemers, Esther E.

    2016-01-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new

  6. 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.

  7. ASpedia: a comprehensive encyclopedia of human alternative splicing.

    Science.gov (United States)

    Hyung, Daejin; Kim, Jihyun; Cho, Soo Young; Park, Charny

    2018-01-04

    Alternative splicing confers the human genome complexity by increasing the diversity of expressed mRNAs. Hundreds or thousands of splicing regions have been identified through differential alternative splicing analysis of high-throughput datasets. However, it is hard to explain the functional impact of each splicing event. Protein domain formation and nonsense-mediated decay are considered the main functional features of splicing. However, other functional features such as miRNA target sites, phosphorylation sites and single-nucleotide variations are directly affected by alternative splicing and affect downstream function. Hence, we established ASpedia: a comprehensive database for human alternative splicing annotation, which encompasses a range of functions, from genomic annotation to isoform-specific function (ASpedia, http://combio.snu.ac.kr/aspedia). The database provides three features: (i) genomic annotation extracted from DNA, RNA and proteins; (ii) transcription and regulation elements analyzed from next-generation sequencing datasets; and (iii) isoform-specific functions collected from known and published datasets. The ASpedia web application includes three components: an annotation database, a retrieval system and a browser specialized in the identification of human alternative splicing events. The retrieval system supports multiple AS event searches resulting from high-throughput analysis and the AS browser comprises genome tracks. Thus, ASpedia facilitates the systemic annotation of the functional impacts of multiple AS events. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Quantitative regulation of alternative splicing in evolution and development

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Roy, Scott W

    2009-01-01

    Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or o...

  9. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  10. A study of alternative splicing in the pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Cirera Salicio, Susanna; Gilchrist, Michael J.

    2010-01-01

    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...... 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...

  11. Detecting Image Splicing Using Merged Features in Chroma Space

    Directory of Open Access Journals (Sweden)

    Bo Xu

    2014-01-01

    Full Text Available Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature.

  12. Some relations between two stages DNA splicing languages

    Science.gov (United States)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-06-01

    A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

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

    Science.gov (United States)

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

    2015-12-01

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

  14. SpliceDetector: a software for detection of alternative splicing events in human and model organisms directly from transcript IDs.

    Science.gov (United States)

    Baharlou Houreh, Mandana; Ghorbani Kalkhajeh, Payam; Niazi, Ali; Ebrahimi, Faezeh; Ebrahimie, Esmaeil

    2018-03-22

    In eukaryotes, different combinations of exons lead to multiple transcripts with various functions in protein level, in a process called alternative splicing (AS). Unfolding the complexity of functional genomics through genome-wide profiling of AS and determining the altered ultimate products provide new insights for better understanding of many biological processes, disease progress as well as drug development programs to target harmful splicing variants. The current available tools of alternative splicing work with raw data and include heavy computation. In particular, there is a shortcoming in tools to discover AS events directly from transcripts. Here, we developed a Windows-based user-friendly tool for identifying AS events from transcripts without the need to any advanced computer skill or database download. Meanwhile, due to online working mode, our application employs the updated SpliceGraphs without the need to any resource updating. First, SpliceGraph forms based on the frequency of active splice sites in pre-mRNA. Then, the presented approach compares query transcript exons to SpliceGraph exons. The tool provides the possibility of statistical analysis of AS events as well as AS visualization compared to SpliceGraph. The developed application works for transcript sets in human and model organisms.

  15. Selective degradation of splicing factor CAPERα by anticancer sulfonamides.

    Science.gov (United States)

    Uehara, Taisuke; Minoshima, Yukinori; Sagane, Koji; Sugi, Naoko Hata; Mitsuhashi, Kaoru Ogawa; Yamamoto, Noboru; Kamiyama, Hiroshi; Takahashi, Kentaro; Kotake, Yoshihiko; Uesugi, Mai; Yokoi, Akira; Inoue, Atsushi; Yoshida, Taku; Mabuchi, Miyuki; Tanaka, Akito; Owa, Takashi

    2017-06-01

    Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4 DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

  16. SRSF1 Prevents DNA Damage and Promotes Tumorigenesis through Regulation of DBF4B Pre-mRNA Splicing

    Directory of Open Access Journals (Sweden)

    Linlin Chen

    2017-12-01

    Full Text Available Dysregulated alternative splicing events have been implicated in many types of cancer, but the underlying molecular mechanisms remain unclear. Here, we observe that the splicing factor SRSF1 regulates DBF4B exon6 splicing by specifically binding and promoting its inclusion. Knockdown of the exon6-containing isoform (DBF4B-FL significantly inhibits the tumorigenic potential of colon cancer cells in vitro and in mice, and SRSF1 inactivation phenocopies DBF4B-FL depletion. DBF4B-FL and SRSF1 are required for cancer cell proliferation and for the maintenance of genomic stability. Overexpression of DBF4B-FL can protect against DNA damage induced by SRSF1 knockdown and rescues growth defects in SRSF1-depleted cells. Increased DBF4B exon6 inclusion parallels SRSF1 upregulation in clinical colorectal cancer samples. Taken together, our findings identify SRSF1 as a key regulator of DBF4B pre-mRNA splicing dysregulation in colon cancer, with possible clinical implications as candidate prognostic factors in cancer patients.

  17. Alternative splicing regulated by butyrate in bovine epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sitao Wu

    Full Text Available As a signaling molecule and an inhibitor of histone deacetylases (HDACs, butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT and control (CT groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001 at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor and Exon#11 (Acceptor in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC

  18. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    OpenAIRE

    Kroll, Jose E.; Kim, Jihoon; Ohno-Machado, Lucila; de Souza, Sandro J.

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many dif...

  19. Global Splicing Pattern Reversion during Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Sho Ohta

    2013-10-01

    Full Text Available Alternative splicing generates multiple transcripts from a single gene, and cell-type-specific splicing profiles are important for the properties and functions of the cells. Recently, somatic cells have been shown to undergo dedifferentiation after the forced expression of transcription factors. However, it remains unclear whether somatic cell splicing is reorganized during reprogramming. Here, by combining deep sequencing with high-throughput absolute qRT-PCR, we show that somatic splicing profiles revert to pluripotent ones during reprogramming. Remarkably, the splicing pattern in pluripotent stem cells resembles that in testes, and the regulatory regions have specific characteristics in length and sequence. Furthermore, our siRNA screen has identified RNA-binding proteins that regulate splicing events in iPSCs. We have then demonstrated that two of the RNA-binding proteins, U2af1 and Srsf3, play a role in somatic cell reprogramming. Our results indicate that the drastic alteration in splicing represents part of the molecular network involved in the reprogramming process.

  20. 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.

  1. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

    NARCIS (Netherlands)

    Borensztajn, Keren; Sobrier, Marie-Laure; Duquesnoy, Philippe; Fischer, Anne-Marie; Tapon-Bretaudière, Jacqueline; Amselem, Serge

    2006-01-01

    Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7

  2. Splicing landscape of the eight collaborative cross founder strains.

    Science.gov (United States)

    Zheng, Christina L; Wilmot, Beth; Walter, Nicole Ar; Oberbeck, Denesa; Kawane, Sunita; Searles, Robert P; McWeeney, Shannon K; Hitzemann, Robert

    2015-02-05

    The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding

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

    Directory of Open Access Journals (Sweden)

    Furuichi Teiichi

    2007-04-01

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

  4. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    International Nuclear Information System (INIS)

    Reddy, A.S.N.

    2008-01-01

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  5. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    A.S.N. Reddy

    2008-11-25

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  6. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2016-10-01

    report. Inventions, patent applications, and/or licenses Nothing to report. Others Nothing to report. 7. Participants & Other... Brand LJ et al: Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines. Cancer Res

  7. Minor class splicing shapes the zebrafish transcriptome during development

    DEFF Research Database (Denmark)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M

    2014-01-01

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities...... known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we...... as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays...

  8. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

    Science.gov (United States)

    Sirand-Pugnet, P; Durosay, P; Brody, E; Marie, J

    1995-09-11

    Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.

  9. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  10. Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-{beta}.

    Science.gov (United States)

    Dreumont, Natacha; Bourgeois, Cyril F; Lejeune, Fabrice; Liu, Yilei; Ehrmann, Ingrid E; Elliott, David J; Stévenin, James

    2010-01-01

    RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.

  11. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA.

    Directory of Open Access Journals (Sweden)

    Stephen H Munroe

    Full Text Available The α-thyroid hormone receptor gene (TRα codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3' end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30 located downstream of the alternative 3'splice site of TRα2 mRNA and antisense to the 3'UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing.

  12. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

    Science.gov (United States)

    Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  13. MYC Regulates α6 Integrin Subunit Expression and Splicing Under Its Pro-Proliferative ITGA6A Form in Colorectal Cancer Cells.

    Science.gov (United States)

    Groulx, Jean-François; Boudjadi, Salah; Beaulieu, Jean-François

    2018-02-03

    The α6 integrin subunit ( ITGA6 ) pre-mRNA undergoes alternative splicing to form two splicing variants, named ITGA6A and ITGA6B. In primary human colorectal cancer cells, the levels of both ITGA6 and β4 integrin subunit (ITGB4) subunits of the α6β4 integrin are increased. We previously found that the upregulation of ITGA6 is a direct consequence of the increase of the pro-proliferative ITGA6A variant. However, the mechanisms that control ITGA6 expression and splicing into the ITGA6A variant over ITGA6B in colorectal cancer cells remain poorly understood. Here, we show that the promoter activity of the ITGA6 gene is regulated by MYC. Pharmacological inhibition of MYC activity with the MYC inhibitor (MYCi) 10058-F4 or knockdown of MYC expression by short hairpin RNA (shRNA) both lead to a decrease in ITGA6 and ITGA6A levels in colorectal cancer cells, while overexpression of MYC enhances ITGA6 promoter activity. We also found that MYC inhibition decreases the epithelial splicing regulatory protein 2 (ESRP2) splicing factor at both the mRNA and protein levels. Chromatin immunoprecipitation revealed that the proximal promoter sequences of ITGA6 and ESRP2 were occupied by MYC and actively transcribed in colorectal cancer cells. Furthermore, expression studies in primary colorectal tumors and corresponding resection margins confirmed that the up-regulation of the ITGA6A subunit can be correlated with the increase in MYC and ESRP2 . Taken together, our results demonstrate that the proto-oncogene MYC can regulate the promoter activation and splicing of the ITGA6 integrin gene through ESRP2 to favor the production of the pro-proliferative ITGA6A variant in colorectal cancer cells.

  14. Width of gene expression profile drives alternative splicing.

    Directory of Open Access Journals (Sweden)

    Daniel Wegmann

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

  15. Splice site mutations in the ATP7A gene.

    Directory of Open Access Journals (Sweden)

    Tina Skjørringe

    Full Text Available Menkes disease (MD is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.

  16. 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

  17. Novel female-specific trans-spliced and alternative splice forms of dsx in the silkworm Bombyx mori.

    Science.gov (United States)

    Duan, Jianping; Xu, Hanfu; Wang, Feng; Ma, Sanyuan; Zha, Xingfu; Guo, Huizhen; Zhao, Ping; Xia, Qingyou

    2013-02-15

    The Bombyx mori doublesex gene (Bmdsx) plays an important role in somatic sexual development. Its pre-mRNA splices in a sex-specific manner to generate two female-specific and one male-specific splice forms. The present study investigated six novel dsx variants generated by trans-splicing between female dsx transcripts and two additional novel genes, dsr1 and dsr2. Expression analysis indicated that Bmdsx-dsr1 represented splicing noise, whereas dsr2, which trans-spliced with dsx to generate five variants, regulated the expression of the female-specific B. mori dsx transcript Bmdsx(F)s. We unexpectedly found a novel exon 2n insertion during Bmdsx transcription, which did not influence the validity of the novel protein, BmDSX(F3). Ectopic expression of BmDSX(F3) repressed the pheromone-binding protein gene and the testis-specific gene A2 in males, and activated of the storage protein 1 gene. Our findings suggest that trans-splicing is a novel regulatory function of Bmdsx, which participates in female sexual development by regulating the expression of three BmDSX(F) proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Method of predicting Splice Sites based on signal interactions

    Directory of Open Access Journals (Sweden)

    Deogun Jitender S

    2006-04-01

    Full Text Available Abstract Background Predicting and proper ranking of canonical splice sites (SSs is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE and Intronic (ISE Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand.

  19. Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.

    Science.gov (United States)

    Derelle, Romain; Momose, Tsuyoshi; Manuel, Michael; Da Silva, Corinne; Wincker, Patrick; Houliston, Evelyn

    2010-04-01

    Replacement of mRNA 5' UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3' of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare.

  20. 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.

  1. Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

    Directory of Open Access Journals (Sweden)

    Arvin B. Tam

    2014-11-01

    Full Text Available An evolutionarily conserved unfolded protein response (UPR component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD; however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

  2. Alternative mRNA splicing creates transcripts encoding soluble proteins from most LILR genes.

    Science.gov (United States)

    Jones, Des C; Roghanian, Ali; Brown, Damien P; Chang, Chiwen; Allen, Rachel L; Trowsdale, John; Young, Neil T

    2009-11-01

    Leucocyte Ig-like receptors (LILR) are a family of innate immune receptors expressed on myeloid and lymphoid cells that influence adaptive immune responses. We identified a common mechanism of alternative mRNA splicing, which generates transcripts that encode soluble protein isoforms of the majority of human LILR. These alternative splice variants lack transmembrane and cytoplasmic encoding regions, due to the transcription of a cryptic stop codon present in an intron 5' of the transmembrane encoding exon. The alternative LILR transcripts were detected in cell types that express their membrane-associated isoforms. Expression of the alternative LILRB1 transcript in transfected cells resulted in the release of a soluble approximately 65 Kd LILRB1 protein into culture supernatants. Soluble LILRB1 protein was also detected in the culture supernatants of monocyte-derived DC. In vitro assays suggested that soluble LILRB1 could block the interaction between membrane-associated LILRB1 and HLA-class I. Soluble LILRB1 may act as a dominant negative regulator of HLA-class I-mediated LILRB1 inhibition. Soluble isoforms of the other LILR may function in a comparable way.

  3. Fusion gene and splice variant analyses in liquid biopsies of lung cancer patients.

    Science.gov (United States)

    Aguado, Cristina; Giménez-Capitán, Ana; Karachaliou, Niki; Pérez-Rosado, Ana; Viteri, Santiago; Morales-Espinosa, Daniela; Rosell, Rafael

    2016-10-01

    Obtaining a biopsy of solid tumors requires invasive procedures that strongly limit patient compliance. In contrast, a blood extraction is safe, can be performed at many time points during the course disease and encourages appropriate therapy modifications, potentially improving the patient's clinical outcome and quality of life. Fusion of the tyrosine kinase genes anaplastic lymphoma kinase ( ALK ), C-ROS oncogen 1 ( ROS 1 ), rearranged during transfection ( RET ) and neurotrophic tyrosine kinase 1 ( NTRK1 ) occur in 1-5% of lung adenocarcinomas and constitute therapeutic targets for tyrosine kinase inhibitors. In addition, a MET splicing variant of exon 14, has been reported in 2-4% of lung adenocarcinoma and recent studies suggests that targeted therapies inhibiting MET signaling would be beneficial for patients with this alteration. In this review, we will summarize the new techniques recently developed to detect ALK , RET , ROS and NTRK1 fusions and MET exon 14 splicing variant in liquid biopsy using plasma, serum, circulating tumor cells (CTCs), platelets and exosomes as starting material.

  4. SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

    Directory of Open Access Journals (Sweden)

    Xiao-Peng Xiong

    2015-08-01

    Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

  5. Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network

    Directory of Open Access Journals (Sweden)

    Ahn R. Lee

    2018-04-01

    Full Text Available While the use of next-generation androgen receptor pathway inhibition (ARPI therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC, several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR indifferent and gain a neuroendocrine (NE-like phenotype. This subtype of castration-resistant prostate cancer has been termed “treatment-induced castration-resistant neuroendocrine prostate cancer” (CRPC-NE. Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4, a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4

  6. Functional Characterization of NIPBL Physiological Splice Variants and Eight Splicing Mutations in Patients with Cornelia de Lange Syndrome

    Directory of Open Access Journals (Sweden)

    María E. Teresa-Rodrigo

    2014-06-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21 or functionally associated factors (NIPBL, HDAC8 of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

  7. Understanding pre-mRNA splicing through crystallography.

    Science.gov (United States)

    Espinosa, Sara; Zhang, Lingdi; Li, Xueni; Zhao, Rui

    2017-08-01

    Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods - the serial femtosecond crystallography and 3D electron crystallography - were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve. This review discusses the theoretical basis, instrument requirements, troubleshooting, and exciting potential of these crystallographic methods to further our understanding of pre-mRNA splicing, a critical event in gene expression of all eukaryotes. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

    Science.gov (United States)

    Preußner, Marco; Goldammer, Gesine; Neumann, Alexander; Haltenhof, Tom; Rautenstrauch, Pia; Müller-McNicoll, Michaela; Heyd, Florian

    2017-08-03

    The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  10. Minor class splicing shapes the zebrafish transcriptome during development.

    Science.gov (United States)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M; Doggett, Karen; Keightley, Maria-Cristina; Boglev, Yeliz; Trotter, Andrew J; Ng, Annie Y; Wilkins, Simon J; Verkade, Heather; Ober, Elke A; Field, Holly A; Grimmond, Sean M; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2014-02-25

    Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

  11. Analysis for Behavior of Reinforcement Lap Splices in Deep Beams

    Directory of Open Access Journals (Sweden)

    Ammar Yaser Ali

    2018-03-01

    Full Text Available The present study includes an experimental and theoretical investigation of reinforced concrete deep beams containing tensile reinforcement lap splices at constant moment zone under static load. The study included two stages: in the first one, an experimental work included testing of eight simply supported RC deep beams having a total length (L = 2000 mm, overall depth (h= 600 mm and width (b = 150 mm. The tested specimens were divided into three groups to study the effect of main variables: lap length, location of splice, internal confinement (stirrups and external confinement (strengthening by CFRP laminates. The experimental results showed that the use of CFRP as external strengthening in deep beam with lap spliced gives best behavior such as increase in stiffness, decrease in deflection, delaying the cracks appearance and reducing the crack width. The reduction in deflection about (14-21 % than the unstrengthened beam and about (5-14 % than the beam with continuous bars near ultimate load. Also, it was observed that the beams with unstrengthened tensile reinforcement lap splices had three types of cracks: flexural, flexural-shear and splitting cracks while the beams with strengthened tensile reinforcement lap splices or continuous bars don’t observe splitting cracks. In the second stage, a numerical analysis of three dimensional finite element analysis was utilized to explore the behavior of the RC deep beams with tensile reinforcement lap splices, in addition to parametric study of many variables. The comparison between the experimental and theoretical results showed reasonable agreement. The average difference of the deflection at service load was less than 5%.

  12. Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Akihiro, E-mail: a-wada@nagasaki-u.ac.jp [Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Wong, Pooi-Fong [Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur (Malaysia); Hojo, Hironobu [Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, Kanagawa 2591292 (Japan); Hasegawa, Makoto [Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Shiga 5260829 (Japan); Ichinose, Akitoyo [Electron Microscopy Shop Central Laboratory, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Llanes, Rafael [Institute Pedro Kouri, Havana (Cuba); Kubo, Yoshinao [Division of Cytokine Signaling, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 8528523 (Japan); Senba, Masachika [Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Ichinose, Yoshio [Kenya Research Station, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan)

    2013-05-03

    Highlights: • Alarin inhibits the growth of E. coli but not S. aureus. • Alarin’s potency is comparable to LL-37 in inhibiting the growth of E. coli. • Alarin can cause bacterial membrane blebbing. • Alalin does not induce hemolysis on erythrocytes. -- Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.

  13. Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

    OpenAIRE

    Bonomi, Serena; Gallo, Stefania; Catillo, Morena; Pignataro, Daniela; Biamonti, Giuseppe; Ghigna, Claudia

    2013-01-01

    Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer ...

  14. A novel splicing mutation in the V2 vasopressin receptor

    DEFF Research Database (Denmark)

    Kamperis, Konstantinos; Siggaard, C; Herlin, Troels

    2000-01-01

    as clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron...... of the gene in both the affected male (hemizygous) and his mother (heterozygous). This mutation is likely to cause aberrant splicing of the terminal intron of the gene, leading to a non-functional AVP receptor. The clinical studies were consistent with such a hypothesis, as the affected subject had a severe...

  15. 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/ .

  16. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    Science.gov (United States)

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  17. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  18. Splicing up” drug discovery. Cell-Based Expression and Screening of Genetically-Encoded Libraries of Backbone Cyclized Polypeptides

    Science.gov (United States)

    Sancheti, Harshkumar; Camarero, Julio A.

    2012-01-01

    The present paper reviews the use of protein splicing for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes thus providing a new way for finding therapeutic agents. PMID:19628015

  19. CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

    KAUST Repository

    Aouida, Mustapha

    2016-02-24

    CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

  20. fruitless alternative splicing and sex behaviour in insects

    Indian Academy of Sciences (India)

    In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila mutant flies with loss-of-function alleles of the fru gene exhibit blocked male courtship behaviour. However, various individual steps in the ...

  1. fruitless alternative splicing and sex behaviour in insects: an ancient ...

    Indian Academy of Sciences (India)

    In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila mutant flies with loss-of-function alleles of the fru gene exhibit blocked male courtship behaviour. However, various individual steps in the ...

  2. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable ...

  3. Multishot diffusion-weighted SPLICE PROPELLER MRI of the abdomen.

    Science.gov (United States)

    Deng, Jie; Omary, Reed A; Larson, Andrew C

    2008-05-01

    Multishot FSE (fast spin echo)-based diffusion-weighted (DW)-PROPELLER (periodically rotated overlapping parallel lines with enhanced reconstruction) MRI offers the potential to reduce susceptibility artifacts associated with single-shot DW-EPI (echo-planar imaging) approaches. However, DW-PROPELLER in the abdomen is challenging due to the large field-of-view and respiratory motion during DW preparation. Incoherent signal phase due to motion will violate the Carr-Purcell-Meiboom-Gill (CPMG) conditions, leading to destructive interference between spin echo and stimulated echo signals and consequent signal cancellation. The SPLICE (split-echo acquisition of FSE signals) technique can mitigate non-CPMG artifacts in FSE-based sequences. For SPLICE, spin echo and stimulated echo are separated by using imbalanced readout gradients and extended acquisition window. Two signal families each with coherent phase properties are acquired at different intervals within the readout window. Separate reconstruction of these two signal families can avoid destructive phase interference. Phantom studies were performed to validate signal phase properties with different initial magnetization phases. This study evaluated the feasibility of combining SPLICE and PROPELLER for DW imaging of the abdomen. It is demonstrated that DW-SPLICE-PROPELLER can effectively mitigate non-CPMG artifacts and improve DW image quality and apparent diffusion coefficient (ADC) map homogeneity. (c) 2008 Wiley-Liss, Inc.

  4. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    Science.gov (United States)

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Long-range RNA pairings contribute to mutually exclusive splicing.

    Science.gov (United States)

    Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

    2016-01-01

    Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks. © 2015 Yue et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. CD44 splice variants as prognostic markers in colorectal cancer

    NARCIS (Netherlands)

    Wielenga, V. J.; van der Voort, R.; Mulder, J. W.; Kruyt, P. M.; Weidema, W. F.; Oosting, J.; Seldenrijk, C. A.; van Krimpen, C.; Offerhaus, G. J.; Pals, S. T.

    1998-01-01

    BACKGROUND: Splice variants of CD44 play a causal role in the metastatic spread of pancreatic carcinoma in the rat. In previous studies we have shown that homologues of these CD44 isoforms (CD44v6) are overexpressed during colorectal tumorigenesis in man and that CD44v6 overexpression is associated

  7. Unusual structure and splicing pattern of the vertebrate ...

    Indian Academy of Sciences (India)

    ROSA CALVELLO

    2018-03-08

    Mar 8, 2018 ... coding section of SLC25A3 which occurred in fish and is conserved in amphibia, birds and mammals. Further, we discuss the way the splicing mechanism compensates. Rosa Calvello and Antonia Cianciulli contributed equally to this work. for the potentially harmful consequences of this 'genomic glitch'.

  8. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    . [Parsam VL, Ali MJ, Honavar SG, Vemuganti GK and Kannabiran C 2011 Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma. J. Biosci. 36 281–287] DOI 10.1007/s12038-011-9062-9. 1. Introduction.

  9. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, .... bilateral Rb. Genomic DNA analysis from peripheral blood was as described by Parsam .... the patterns are not always the same in different studies (Klutz et al. 2002; Taylor et al.

  10. The splicing fate of plant SPO11 genes

    Directory of Open Access Journals (Sweden)

    Thorben eSprink

    2014-05-01

    Full Text Available Towards the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in plants are essential for the initiation of double strand breaks (DSBs during the meiotic prophase. In nearly all eukaryotic organisms DSB induction by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO as physical connections between the allelic chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of aberrant splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non functional as they either showed intron retention or shortened exons accompanied by a frameshift leading to premature termination codons (PTCs in most cases. Nevertheless, we could detect one putative functional alternatively spliced mRNA for SPO11-1 and -2 each, indicating that splicing of SPO11 does not depend only on the gene sequence but also on the plant species and that it might play a regulatory role.

  11. Alternative Splicing of NOX4 in the Failing Human Heart

    Directory of Open Access Journals (Sweden)

    Zoltán V. Varga

    2017-11-01

    Full Text Available Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure.

  12. A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize

    Directory of Open Access Journals (Sweden)

    Wenbin Mei

    2017-05-01

    Full Text Available Identifying and characterizing alternative splicing (AS enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs identified splicing QTL (sQTL. The 43.3% of cis-sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans-sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

  13. Rapid screening of yeast mutants with reporters identifies new splicing phenotypes.

    Science.gov (United States)

    Dreumont, Natacha; Séraphin, Bertrand

    2013-06-01

    Nuclear precursor mRNA splicing requires the stepwise assembly of a large complex, the spliceosome. Recent large-scale analyses, including purification of splicing complexes, high-throughput genetic screens and interactomic studies, have linked numerous factors to this dynamic process, including a well-defined core conserved from yeast to human. Intriguingly, despite extensive studies, no splicing defects were reported for some of the corresponding yeast mutants. To resolve this paradox, we screened a collection of viable yeast strains carrying mutations in splicing-related factors with a set of reporters including artificial constructs carrying competing splice sites. Previous analyses have indeed demonstrated that this strategy identifies yeast factors able to regulate alternative splicing and whose properties are conserved in human cells. The method, sensitive to subtle defects, revealed new splicing phenotypes for most analyzed factors such as the Urn1 protein. Interestingly, a mutant of PRP8 specifically lacking an N-terminal proline-rich region stimulated the splicing of a reporter containing competing branchpoint/3' splice site regions. Thus, using appropriate reporters, yeast can be used to quickly delineate the effect of various factors on splicing and identify those with the propensity to regulate alternative splicing events. © 2013 FEBS.

  14. Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast.

    Science.gov (United States)

    Aslanzadeh, Vahid; Huang, Yuanhua; Sanguinetti, Guido; Beggs, Jean D

    2018-02-01

    The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when cotranscriptional. Moreover, we demonstrate that altering the RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimize the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the nonribosomal protein transcripts. © 2018 Aslanzadeh et al.; Published by Cold Spring Harbor Laboratory Press.

  15. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

    2008-01-01

    Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs, harb...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly.......Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs......, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...

  16. Effect of Chord Splice Joints on Force Distribution and Deformations in Trusses with Punched Metal Plate Fasteners

    DEFF Research Database (Denmark)

    Ellegaard, Peter

    2007-01-01

    - their real behaviour is semi-rigid. The influence of splice joints on the distribution of member forces and rotations in the splice joints is investigated in this paper. A finite element program, TrussLab, where the splice joints are given semi-rigid properties is used to analyse the effect of splice joints...

  17. Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

    DEFF Research Database (Denmark)

    Weischenfeldt, Joachim Lütken; Waage, Johannes Eichler; Tian, Geng

    2012-01-01

    ABSTRACT: BACKGROUND: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation...... of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. RESULTS: We developed...... importance, the latter events are associated with high intronic conservation. CONCLUSIONS: Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel...

  18. Effect of fiber blending ratios of cotton/polyester yarns on retained splice diameter

    Science.gov (United States)

    Çelik, H. İ.; Kaynak, H. K.

    2017-10-01

    The most important performance parameters of splicing are obtaining adequate strength and appearance at the splice point for all processing requirements. The diameter of spliced portion effects not only appearance of the splice joints but also physical characteristics such as packing density, strength, specific volume of the yarn. In this study, the effect of cotton/polyester fiber blend ratios on spliced portion diameter at different slicing air pressures was investigated. For this aim, three yarn samples 100% cotton, 80-20% CO-PES and 50- 50% CO-PES were produced with 40/1 Ne. Each yarn samples was spliced at three different pressures; 4 bar, 5 bar and 6 bar. The diameters of spliced portion and retained yarns were measured by using ImageJ program and the results were analyzed statistically.

  19. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals.

    Directory of Open Access Journals (Sweden)

    Keren Borensztajn

    2006-09-01

    Full Text Available Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7 a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.

  20. Dynamic changes in neurexins' alternative splicing: role of Rho-associated protein kinases and relevance to memory formation.

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    Gabriela Rozic

    Full Text Available The three neurexins genes (NRXN1/2/3 encode polymorphic synaptic membrane proteins that are involved in cognitive functioning. Neurexins' selectivity of function is presumably conferred through differential use of 2 promoters and 5 alternative splicing sites (SS#1/2/3/4/5. In day-old rat brain neurons grown in culture, activation (depolarization induces reversible, calcium dependent, repression of NRXN2α SS#3 insert. The effects of depolarization on NRXN1/2/3α splicing and biochemical pathways mediating them were further studied in these neurons. NRXN1/2/3α splicing in the course of memory formation in vivo was also explored, using fear conditioning paradigm in rats in which the animals were trained to associate an aversive stimulus (electrical shock with a neutral context (a tone, resulting in the expression of fear responses to the neutral context.In the cultured neurons depolarization induced, beside NRXN2α SS#3, repression of SS#3 and SS#4 exons in NRXN3α but not NRXN1α. The repressions were mediated by the calcium/protein kinase C/Rho-associated protein kinase (ROCK pathway. Fear conditioning induced significant and transient repressions of the NRXN1/2/3α SS#4 exons in the rat hippocampus. ROCK inhibition prior to training attenuated the behavioral fear response, the NRXN1/2/3α splicing repressions and subsequent recovery and the levels of excitatory (PSD95 and inhibitory (gephyrin synaptic proteins in the hippocampus. No such effects were observed in the prefrontal cortex. Significant correlations existed between the fear response and hippocampal NRXN3α and NRXN2α SS#4 inserts as well as PSD95 protein levels. Hippocampal NRXN1α SS#4 insert and gephyrin levels did not correlate with the behavioral response but were negatively correlated with each other.These results show for the first time dynamic, experience related changes in NRXN1/2/3α alternative splicing in the rat brain and a role for ROCK in them. Specific neurexins

  1. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Finley, Jahahreeh

    2015-09-01

    Although the use of antiretroviral therapy (ART) has proven highly effective in controlling and suppressing HIV-1 replication, the persistence of latent but replication-competent proviruses in a small subset of CD4(+) memory T cells presents significant challenges to viral eradication from infected individuals. Attempts to eliminate latent reservoirs are epitomized by the 'shock and kill' approach, a strategy involving the combinatorial usage of compounds that influence epigenetic modulation and initiation of proviral transcription. However, efficient regulation of viral pre-mRNA splicing through manipulation of host cell splicing machinery is also indispensible for HIV-1 replication. Interestingly, aberrant alternative splicing of the LMNA gene via the usage of a cryptic splice site has been shown to be the cause of most cases of Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic condition characterized by an accelerated aging phenotype due to the accumulation of a truncated form of lamin A known as progerin. Recent evidence has shown that inhibition of the splicing factors ASF/SF2 (or SRSF1) and SRp55 (or SRSF6) leads to a reduction or an increase in progerin at both the mRNA and protein levels, respectively, thus altering the LMNA pre-mRNA splicing ratio. It is also well-established that during the latter stages of HIV-1 infection, an increase in the production and nuclear export of unspliced viral mRNA is indispensible for efficient HIV-1 replication and that the presence of ASF/SF2 leads to excessive viral pre-mRNA splicing and a reduction of unspliced mRNA, while the presence of SRp55 inhibits viral pre-mRNA splicing and aids in the generation and translation of unspliced HIV-1 mRNAs. The splicing-factor associated protein and putative mitochondrial chaperone p32 has also been shown to inhibit ASF/SF2, increase unspliced HIV-1 viral mRNA, and enhance mitochondrial DNA replication and oxidative phosphorylation. It is our hypothesis that activation of

  2. Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

    Science.gov (United States)

    Bhat-Nakshatri, Poornima; Song, Eun-Kyung; Collins, Nikail R; Uversky, Vladimir N; Dunker, A Keith; O'Malley, Bert W; Geistlinger, Tim R; Carroll, Jason S; Brown, Myles; Nakshatri, Harikrishna

    2013-06-11

    Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ER

  3. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien

    2017-04-11

    The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

  4. Features generated for computational splice-site prediction correspond to functional elements

    Directory of Open Access Journals (Sweden)

    Wilbur W John

    2007-10-01

    Full Text Available Abstract Background Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals. Results We present examples from the observed features that correspond to known signals, both core signals (including the branch site and pyrimidine tract and auxiliary signals (including GGG triplets and exon splicing enhancers. We present evidence that features identified by FGA include splicing signals not found by other methods. Conclusion Our generated features capture known biological signals in the expected sequence interval flanking splice sites. The method can be easily applied to other species and to similar classification problems, such as tissue-specific regulatory elements, polyadenylation sites, promoters, etc.

  5. The Role of Canonical and Noncanonical Pre-mRNA Splicing in Plant Stress Responses

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    A. S. Dubrovina

    2013-01-01

    Full Text Available Plants are sessile organisms capable of adapting to various environmental constraints, such as high or low temperatures, drought, soil salinity, or pathogen attack. To survive the unfavorable conditions, plants actively employ pre-mRNA splicing as a mechanism to regulate expression of stress-responsive genes and reprogram intracellular regulatory networks. There is a growing evidence that various stresses strongly affect the frequency and diversity of alternative splicing events in the stress-responsive genes and lead to an increased accumulation of mRNAs containing premature stop codons, which in turn have an impact on plant stress response. A number of studies revealed that some mRNAs involved in plant stress response are spliced counter to the traditional conception of alternative splicing. Such noncanonical mRNA splicing events include trans-splicing, intraexonic deletions, or variations affecting multiple exons and often require short direct repeats to occur. The noncanonical alternative splicing, along with common splicing events, targets the spliced transcripts to degradation through nonsense-mediated mRNA decay or leads to translation of truncated proteins. Investigation of the diversity, biological consequences, and mechanisms of the canonical and noncanonical alternative splicing events will help one to identify those transcripts which are promising for using in genetic engineering and selection of stress-tolerant plants.

  6. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

    Directory of Open Access Journals (Sweden)

    Mostafa Waly

    2016-01-01

    Full Text Available The folate and cobalamin (Cbl- dependent enzyme methionine synthase (MS is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl or the combination of hydroxocobalamin (OHCbl and S-adenosylmethionine (SAM. OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH but could be rescued by provision of either glutathionylcobalamin (GSCbl or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

  7. A Feature-Based Forensic Procedure for Splicing Forgeries Detection

    Directory of Open Access Journals (Sweden)

    Irene Amerini

    2015-01-01

    Full Text Available Nowadays, determining if an image appeared somewhere on the web or in a magazine or is authentic or not has become crucial. Image forensics methods based on features have demonstrated so far to be very effective in detecting forgeries in which a portion of an image is cloned somewhere else onto the same image. Anyway such techniques cannot be adopted to deal with splicing attack, that is, when the image portion comes from another picture that then, usually, is not available anymore for an operation of feature match. In this paper, a procedure in which these techniques could also be employed will be shown to get rid of splicing attack by resorting to the use of some repositories of images available on the Internet like Google Images or TinEye Reverse Image Search. Experimental results are presented on some real case images retrieved on the Internet to demonstrate the capacity of the proposed procedure.

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

    Science.gov (United States)

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

    2016-10-01

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

  9. 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...

  10. Exon expression and alternatively spliced genes in Tourette Syndrome.

    Science.gov (United States)

    Tian, Yingfang; Liao, Isaac H; Zhan, Xinhua; Gunther, Joan R; Ander, Bradley P; Liu, Dazhi; Lit, Lisa; Jickling, Glen C; Corbett, Blythe A; Bos-Veneman, Netty G P; Hoekstra, Pieter J; Sharp, Frank R

    2011-01-01

    Tourette Syndrome (TS) is diagnosed based upon clinical criteria including motor and vocal tics. We hypothesized that differences in exon expression and splicing might be useful for pathophysiology and diagnosis. To demonstrate exon expression and alternatively spliced gene differences in blood of individuals with TS compared to healthy controls (HC), RNA was isolated from the blood of 26 un-medicated TS subjects and 23 HC. Each sample was run on Affymetrix Human Exon 1.0 ST (HuExon) arrays and on 3' biased U133 Plus 2.0 (HuU133) arrays. To investigate the differentially expressed exons and transcripts, analyses of covariance (ANCOVA) were performed, controlling for age, gender, and batch. Differential alternative splicing patterns between TS and HC were identified using analyses of variance (ANOVA) models in Partek. Three hundred and seventy-six exon probe sets were differentially expressed between TS and HC (raw P |1.2|) that separated TS and HC subjects using hierarchical clustering and Principal Components Analysis. The probe sets predicted TS compared to HC with a >90% sensitivity and specificity using a 10-fold cross-validation. Ninety genes (transcripts) had differential expression of a single exon (raw P < 0.005) and were predicted to be alternatively spliced (raw P < 0.05) in TS compared to HC. These preliminary findings might provide insight into the pathophysiology of TS and potentially provide prognostic and diagnostic biomarkers. However, the findings are tempered by the small sample size and multiple comparisons and require confirmation using PCR or deep RNA sequencing and a much larger patient population. Copyright © 2010 Wiley-Liss, Inc.

  11. The plethora of PMCA isoforms: Alternative splicing and differential expression.

    Science.gov (United States)

    Krebs, Joachim

    2015-09-01

    In this review the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to their tissue distribution, their differences during development and their importance for regulating Ca²⁺ homeostasis under different conditions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Conserved and species-specific alternative splicing in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Favorov Alexander V

    2007-12-01

    Full Text Available Abstract Background Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants. Results We analyzed conservation of human alternative splice sites and cassette exons in the mouse and dog genomes. Alternative exons, especially minor-isofom ones, were shown to be less conserved than constitutive exons. Frame-shifting alternatives in the protein-coding regions are less conserved than frame-preserving ones. Similarly, the conservation of alternative sites is highest for evenly used alternatives, and higher when the distance between the sites is divisible by three. The rate of alternative-exon and site loss in mouse is slightly higher than in dog, consistent with faster evolution of the former. The evolutionary dynamics of alternative sites was shown to be consistent with the model of random activation of cryptic sites. Conclusion Consistent with other studies, our results show that minor cassette exons are less conserved than major-alternative and constitutive exons. However, our study provides evidence that this is caused not only by exon birth, but also lineage-specific loss of alternative exons and sites, and it depends on exon functionality.

  13. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  14. VEGF Spliced Variants: Possible Role of Anti-Angiogenesis Therapy

    Directory of Open Access Journals (Sweden)

    Caroline Hilmi

    2012-01-01

    Full Text Available Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney. Among these tumour types, clear cell renal cell carcinomas (RCCs are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF. Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich proteins implicated in VEGF splicing.

  15. Diverse splicing patterns of exonized Alu elements in human tissues.

    Directory of Open Access Journals (Sweden)

    Lan Lin

    2008-10-01

    Full Text Available Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu-derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu-derived exons in 11 human tissues and detailed RT-PCR analyses of 38 exons, we show that some Alu-derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue-specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle-specific inclusion of an Alu-derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human-specific increase in its transcript inclusion level and muscle specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

  16. Changes in RNA Splicing in Developing Soybean (Glycine max Embryos

    Directory of Open Access Journals (Sweden)

    Delasa Aghamirzaie

    2013-11-01

    Full Text Available Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

  17. Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy.

    Science.gov (United States)

    Chan, Hon; Pearson, C Seth; Green, Cathleen M; Li, Zhong; Zhang, Jing; Belfort, Georges; Shekhtman, Alex; Li, Hongmin; Belfort, Marlene

    2016-10-21

    Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH 3 ) 2 Cl 2 ], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. An alternative splicing isoform of MITA antagonizes MITA-mediated induction of type I IFNs.

    Science.gov (United States)

    Chen, Honghe; Pei, Rongjuan; Zhu, Wandi; Zeng, Rui; Wang, Yun; Wang, Yanyi; Lu, Mengji; Chen, Xinwen

    2014-02-01

    Mediator of IFN regulatory transcription factor 3 activation (MITA) is an important adaptor protein to mediate the induction of type I IFNs. In this study, we identified an alternatively spliced isoform of MITA lacking exon 7, termed MITA-related protein (MRP). MRP shares the N-terminal portion aa 1-253 with MITA but possesses a unique 30-aa sequence at the carboxyl terminal part, therefore lacking the conserved domains including TANK-binding kinase 1 (TBK1) and cyclic diguanylate binding domain. MRP is expressed in multiple tissues and distinct cell lines. Overexpression of MRP inhibited MITA-mediated activation of IFN-β promoter by sendai virus infection and cyclic diguanylate treatment but enhanced that in HSV-1 infection. Interestingly, MRP expression was reduced after Sendai virus infection but was upregulated after HSV-1 infection. Overexpression of MRP inhibited MITA-mediated induction of IFN-β via TBK1-IFN regulatory transcription factor 3 by disrupting the MITA-TBK1 interaction. However, NF-κB pathway was still activated by MRP, as MRP retained the ability to interact with inducible inhibitor of NF-κB (iκB) kinase. Thus, MRP acts as a dominant negative regulator of MITA-mediated induction of IFN production.

  19. MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

    Science.gov (United States)

    Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

    2017-01-01

    An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

  20. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites...... and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice...... in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-.3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also...

  1. 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.

  2. Identification of genome-wide non-canonical spliced regions and analysis of biological functions for spliced sequences using Read-Split-Fly.

    Science.gov (United States)

    Bai, Yongsheng; Kinne, Jeff; Ding, Lizhong; Rath, Ethan C; Cox, Aaron; Naidu, Siva Dharman

    2017-10-03

    It is generally thought that most canonical or non-canonical splicing events involving U2- and U12 spliceosomes occur within nuclear pre-mRNAs. However, the question of whether at least some U12-type splicing occurs in the cytoplasm is still unclear. In recent years next-generation sequencing technologies have revolutionized the field. The "Read-Split-Walk" (RSW) and "Read-Split-Run" (RSR) methods were developed to identify genome-wide non-canonical spliced regions including special events occurring in cytoplasm. As the significant amount of genome/transcriptome data such as, Encyclopedia of DNA Elements (ENCODE) project, have been generated, we have advanced a newer more memory-efficient version of the algorithm, "Read-Split-Fly" (RSF), which can detect non-canonical spliced regions with higher sensitivity and improved speed. The RSF algorithm also outputs the spliced sequences for further downstream biological function analysis. We used open access ENCODE project RNA-Seq data to search spliced intron sequences against the U12-type spliced intron sequence database to examine whether some events could occur as potential signatures of U12-type splicing. The check was performed by searching spliced sequences against 5'ss and 3'ss sequences from the well-known orthologous U12-type spliceosomal intron database U12DB. Preliminary results of searching 70 ENCODE samples indicated that the presence of 5'ss with U12-type signature is more frequent than U2-type and prevalent in non-canonical junctions reported by RSF. The selected spliced sequences have also been further studied using miRBase to elucidate their functionality. Preliminary results from 70 samples of ENCODE datasets show that several miRNAs are prevalent in studied ENCODE samples. Two of these are associated with many diseases as suggested in the literature. Specifically, hsa-miR-1273 and hsa-miR-548 are associated with many diseases and cancers. Our RSF pipeline is able to detect many possible junctions

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

    Science.gov (United States)

    2013-07-01

    the dual role of these proteins can be exploited by splicing re-direction approaches to manipulate their expression, in order to simultaneously...55 Wang, Z. et al. (2012) Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides. Open Biol 2 (10), 120133 56...an antisense-mediated shift of Bcl-x pre-mRNA splicing and antineoplastic agents. J Biol Chem 277 (51), 49374-49382 64 Bauman, J.A. et al. (2010

  5. Antagonistic factors control the unproductive splicing of SC35 terminal intron

    OpenAIRE

    Dreumont, Natacha; Hardy, Sara; Behm-Ansmant, Isabelle; Kister, Liliane; Branlant, Christiane; St?venin, James; Bourgeois, Cyril F.

    2009-01-01

    Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3? untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements respon...

  6. Clinical significance of intronic variants in BRAF inhibitor resistant melanomas with altered BRAF transcript splicing

    OpenAIRE

    Pupo, Gulietta M.; Boyd, Suzanah C.; Fung, Carina; Carlino, Matteo S.; Menzies, Alexander M.; Pedersen, Bernadette; Johansson, Peter; Hayward, Nicholas K.; Kefford, Richard F.; Scolyer, Richard A.; Long, Georgina V.; Rizos, Helen

    2017-01-01

    Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4?8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibito...

  7. 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....

  8. Splice of photonic crystal fibres by use of double phase-conjugate mirror

    Science.gov (United States)

    Nakayama, Yusuke; Okamoto, Atsushi; Takayama, Yoshihisa; Hayano, Yutaka

    2007-05-01

    We present a novel splicing method for photonic crystal fibres (PCFs) with a double phase-conjugate mirror (DPCM). The DPCM is an optical device with photorefractive crystal (PRC) which generates phase-conjugate beams easily. In this report, we experimentally measure the splice losses of the DPCM for transverse PCF offset. We numerically estimate the splice losses in the case that butt coupled PCFs without DPCM. Comparing the experimental and numerical values of the splice loss of PCFs, we discuss the tolerance of the DPCM for the PCF displacement. Also, we discuss the causes of loss inside the DPCM module.

  9. Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

    Directory of Open Access Journals (Sweden)

    Michael Seiler

    2018-04-01

    Full Text Available Summary: Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA, and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like, or hotspot mutation profile (oncogene-like. Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis. : Seiler et al. report that 119 splicing factor genes carry putative driver mutations over 33 tumor types in TCGA. The most common mutations appear to be mutually exclusive and are associated with lineage-independent altered splicing. Samples with these mutations show deregulation of cell-autonomous pathways and immune infiltration. Keywords: splicing, SF3B1, U2AF1, SRSF2, RBM10, FUBP1, cancer, mutation

  10. Quantification of pre-mRNA escape rate and synergy in splicing.

    Science.gov (United States)

    Bonde, Marie Mi; Voegeli, Sylvia; Baudrimont, Antoine; Séraphin, Bertrand; Becskei, Attila

    2014-11-10

    Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. ulfasQTL: an ultra-fast method of composite splicing QTL analysis.

    Science.gov (United States)

    Yang, Qian; Hu, Yue; Li, Jun; Zhang, Xuegong

    2017-01-25

    Alternative splicing plays important roles in many regulatory processes and diseases in human. Many genetic variants contribute to phenotypic differences in gene expression and splicing that determine variations in human traits. Detecting genetic variants that affect splicing phenotypes is essential for understanding the functional impact of genetic variations on alternative splicing. For many situations, the key phenotype is the relative splicing ratios of alternative isoforms rather than the expression values of individual isoforms. Splicing quantitative trait loci (sQTL) analysis methods have been proposed for detecting associations of genetic variants with the vectors of isoform splicing ratios of genes. We call this task as composite sQTL analysis. Existing methods are computationally intensive and cannot scale up for whole genome analysis. We developed an ultra-fast method named ulfasQTL for this task based on a previous method sQTLseekeR. It transforms tests of splicing ratios of multiple genes to a matrix form for efficient computation, and therefore can be applied for sQTL analysis at whole-genome scales at the speed thousands times faster than the existing method. We tested ulfasQTL on the data from the GEUVADIS project and compared it with an existing method. ulfasQTL is a very efficient tool for composite splicing QTL analysis and can be applied on whole-genome analysis with acceptable time.

  12. The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

    Directory of Open Access Journals (Sweden)

    Scott P Kallgren

    Full Text Available Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres.

  13. Trans-Splicing Improvement by the Combined Application of Antisense Strategies

    Directory of Open Access Journals (Sweden)

    Ulrich Koller

    2015-01-01

    Full Text Available Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs, presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB. We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG, lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  14. Non-sequential and multi-step splicing of the dystrophin transcript.

    Science.gov (United States)

    Gazzoli, Isabella; Pulyakhina, Irina; Verwey, Nisha E; Ariyurek, Yavuz; Laros, Jeroen F J; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2016-01-01

    The dystrophin protein encoding DMD gene is the longest human gene. The 2.2 Mb long human dystrophin transcript takes 16 hours to be transcribed and is co-transcriptionally spliced. It contains long introns (24 over 10kb long, 5 over 100kb long) and the heterogeneity in intron size makes it an ideal transcript to study different aspects of the human splicing process. Splicing is a complex process and much is unknown regarding the splicing of long introns in human genes. Here, we used ultra-deep transcript sequencing to characterize splicing of the dystrophin transcripts in 3 different human skeletal muscle cell lines, and explored the order of intron removal and multi-step splicing. Coverage and read pair analyses showed that around 40% of the introns were not always removed sequentially. Additionally, for the first time, we report that non-consecutive intron removal resulted in 3 or more joined exons which are flanked by unspliced introns and we defined these joined exons as an exon block. Lastly, computational and experimental data revealed that, for the majority of dystrophin introns, multistep splicing events are used to splice out a single intron. Overall, our data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.

  15. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    Science.gov (United States)

    Straniero, Letizia; Rimoldi, Valeria; Soldà, Giulia; Mauri, Lucia; Manfredini, Emanuela; Andreucci, Elena; Bargiacchi, Sara; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Asselta, Rosanna; Primignani, Paola

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

  16. Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle.

    Science.gov (United States)

    Bougé, Anne-Laure; Murauer, Eva; Beyne, Emmanuelle; Miro, Julie; Varilh, Jessica; Taulan, Magali; Koenig, Michel; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2017-01-03

    We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3' splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

  17. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

    Science.gov (United States)

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J; Setó-Salvia, Núria; Ryan, Natalie S; Morris, Huw R; Arber, Charles; Devine, Michael J; Houlden, Henry; Warner, Thomas T; Bushell, Trevor J; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J; Fox, Nick C; Rossor, Martin N; Hardy, John; Wray, Selina

    2015-09-15

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. © The Author 2015. Published by Oxford University Press.

  18. Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

    Science.gov (United States)

    Valdés, Jesús; Nozaki, Tomoyoshi; Sato, Emi; Chiba, Yoko; Nakada-Tsukui, Kumiko; Villegas-Sepúlveda, Nicolás; Winkler, Robert; Azuara-Liceaga, Elisa; Mendoza-Figueroa, María Saraí; Watanabe, Natsuki; Santos, Herbert J; Saito-Nakano, Yumiko; Galindo-Rosales, José Manuel

    2014-12-05

    The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2-GST-sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses. Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities. Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite. We have found numerous new splicing-related factors which could have therapeutic benefit. We

  19. Characterization of a splicing mutation in group A xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Satokata, Ichiro; Tanaka, Kiyoji; Miura, Naoyuki; Miyamoto, Iwai; Okada, Yoshio; Satoh, Yoshiaki; Kondo, Seiji

    1990-01-01

    The molecular basis of group A xeroderma pigmentosum (WP) was investigated by comparison of the nucleotide sequences of multiple clones of the XP group A complementing gene (XPAC) from a patient with group A XP with that of a normal gene. The clones showed a G → C substitution at the 3' splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotide to AC. Nucleotide sequencing of cDNAs amplified by the polymerase chain reaction revealed that this single base substitution abolishes the canonical 3' splice site, thus creating two abnormally spliced mRNA forms. The larger form is identical with normal mRNA except for a dinucleotide deletion at the 5' end of exon 4. This deletion results in a frameshift with premature translation termination in exon 4. The smaller form has a deletion of the entire exon 3 and the dinucleotide at the 5' end of exon 4. The result of a transfection study provided additional evidence that this single base substitution is the disease-causing mutation. This single base substitution creates a new cleavage site for the restriction nuclease AlwNI. Analysis of AlwNI restriction fragment length polymorphism showed a high frequency of this mutation in Japanese patients with group A XP: 16 of 21 unrelated Japanese patients were homozygous and 4 were heterozygous for this mutation. However, 11 Caucasians and 2 Blacks with group A XP did not have this mutant allele. The polymorphic AlwNI restriction fragments are concluded to be useful for diagnosis of group A XP in Japanese subjects, including prenatal cases and carriers

  20. Performance of Grouted Splice Sleeve Connector under Tensile Load

    Directory of Open Access Journals (Sweden)

    A. Alias

    2016-05-01

    Full Text Available The grouted splice sleeve connector system takes advantage of the bond-slip resistance of the grout and the mechanical gripping of reinforcement bars to provide resistance to tensile force. In this system, grout acts as a load-transferring medium and bonding material between the bars and sleeve. This study adopted the end-to-end rebars connection method to investigate the effect of development length and sleeve diameter on the bonding performance of the sleeve connector. The end-to-end method refers to the condition where reinforcement bars are inserted into the sleeve from both ends and meet at the centre before grout is filled. Eight specimens of grouted splice sleeve connector were tested under tensile load to determine their performance. The sleeve connector was designed using 5 mm thick circular hollow section (CHS steel pipe and consisted of one external and two internal sleeves. The tensile test results show that connectors with a smaller external and internal sleeve diameter appear to provide better bonding performance. Three types of failure were observed in this research, which are bar fracture (outside the sleeve, bar pullout, and internal sleeve pullout. With reference to these failure types, the development length of 200 mm is the optimum value due to its bar fracture type, which indicates that the tensile capacity of the connector is higher than the reinforcement bar. It is found that the performance of the grouted splice sleeve connector is influenced by the development length of the reinforcement bar and the diameter of the sleeve.

  1. Periostin shows increased evolutionary plasticity in its alternatively spliced region

    Directory of Open Access Journals (Sweden)

    Hoersch Sebastian

    2010-01-01

    Full Text Available Abstract Background Periostin (POSTN is a secreted extracellular matrix protein of poorly defined function that has been related to bone and heart development as well as to cancer. In human and mouse, it is known to undergo alternative splicing in its C-terminal region, which is devoid of known protein domains. Differential expression of periostin, sometimes of specific splicing isoforms, is observed in a broad range of human cancers, including breast, pancreatic, and colon cancer. Here, we combine genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C-terminal region. Results We found that the C-terminal part of periostin is markedly more variable among vertebrates than the rest of periostin in terms of exon count, length, and splicing pattern, which we interpret as a consequence of neofunctionalization after the split between periostin and its paralog transforming growth factor, beta-induced (TGFBI. We also defined periostin's sequential 13-amino acid repeat units - well conserved in teleost fish, but more obscure in higher vertebrates - whose secondary structure is predicted to be consecutive beta strands. We suggest that these beta strands may mediate binding interactions with other proteins through an extended beta-zipper in a manner similar to the way repeat units in bacterial cell wall proteins have been reported to bind human fibronectin. Conclusions Our results, obtained with the help of the increasingly large collection of complete vertebrate genomes, document the evolutionary plasticity of periostin's C-terminal region, and for the first time suggest a basis for its functional role.

  2. Recurrent Hyperparathyroidism Due to a Novel CDC73 Splice Mutation.

    Science.gov (United States)

    Hattangady, Namita Ganesh; Wilson, Tremika Le-Shan; Miller, Barbra Sue; Lerario, Antonio Marcondes; Giordano, Thomas James; Choksi, Palak; Else, Tobias

    2017-08-01

    The recognition of hereditary causes of primary hyperparathyroidism (pHPT) is important because clinical care and surveillance differ significantly between sporadic and hereditary pHPT. In addition, the increasing number of genetic tests poses a challenge to classify mutations as benign or pathogenic. Functional work-up of variants remains a mainstay to provide evidence for pathogenicity. We describe a 52-year-old male patient with recurrent pHPT since age 35 years. Despite several neck surgeries with complete parathyroidectomy, he experienced persistent pHPT, necessitating repeated surgery for a forearm autotransplant, which finally resulted in unmeasurable parathyroid hormone (PTH) levels. Genetic testing revealed a new CDC73 variant (c.238-8G>A [IVS2-8G>A]), initially classified as a variant of uncertain significance. Parathyroid tissue from the initial surgeries showed loss of heterozygosity. Using an RT-PCR approach, we show that the mutation leads to the use of a cryptic splice site in peripheral mononuclear cells. In addition, a minigene approach confirms the use of the cryptic splice site in a heterologous cell system. The novel c.238-8G>A CDC73 variant activates a cryptic splice site, and the functional data provided justify the classification as a likely pathogenic variant. Our results underscore the importance of functional work-up for variant classification in the absence of other available data, such as presence in disease-specific databases, other syndromic clinical findings, or family history. In addition, the presented case exemplifies the importance to consider a hereditary condition in young patients with pHPT, particularly those with multi-gland involvement. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  3. G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

    Science.gov (United States)

    Verma, Shiv Prakash; Das, Parimal

    2018-03-01

    Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. hnRNP A1 and hnRNP F modulate the alternative splicing of exon 11 of the insulin receptor gene.

    Directory of Open Access Journals (Sweden)

    Indrani Talukdar

    Full Text Available Exon 11 of the insulin receptor gene (INSR is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5' GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3' end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5' splice site of intron 11. The SR protein SRSF1 (SF2/ASF co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

  5. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

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

    Directory of Open Access Journals (Sweden)

    Anamika Krishanpal

    2009-12-01

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    , PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three...

  8. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  9. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics

    DEFF Research Database (Denmark)

    Roca, Xavier; Olson, Andrew J; Rao, Atmakuri R

    2008-01-01

    Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies...

  10. Conservation and sex-specific splicing of the doublesex gene in the ...

    Indian Academy of Sciences (India)

    Genetic control of sex determination in insects has been best characterized in Drosophila melanogaster, where the master gene Sxl codes for RNA that is sex specifically spliced to produce a functional protein only in females. SXL regulates the sex-specific splicing of transformer (tra) RNA which, in turn, regulates the ...

  11. Differential dynamics of splicing factor SC35 during the cell cycle

    Indian Academy of Sciences (India)

    Srinivas

    We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized ... Cell cycle dynamics; FRAP analysis; mitotic interchromatin granules; splicing factor SC35 .... for 1 h at room temperature for single labelling experiments.

  12. Effect of tension lap splice on the behavior of high strength concrete (HSC beams

    Directory of Open Access Journals (Sweden)

    Ahmed El-Azab

    2014-12-01

    Full Text Available In the recent years, many research efforts have been carried out on the bond strength between normal strength concrete (NSC and reinforcing bars spliced in tension zones in beams. Many codes gave a minimum splice length for tension and compression reinforcement as a factor of the bar diameter depending on many parameters such as concrete strength, steel yield stress, shape of bar end, shape of bar surface and also bar location. Also, codes gave another restriction about the percentage of total reinforcement to be spliced at the same time. Comparatively limited attention has been directed toward the bond between high strength concrete (HSC and reinforcing bars spliced in tension zones in beams. HSC has high modulus of elasticity, high density and long-term durability. This research presents an experimental study on the bond between high strength concrete (HSC and reinforcing bars spliced in tension zones in beams. It reports the influence of several parameters on bond in splices. The parameters covered are casting position, splice length as a factor of bar diameter, bar diameter and reinforcement ratio. The research involved tests on sixteen simply-supported beams of 1800 mm span, 200 mm width and 400 mm thickness made of HSC. In each beam, the total tensile steel bars were spliced in the constant moment zone. Crack pattern, crack propagation, cracking load, failure load and mi span deflection were recorded and analyzed to study the mentioned parameters effect.

  13. Measurement of Resistance and Strength of Conductor Splices in the MICE Coupling Magnets

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Feng Yu; Pan, Heng; Wu, Hong; Lui, X. K.; Li, E.; Dietderich, Dan; Higley, Hugh; Tam, D. G.; Trillaud, Fredric; Wang, Li; Green, M.A.

    2009-08-19

    The superconducting magnets for the Muon Ionization Cooling Experiment [1] (MICE) use a copper based Nb-Ti conductor with un-insulated dimensions of 0.95 by 1.60 mm. There may be as many as twelve splices in one MICE superconducting coupling coil. These splices are to be wound in the coil. The conductor splices produce Joule heating, which may cause the magnet to quench. A technique of making conductor splices was developed by ICST. Two types of 1-meter long of soldered lap-joints have been tested. Side-by-side splices and up-down one splices were studied theoretically and experimentally using two types of soft solder made of eutectic tin-lead solder and tin-silver solder. The resistances of the splices made by ICST were tested at LBNL at liquid helium temperatures over a range of magnetic fields up to 5 T. The breaking strength of 250 mm long splices was also measured at room temperature and liquid nitrogen temperature.

  14. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    Energy Technology Data Exchange (ETDEWEB)

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

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

    African Journals Online (AJOL)

    user6

    2012-05-15

    May 15, 2012 ... provide some useful information for further studies into the function of the bovine ATP1B2 gene. Alternative splicing (AS) is recognized as the major contributor to protein diversity from limited gene pool. ATP1B2-AS2 was the splice of intron retention found from ATP1B2 in liver, kidney, muscle and.

  16. Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ...

    Indian Academy of Sciences (India)

    Unknown

    structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in certain pathological conditions. ..... show the genomic structures of the normal (diagram “a”) and abnormal (diagram “b” and “c”) splicing forms. Inserted and deleted sequences are indicated ...

  17. A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni.

    Science.gov (United States)

    Mourão, Marina de Moraes; Bitar, Mainá; Lobo, Francisco Pereira; Peconick, Ana Paula; Grynberg, Priscila; Prosdocimi, Francisco; Waisberg, Michael; Cerqueira, Gustavo Coutinho; Macedo, Andréa Mara; Machado, Carlos Renato; Yoshino, Timothy; Franco, Glória Regina

    2013-09-01

    Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

  18. A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Marina de Moraes Mourao

    2013-09-01

    Full Text Available Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779, (ii female adult worms (LIBEST_028000: JZ139780 - JZ140379, (iii male adult worms (LIBEST_028001: JZ140380 - JZ141002, (iv eggs (LIBEST_028002: JZ141003 - JZ141497 and (v schistosomula (LIBEST_028003: JZ141498 - JZ141974.

  19. Self-splicing of a group IIC intron: 5? exon recognition and alternative 5? splicing events implicate the stem?loop motif of a transcriptional terminator

    OpenAIRE

    Toor, Navtej; Robart, Aaron R.; Christianson, Joshua; Zimmerly, Steven

    2006-01-01

    Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interesting...

  20. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    Science.gov (United States)

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  1. Towards a Consolidation of LHC Superconducting Splices for 7 TeV Operation

    CERN Document Server

    Bertinelli, F; Fessia, P; Garion, C; Mathot, S; Perin, A; Scheuerlein, C; Sgobba, S; ten Kat, H; Tock, J P; Verweij, A; Willering, G

    2010-01-01

    Following the analysis of the September 2008 LHC incident, the assembly process and the quality assurance of the main 13 kA interconnection splices were improved, with new measurement and diagnostics methods introduced. During the 2008-2009 shutdown ~5% of these 10 000 splices were newly assembled with these improvements implemented, but essentially maintaining the original design. It is known today that a limiting factor towards 7 TeV operation is the normal conducting resistance of ~15% of the original main 13 kA interconnection splices, associated to the electrical continuity of the copper stabiliser. A “Splices Task Force” has been set up at CERN to evaluate the need for, develop and test design improvements and prepare the implementation of a consolidation campaign. Important issues of splice design, process choice, resources and time requirements are considered.

  2. An experimental investigation on contact compression lap splice in circular columns

    Directory of Open Access Journals (Sweden)

    Hamed S. Askar

    2016-08-01

    The objective of the present investigation is to study the influence of splice length, volume of transverse reinforcement and end bearing condition on the behavior of compression lap splice. The conducted investigation included experimental tests of nine circular columns under uniaxial compression loads. All spliced bars were in contact with each other and with constant concrete cover. Based on the experimental investigation and test results, concluding remarks have been drawn, based on which a design simplified equation for splice length in compression has been developed. A correlation between the experimental and calculated results of the author specimens and other results available in literature, showed a good agreement. Also, the formulas adapted by different codes for predicting the compression lap splice length have been checked with the proposed equation.

  3. Fractional Differential Texture Descriptors Based on the Machado Entropy for Image Splicing Detection

    Directory of Open Access Journals (Sweden)

    Rabha W. Ibrahim

    2015-07-01

    Full Text Available Image splicing is a common operation in image forgery. Different techniques of image splicing detection have been utilized to regain people’s trust. This study introduces a texture enhancement technique involving the use of fractional differential masks based on the Machado entropy. The masks slide over the tampered image, and each pixel of the tampered image is convolved with the fractional mask weight window on eight directions. Consequently, the fractional differential texture descriptors are extracted using the gray-level co-occurrence matrix for image splicing detection. The support vector machine is used as a classifier that distinguishes between authentic and spliced images. Results prove that the achieved improvements of the proposed algorithm are compatible with other splicing detection methods.

  4. Controlled synthesis of target strings in a class of splicing systems.

    Science.gov (United States)

    Chen, Peter C Y

    2005-08-01

    This article presents an approach for synthesizing target strings in a class of computational models of DNA recombination. The computational models are formalized as splicing systems in the context of formal languages. Given a splicing system (of a restricted type) and a target string to be synthesized, we construct (i) a rule-embedded splicing automaton that recognizes languages containing strings embedded with symbols representing splicing rules, and (ii) an automaton that implicitly recognizes the target string. By manipulating these two automata, we extract all rule sequences that lead to the production of the target string (if that string belongs to the splicing language). An algorithm for synthesizing a certain type of target strings based on such rule sequences is presented.

  5. Genetic variations and alternative splicing. The Glioma associated oncogene 1, GLI1.

    Directory of Open Access Journals (Sweden)

    Peter eZaphiropoulos

    2012-07-01

    Full Text Available Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation.

  6. Coding potential of the products of alternative splicing in human.

    KAUST Repository

    Leoni, Guido

    2011-01-20

    BACKGROUND: Analysis of the human genome has revealed that as much as an order of magnitude more of the genomic sequence is transcribed than accounted for by the predicted and characterized genes. A number of these transcripts are alternatively spliced forms of known protein coding genes; however, it is becoming clear that many of them do not necessarily correspond to a functional protein. RESULTS: In this study we analyze alternative splicing isoforms of human gene products that are unambiguously identified by mass spectrometry and compare their properties with those of isoforms of the same genes for which no peptide was found in publicly available mass spectrometry datasets. We analyze them in detail for the presence of uninterrupted functional domains, active sites as well as the plausibility of their predicted structure. We report how well each of these strategies and their combination can correctly identify translated isoforms and derive a lower limit for their specificity, that is, their ability to correctly identify non-translated products. CONCLUSIONS: The most effective strategy for correctly identifying translated products relies on the conservation of active sites, but it can only be applied to a small fraction of isoforms, while a reasonably high coverage, sensitivity and specificity can be achieved by analyzing the presence of non-truncated functional domains. Combining the latter with an assessment of the plausibility of the modeled structure of the isoform increases both coverage and specificity with a moderate cost in terms of sensitivity.

  7. Comparative cross-species alternative splicing in plants.

    Science.gov (United States)

    Ner-Gaon, Hadas; Leviatan, Noam; Rubin, Eitan; Fluhr, Robert

    2007-07-01

    Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less AS than animals. An algorithm, based on expressed sequence tag (EST) pairs gapped alignment, was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for AS. EST pairs gapped alignment was first evaluated in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum) for which annotated genome sequence is available and was shown to accurately predict splicing events. The method was then applied to 11 plant species that include 17 cultivars for which enough ESTs are available. The results show a large, 3.7-fold difference in AS rates between plant species with Arabidopsis and rice in the lower range and lettuce (Lactuca sativa) and sorghum (Sorghum bicolor) in the upper range. Hence, compared to higher animals, plants show a much greater degree of variety in their AS rates and in some plant species the rates of animal and plant AS are comparable although the distribution of AS types may differ. In eudicots but not monocots, a correlation between genome size and AS rates was detected, implying that in eudicots the mechanisms that lead to larger genomes are a driving force for the evolution of AS.

  8. The emergence of alternative 3' and 5' splice site exons from constitutive exons.

    Directory of Open Access Journals (Sweden)

    Eli Koren

    2007-05-01

    Full Text Available Alternative 3' and 5' splice site (ss events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving, similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.

  9. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG).

    Science.gov (United States)

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-08-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations.

  10. Splice Expression Variation Analysis (SEVA) for Inter-tumor Heterogeneity of Gene Isoform Usage in Cancer.

    Science.gov (United States)

    Afsari, Bahman; Guo, Theresa; Considine, Michael; Florea, Liliana; Kagohara, Luciane T; Stein-O'Brien, Genevieve L; Kelley, Dylan; Flam, Emily; Zambo, Kristina D; Ha, Patrick K; Geman, Donald; Ochs, Michael F; Califano, Joseph A; Gaykalova, Daria A; Favorov, Alexander V; Fertig, Elana J

    2018-01-12

    Current bioinformatics methods to detect changes in gene isoform usage in distinct phenotypes compare the relative expected isoform usage in phenotypes. These statistics model differences in isoform usage in normal tissues, which have stable regulation of gene splicing. Pathological conditions, such as cancer, can have broken regulation of splicing that increases the heterogeneity of the expression of splice variants. Inferring events with such differential heterogeneity in gene isoform usage requires new statistical approaches. We introduce Splice Expression Variability Analysis (SEVA) to model increased heterogeneity of splice variant usage between conditions (e.g., tumor and normal samples). SEVA uses a rank-based multivariate statistic that compares the variability of junction expression profiles within one condition to the variability within another. Simulated data show that SEVA is unique in modeling heterogeneity of gene isoform usage, and benchmark SEVA's performance against EBSeq, DiffSplice, and rMATS that model differential isoform usage instead of heterogeneity. We confirm the accuracy of SEVA in identifying known splice variants in head and neck cancer and perform cross-study validation of novel splice variants. A novel comparison of splice variant heterogeneity between subtypes of head and neck cancer demonstrated unanticipated similarity between the heterogeneity of gene isoform usage in HPV-positive and HPV-negative subtypes and anticipated increased heterogeneity among HPV-negative samples with mutations in genes that regulate the splice variant machinery. These results show that SEVA accurately models differential heterogeneity of gene isoform usage from RNA-seq data. SEVA is implemented in the R/Bioconductor package GSReg. bahman@jhu.edu, ejfertig@jhmi.edu. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  11. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    Science.gov (United States)

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  12. Ultraviolet B (UVB) induced DNA damage affects alternative splicing in skin cells

    International Nuclear Information System (INIS)

    Munoz, M.J.; Nieto Moreno, N.; Kornblihtt, A.R.

    2010-01-01

    The ultraviolet (UV) radiation from the Sun that reaches the Earth's surface is a combination of low (UVA, 320-400 nm) and high (UVB, 290-320 nm) energy light. UVB light causes two types of mutagenic DNA lesions: thymine dimers and (6-4) photo-products. UVB mutagenesis is a critical step in the generation of different forms of skin cancer, which develops almost exclusively in sun exposed areas. We have previously shown that RNA polymerase II (pol II) hyperphosphorylation induced by UVC (254 nm) irradiation of non-skin cells inhibits pol II elongation rates which in turn affects alternative splicing (AS) patterns, altering the synthesis of pro- and anti-apoptotic isoforms of key proteins like Bcl-x or Caspase 9 (C9). Since the UVC radiation is fully filtered by the ozone layer and AS regulation in skin pathologies has been poorly studied, we decided to extend our studies to human keratinocytes in culture treated with UVB (302 nm) light. We observed that pol II hyperphosphorylation is increased upon UVB irradiation, being this modification necessary for the observed change in AS of a model cassette exon. Moreover, UVB irradiation induces the proapoptotic mRNA isoforms of Bcl-x and C9 consistently with a key role of AS in skin response to DNA damage. (authors)

  13. 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.

  14. Ultraviolet B (UVB) induced DNA damage affects alternative splicing in skin cells

    International Nuclear Information System (INIS)

    Nieto Moreno, N.; Dujardin, G.; Kornblihtt, A.R.; Muñoz, M.J.

    2011-01-01

    The ultraviolet (UV) radiation from the Sun that reaches the Earth’s surface is a combination of low (UVA, 320-400nm) and high (UVB, 290-320nm) energy light. UVB light causes two types of mutagenic DNA lesions: thymine dimers and (6-4)photo-products. UVB mutagenesis is critical in the generation of skin cancer. We have previously shown that RNA polymerase II (pol II) hyperphosphorylation induced by UVC (254 nm) irradiation of non-skin cells inhibits pol II elongation rates which in turn affects alternative splicing (AS) patterns favouring the synthesis of proapoptotic isoforms of key proteins like Bcl-x or Caspase 9 (C9). As UVC radiation is fully filtered by the ozone layer and AS regulation in skin pathologies has been poorly studied, we decided to extend our studies to human keratinocytes in culture treated with UVB (302nm) light. We observed an increase in pol II hyperphosphorylation, being this modification necessary for the change in AS of a model cassette exon. Moreover, UVB irradiation induces the pro-apoptotic mRNA isoforms of Bcl-x and C9 being these consistent with a key role of AS in response to DNA damage. Our results suggest that UVC and UVB light affect AS decisions through a similar mechanism. This indicates that lower energy irradiation, causing more limited DNA damage than UVC light, is sufficient to alter qualitatively patterns of gene expression in skin cells. (authors)

  15. CRISPR/Cas9-mediated target validation of the splicing inhibitor Pladienolide B

    Directory of Open Access Journals (Sweden)

    Mustapha Aouida

    2016-12-01

    Full Text Available CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

  16. 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

  17. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

  18. Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome.

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    Ronghui Li

    2016-06-01

    Full Text Available Mutations in the human MECP2 gene cause Rett syndrome (RTT, a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.

  19. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina

    Science.gov (United States)

    Murphy, Daniel; Carstens, Russ

    2016-01-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such “switch-like” exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  20. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

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    Nathan Salomonis

    2009-11-01

    Full Text Available The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

  1. Benefits of CO2 laser heating for high reliability fiber splicing

    Science.gov (United States)

    Duke, Douglas M.; Nasir, Usman; Saravanos, Elli

    2016-03-01

    The use of a CO2 laser as a heat source became commercially available for optical fiber splicing and component fabrication only in recent years. In addition to long-term trouble-free and low-maintenance heat source operation, laser fusion splicing offers unique benefits for fabricating high-power optical components, as well as for splice reliability. When used as the heating method for fiber splicing, the energy of the CO2 laser beam is efficiently absorbed by the outer layer of the glass, and is then conducted inwards. This heating method is well controlled, and results in a smooth and contamination-free glass surface. Other heating methods, such as arc fusion or resistive heating, may leave tungsten, graphite, or metal oxide deposits on the spliced fiber surface. By contrast, with CO2 laser splicing, the lack of surface irregularities and contamination enables remarkable spliced-fiber strength results, with some strength results nearly within the range of coated fiber breaking strength.

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

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    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.

  3. Alternative RNA Splicing in the Pathogenesis of Liver Disease

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    Nicholas J. G. Webster

    2017-06-01

    Full Text Available Non-alcoholic fatty liver disease (NAFLD is becoming increasingly prevalent due to the worldwide obesity epidemic and currently affects one-third of adults or about one billion people worldwide. NAFLD is predicted to affect over 50% of the world’s population by the end of the next decade. It is the most common form of liver disease and is associated with increased risk for progression to a more severe form non-alcoholic steatohepatitis, as well as insulin resistance, type 2 diabetes mellitus, cirrhosis, and eventually hepatocellular carcinoma. This review article will focus on the role of alternative splicing in normal liver physiology and dysregulation in liver disease.

  4. Splice variants of porcine PPHLN1 encoding periphilin-1

    DEFF Research Database (Denmark)

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

    2017-01-01

    splice variants hereof. RT-PCR cloning using oligonucleotide primers derived from in silico sequences resulted in three PPHLN1 transcripts: a full-length mRNA and two transcript variant resulting in shorter proteins. The longest encoded periphilin-1, consisting of 373 amino acids, displays a high......The periphilin-1 protein is encoded by the PPHLN1 gene. Periphilin-1 is found in the cornified cell envelope during the terminal differentiation of keratinocyte at the outer layer of epidermis. In the current study we report on the cloning and characterization of the porcine PPHLN1 cDNA and two...... homology to the human periphilin-1 protein coded by the transcript variant 2 (91%). A shorter transcript variant (PPHLN1Sp1) contains a 1065-codon ORF, which is consistent with that of the authentic PPHLN1, but lacks a region of 57 bp spanning exon 7. Hence, the encoded polypeptide periphilin-1Sp1 consists...

  5. Novel Alternative Splice Variants of Mouse Cdk5rap2.

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    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.

  6. Splice-Switching Therapy for Spinal Muscular Atrophy

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    Katharina E. Meijboom

    2017-06-01

    Full Text Available Spinal muscular atrophy (SMA is a genetic disorder with severity ranging from premature death in infants to restricted motor function in adult life. Despite the genetic cause of this disease being known for over twenty years, only recently has a therapy been approved to treat the most severe form of this disease. Here we discuss the genetic basis of SMA and the subsequent studies that led to the utilization of splice switching oligonucleotides to enhance production of SMN protein, which is absent in patients, through a mechanism of exon inclusion into the mature mRNA. Whilst approval of oligonucleotide-based therapies for SMA should be celebrated, we also discuss some of the limitations of this approach and alternate genetic strategies that are currently underway in clinical trials.

  7. IMAGE SPLICING DETECTION BASED ON DEMOSAICKING AND WAVELET TRANSFORMATION

    Directory of Open Access Journals (Sweden)

    Endina Putri Purwandari

    2015-03-01

    Full Text Available Image splicing is a form of digital image manipulation by combining two or more image into a new image. The application was developed through a passive approach using demosaicking and wavelet transformation method. This research purposed a method to implement the demosaicking and wavelet transform for digital image forgery detection with a passive approach. This research shows that (1 demosaicking can be used as a comparison image in forgery detection; (2 the application of demosaicking and wavelet transformation can improve the quality of the input image (3 demosaicking and wavelet algorithm are able to estimate whether the input image is real or fake image with a passive approach and estimate the manipulation area from the input image.

  8. BBMap: A Fast, Accurate, Splice-Aware Aligner

    Energy Technology Data Exchange (ETDEWEB)

    Bushnell, Brian

    2014-03-17

    Alignment of reads is one of the primary computational tasks in bioinformatics. Of paramount importance to resequencing, alignment is also crucial to other areas - quality control, scaffolding, string-graph assembly, homology detection, assembly evaluation, error-correction, expression quantification, and even as a tool to evaluate other tools. An optimal aligner would greatly improve virtually any sequencing process, but optimal alignment is prohibitively expensive for gigabases of data. Here, we will present BBMap [1], a fast splice-aware aligner for short and long reads. We will demonstrate that BBMap has superior speed, sensitivity, and specificity to alternative high-throughput aligners bowtie2 [2], bwa [3], smalt, [4] GSNAP [5], and BLASR [6].

  9. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.

    Science.gov (United States)

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-07-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. © 2015 Marquez et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Diffusion MR imaging with PSIF and SPLICE. Experiences in phantom studies and the central nervous system

    International Nuclear Information System (INIS)

    Uchikoshi, Masato; Ueda, Takashi; Kaji, Yasushi

    2001-01-01

    Studies have shown that diffusion MR imaging is a reliable method for the diagnosis of central nervous system diseases, especially acute cerebral infarction. Although echo planar imaging (EPI) is a promising tool for that purpose, it is vulnerable to susceptibility artifacts that are responsible for image distortion or signal loss. Our purpose in this study was to evaluate the usefulness of diffusion MR imaging with PSIF (reversed fast imaging SSFP) and split acquisition of fast-spin-echo signals for diffusion imaging (SPLICE) in the central nervous system (CNS). First, PSIF and SPLICE were applied to the phantoms. Each phantom, including acetone, acetic acid, and water, was analyzed for apparent diffusion coefficient (ADC) based on SPLICE and for diffusion-related coefficient (DRC) based on PSIF. The ADCs based on SPLICE were 4.36±0.89 x 10 -3 mm 2 /sec, 1.25±0.04 x 10 -3 mm 2 /sec, and 2.35±0.04 x 10 -3 mm 2 /sec, and the DRCs based on PSIF were 0.353±0.25, 0.178±0.07, and 0.273±0.018 for acetone, acetic acid, and water, respectively. These calculated ADCs based on SPLICE were well correlated with known diffusion coefficients, showing a correlation coefficient of 0.995. Second, PSIF and SPLICE were applied to the CNS. The advantage of PSIF and SPLICE was that susceptibility artifacts were reduced in the images of spinal cord and brain stem. PSIF was especially useful for diffusion MR imaging in the spinal cord. The disadvantage of SPLICE was the decreased SN ratio. We conclude that PSIF or SPLICE may be helpful when EPI diffusion MR imaging is insufficient. (author)

  11. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  12. Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans

    DEFF Research Database (Denmark)

    Heintz, Caroline; Doktor, Thomas K; Lanjuin, Anne

    2017-01-01

    via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also...... homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction...

  13. A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

    OpenAIRE

    Hebbar, S K; Belcher, S M; Perlman, P S

    1992-01-01

    Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or...

  14. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    Science.gov (United States)

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  15. 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.

  16. Systematic profiling of poly(A+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

    Directory of Open Access Journals (Sweden)

    Wencheng Li

    2015-04-01

    Full Text Available Alternative cleavage and polyadenylation (APA results in mRNA isoforms containing different 3' untranslated regions (3'UTRs and/or coding sequences. How core cleavage/polyadenylation (C/P factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A sites (pAs, CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

  17. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    Science.gov (United States)

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  18. Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene.

    Science.gov (United States)

    Ding, Qiulan; Wu, Wenman; Fu, Qihua; Wang, Xuefeng; Hu, Yiqun; Wang, Hongli; Wang, Zhenyi

    2005-06-01

    Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype.

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

    Science.gov (United States)

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

    2016-07-01

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

  20. The Integrity of ACSR Full Tension Single-Stage Splice Connector at Higher Operation Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jy-An John [ORNL; Lara-Curzio, Edgar [ORNL; King Jr, Thomas J [ORNL

    2008-10-01

    Due to increases in power demand and limited investment in new infrastructure, existing overhead power transmission lines often need to operate at temperatures higher than those used for the original design criteria. This has led to the accelerated aging and degradation of splice connectors. It is manifested by the formation of hot-spots that have been revealed by infrared imaging during inspection. The implications of connector aging is two-fold: (1) significant increases in resistivity of the splice connector (i.e., less efficient transmission of electricity) and (2) significant reductions in the connector clamping strength, which could ultimately result in separation of the power transmission line at the joint. Therefore, the splice connector appears to be the weakest link in electric power transmission lines. This report presents a protocol for integrating analytical and experimental approaches to evaluate the integrity of full tension single-stage splice connector assemblies and the associated effective lifetime at high operating temperature.

  1. Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation.

    Science.gov (United States)

    Runfola, Valeria; Sebastian, Soji; Dilworth, F Jeffrey; Gabellini, Davide

    2015-02-15

    Among the Mef2 family of transcription factors, Mef2D is unique in that it undergoes tissue-specific splicing to generate an isoform that is essential for muscle differentiation. However, the mechanisms mediating this muscle-specific processing of Mef2D remain unknown. Using bioinformatics, we identified Rbfox proteins as putative modulators of Mef2D muscle-specific splicing. Accordingly, we found direct and specific Rbfox1 and Rbfox2 binding to Mef2D pre-mRNA in vivo. Gain- and loss-of-function experiments demonstrated that Rbfox1 and Rbfox2 cooperate in promoting Mef2D splicing and subsequent myogenesis. Thus, our findings reveal a new role for Rbfox proteins in regulating myogenesis through activation of essential muscle-specific splicing events. © 2015. Published by The Company of Biologists Ltd.

  2. The Role of the Polypyrimidine Tract Binding Protein on CD44 Alternative Splicing in Breast Cancer

    National Research Council Canada - National Science Library

    Wagner, Eric

    2001-01-01

    ... of changes seen in breast cancer cells during tumor progression. Thus far, a strong connection between the splicing machinery and these subtle, yet significant, changes in gene expression has yet to be documented...

  3. Splice performance evaluation of enamel-coated rebar for structural safety.

    Science.gov (United States)

    2014-07-01

    This report summarizes the findings and results from an experimental study of vitreous enamel coating effects on the bond : strength between deformed rebar and normal strength concrete. A total of 24 beam splice specimens were tested under four-point...

  4. Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR

    Directory of Open Access Journals (Sweden)

    Yongchang Chang

    2012-04-01

    Full Text Available Endothelial cells (ECs that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2/endothelial cell-specific chemotaxis regulator (ECSCR. In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2, differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293. Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF-induced cell migration. Taken together, we have provided several lines

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

    Science.gov (United States)

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

    2017-09-15

    Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  6. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    Energy Technology Data Exchange (ETDEWEB)

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  7. 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...

  8. Temperature induced alternative splicing is affected in sdg8 and sdg26

    OpenAIRE

    Pajoro, A.; Severing, E.I.; Immink, G.H.

    2017-01-01

    Plants developed a plasticity to environmental conditions, such as temperature, that allows their adaptation. A change in ambient temperature leads to changes in the transcriptome in plants, such as the production of different splicing isoforms. Here we study temperature induced alternative splicing events in Arabidopsis thaliana wild-type and two epigenetic mutants, sdg8-2 and sdg26-1 using an RNA-seq approach.

  9. The influence of calcium signaling on the regulation of alternative splicing.

    Science.gov (United States)

    Krebs, Joachim

    2009-06-01

    In this review the influence of calcium signaling on the regulation of alternative splicing is discussed with respect to its influence on cell- and developmental-specific expression of different isoforms of the plasma membrane calcium pump (PMCA). In a second part the possibility is discussed that due to the interaction of the calcium-binding protein ALG-2 with a spliceosomal regulator of alternative splicing, RBM22, Ca2+-signaling may thus influence its regulatory property.

  10. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

    Directory of Open Access Journals (Sweden)

    Noam Leviatan

    Full Text Available Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  11. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

    Science.gov (United States)

    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  12. Group-II intron splicing factors in higher-plants mitochondria

    Directory of Open Access Journals (Sweden)

    Gregory G. Brown

    2014-02-01

    Full Text Available Group-II introns are large catalytic RNAs (ribozymes which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group-II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has retained in the mtDNA of angiosperms: matR encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4. Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4, which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory

  13. SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.

    Science.gov (United States)

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-08-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5' and 3' splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.

  14. Histone H3 lysine 36 methylation affects temperature-induced alternative splicing and flowering in plants.

    Science.gov (United States)

    Pajoro, A; Severing, E; Angenent, G C; Immink, R G H

    2017-06-01

    Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time. We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.

  15. Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

    Directory of Open Access Journals (Sweden)

    Robert M. Martin

    2013-09-01

    Full Text Available Removal of introns from pre-messenger RNAs (pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py tract in mouse immunoglobulin μ (IgM pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.

  16. Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

    Directory of Open Access Journals (Sweden)

    Yanqi Hao

    2015-07-01

    Full Text Available Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse, which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing.

  17. Antagonistic factors control the unproductive splicing of SC35 terminal intron.

    Science.gov (United States)

    Dreumont, Natacha; Hardy, Sara; Behm-Ansmant, Isabelle; Kister, Liliane; Branlant, Christiane; Stévenin, James; Bourgeois, Cyril F

    2010-03-01

    Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3' untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3' splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.

  18. Experimental Investigation for Behavior of Spliced Continuous RC Girders Strengthened with CFRP Laminates

    Directory of Open Access Journals (Sweden)

    Ammar Yasir Ali

    2016-03-01

    Full Text Available In this paper, the behavior of spliced continuous reinforced concrete girders was experimentally investigated. The main objective was to examine the contribution of the carbon fiber reinforced polymer (CFRP laminates in strengthening the spliced continuous reinforced concrete girders. Eight models of continuous reinforced concrete girder were constructed and tested. The test variables were strengthening the splice joints by different schemes of CFRP laminates, presence of horizontal stirrups through the interfaces of the joints and using binder material at the interfaces of the joints. The results showed that strengthening the continuous spliced girders with 45° inclined CFRP laminates led to an increase in the ultimate load in a range of (47 to 74%. Besides, strengthening the continuous spliced girder with horizontal CFRP laminates bonded at its lateral faces could increase the ultimate load by 70%. Additionally, the ultimate load of the continuous spliced girder was increased by (30% due to presence of the horizontal steel stirrups through the interfaces of the joints

  19. Gene Therapy via Trans-Splicing for LMNA-Related Congenital Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Feriel Azibani

    2018-03-01

    Full Text Available We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT transcripts. We developed 5′-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

  20. Electrical Resistance of the Solder Connections for the Consolidation of the LHC Main Interconnection Splices

    CERN Document Server

    Lutum, R; Scheuerlein, C

    2013-01-01

    For the consolidation of the LHC 13 kA main interconnection splices, shunts will be soldered onto each of the 10170 splices. The solder alloy selected for this purpose is Sn60Pb40. In this context the electrical resistance of shunt to busbar lap splices has been measured in the temperature range from RT to 20 K. A cryocooler set-up has been adapted such that a test current of 150 A could be injected for accurate resistance measurements in the low nΩ range. To study the influence of the solder bulk resistivity on the overall splice resistance, connections produced with Sn96Ag4 and Sn77.2In20Ag2.8 have been studied as well. The influence of the Sn60Pb40 solder resistance is negligible when measuring the splice resistance in a longitudinal configuration over a length of 6 cm. In a transverse measurement configuration the splice resistance is significantly influenced by the solder. The connections prepared with Sn77.2In20Ag2.8 show significantly higher resistance values, as expected from the relatively high sol...

  1. FUBP1: a new protagonist in splicing regulation of the DMD gene.

    Science.gov (United States)

    Miro, Julie; Laaref, Abdelhamid Mahdi; Rofidal, Valérie; Lagrafeuille, Rosyne; Hem, Sonia; Thorel, Delphine; Méchin, Déborah; Mamchaoui, Kamel; Mouly, Vincent; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2015-02-27

    We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot.

    Science.gov (United States)

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin'ichi; Tsukahara, Toshifumi

    2016-10-13

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

  3. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

    Directory of Open Access Journals (Sweden)

    Delphine Laustriat

    2015-01-01

    Full Text Available Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1, a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

  4. 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.

  5. The evolutionary landscape of intergenic trans-splicing events in insects

    Science.gov (United States)

    Kong, Yimeng; Zhou, Hongxia; Yu, Yao; Chen, Longxian; Hao, Pei; Li, Xuan

    2015-01-01

    To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of ‘breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes. PMID:26521696

  6. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    Science.gov (United States)

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

  7. Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

    Energy Technology Data Exchange (ETDEWEB)

    Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

    2006-06-15

    A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

  8. DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation

    DEFF Research Database (Denmark)

    Close, Pierre; East, Philip; Dirac-Svejstrup, A Barbara

    2012-01-01

    Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre-mRNA em...... elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.......Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre...... and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD...

  9. Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

    Science.gov (United States)

    Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

    2017-10-01

    Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

  10. Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha; Conboy, John G.

    2009-02-03

    Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

  11. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    Science.gov (United States)

    Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H; Selbach, Matthias; Barton, Paul J R; Cook, Stuart A; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

    2014-08-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.

  12. A splice isoform of DNedd4, DNedd4-long, negatively regulates neuromuscular synaptogenesis and viability in Drosophila.

    Directory of Open Access Journals (Sweden)

    Yunan Zhong

    Full Text Available Neuromuscular (NM synaptogenesis is a tightly regulated process. We previously showed that in flies, Drosophila Nedd4 (dNedd4/dNedd4S is required for proper NM synaptogenesis by promoting endocytosis of commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNedd4 is an E3 ubiquitin ligase comprised of a C2-WW(x3-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNedd4-short (dNedd4S and dNedd4-long (dNedd4Lo.We show here that while dNedd4S is essential for NM synaptogenesis, the dNedd4Lo isoform inhibits this process and causes lethality. Our results reveal that unlike dNedd4S, dNedd4Lo cannot rescue the lethality of dNedd4 null (DNedd4(T121FS flies. Moreover, overexpression of UAS-dNedd4Lo specifically in wildtype muscles leads to NM synaptogenesis defects, impaired locomotion and larval lethality. These negative effects of dNedd4Lo are ameliorated by deletion of two regions (N-terminus and Middle region unique to this isoform, and by inactivating the catalytic activity of dNedd4Lo, suggesting that these unique regions, as well as catalytic activity, are responsible for the inhibitory effects of dNedd4Lo on synaptogenesis. In accord with these findings, we demonstrate by sqRT-PCR an increase in dNedd4S expression relative to the expression of dNedd4Lo during embryonic stages when synaptogenesis takes place.Our studies demonstrate that splice isoforms of the same dNedd4 gene can lead to opposite effects on NM synaptogenesis.

  13. MAp19, the alternative splice product of the MASP2 gene.

    Science.gov (United States)

    Degn, Søren E; Thiel, Steffen; Nielsen, Ole; Hansen, Annette G; Steffensen, Rudi; Jensenius, Jens C

    2011-10-28

    The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

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

  15. Patterns of alternative splicing vary between species during heat stress.

    Science.gov (United States)

    Kannan, Sumetha; Halter, Gillian; Renner, Tanya; Waters, Elizabeth R

    2018-03-01

    Plants have evolved a variety of mechanisms to respond and adapt to abiotic stress. High temperature stress induces the heat shock response. During the heat shock response a large number of genes are up-regulated, many of which code for chaperone proteins that prevent irreversible protein aggregation and cell death. However, it is clear that heat shock is not the only mechanism involved in the plant heat stress response. Alternative splicing (AS) is also important during heat stress since this post-transcriptional regulatory mechanism can produce significant transcriptome and proteome variation. In this study, we examine AS during heat stress in the model species Arabidopsis thaliana and in the highly thermotolerant native California mustard Boechera depauperata . Analyses of AS during heat stress revealed that while a significant number of genes undergo AS and are differentially expressed (DE) during heat stress, some undergo both AS and DE. Analysis of the functional categories of genes undergoing AS indicated that enrichment patterns are different in the two species. Categories enriched in B. depauperata included light response genes and numerous abiotic stress response genes. Categories enriched in A. thaliana , but not in B. depauperata , included RNA processing and nucleotide binding. We conclude that AS and DE are largely independent responses to heat stress. Furthermore, this study reveals significant differences in the AS response to heat stress in the two related mustard species. This indicates AS responses to heat stress are species-specific. Future studies will explore the role of AS of specific genes in organismal thermotolerance.

  16. Ancient nature of alternative splicing and functions of introns

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

    2011-03-21

    Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

  17. Identifying alternative hyper-splicing signatures in MG-thymoma by exon arrays.

    Directory of Open Access Journals (Sweden)

    Lilach Soreq

    Full Text Available BACKGROUND: The vast majority of human genes (>70% are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. METHODOLOGY/PRINCIPAL FINDINGS: We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. CONCLUSIONS/SIGNIFICANCE: Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatibility gene HLA-DRB1 and interleukin (IL19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF(2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH, Reverse Transcription-Polymerase Chain Reaction (RT-PCR and mass spectrometry (MS followed by peptide sequencing. Our findings

  18. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

    Directory of Open Access Journals (Sweden)

    Janna eBlechman

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  19. Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status

    Directory of Open Access Journals (Sweden)

    Wei Jun S

    2011-04-01

    Full Text Available Abstract Background Neuroblastoma (NB tumors are well known for their pronounced clinical and molecular heterogeneity. The global gene expression and DNA copy number alterations have been shown to have profound differences in tumors of low or high stage and those with or without MYCN amplification. RNA splicing is an important regulatory mechanism of gene expression, and differential RNA splicing may be associated with the clinical behavior of a tumor. Methods In this study, we used exon array profiling to investigate global alternative splicing pattern of 47 neuroblastoma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4- and 4+. The ratio of exon-level expression to gene-level expression was used to detect alternative splicing events, while the gene-level expression was applied to characterize whole gene expression change. Results Principal component analysis (PCA demonstrated distinct splicing pattern in three groups of samples. Pairwise comparison identified genes with splicing changes and/or whole gene expression changes in high stage tumors. In stage 4- compared with stage 1- tumors, alternatively spliced candidate genes had little overlap with genes showing whole gene expression changes, and most of them were involved in different biological processes. In contrast, a larger number of genes exhibited either exon-level splicing, gene-level expression or both changes in stage 4+ versus stage 1- tumors. Those biological processes involved in stage 4- tumors were disrupted to a greater extent by both splicing and transcription regulations in stage 4+ tumors. Conclusions Our results demonstrated a significant role of alternative splicing in high stage neuroblastoma, and suggested a MYCN-associated splicing regulation pathway in stage 4+ tumors. The identification of differentially spliced genes and pathways in neuroblastoma tumors of different stages and molecular subtypes may be important to the

  20. Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones

    NARCIS (Netherlands)

    Highley, J. Robin; Kirby, Janine; Jansweijer, Joeri A.; Webb, Philip S.; Hewamadduma, Channa A.; Heath, Paul R.; Higginbottom, Adrian; Raman, Rohini; Ferraiuolo, Laura; Cooper-Knock, Johnathan; McDermott, Christopher J.; Wharton, Stephen B.; Shaw, Pamela J.; Ince, Paul G.

    2014-01-01

    Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor

  1. Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site

    DEFF Research Database (Denmark)

    Martínez-Pizarro, Ainhoa; Dembic, Maja; Pérez, Belén

    2018-01-01

    Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3' splice site, with different exonic mutations affecting exon 11...

  2. Insertion of a T next to the donor splice site of intron 1 causes aberrantly spliced mRNA in a case of infantile GM1-gangliosidosis.

    Science.gov (United States)

    Morrone, A; Morreau, H; Zhou, X Y; Zammarchi, E; Kleijer, W J; Galjaard, H; d'Azzo, A

    1994-01-01

    The lysosomal storage disorders GM1-gangliosidosis and Morquio B syndrome are caused by a complete or partial deficiency of acid beta-galactosidase. Here, we have characterized the mutation segregating in a family with two siblings affected by the severe infantile form of GM1-gangliosidosis. In total mRNA preparations derived from the patients' fibroblasts at least two aberrantly spliced beta-galactosidase transcripts (1 and 2) have been identified. Both transcripts contain a 20 nucleotide (nt) insertion derived from the 5' end of intron 1 of the beta-galactosidase gene. Furthermore, in transcript 2 sequences encoded by exon II are deleted during the splicing process. Comparison of the 20-nt insertion with wild-type intronic sequences indicated that in the genomic DNA of the patients an extra T nucleotide is present immediately downstream of the conserved GT splice donor dinucleotide of intron 1. Both patients are homozygous for the T nucleotide insertion. We propose that this single base insertion is the mutation responsible for aberrant splicing of beta-galactosidase pre-mRNA, giving rise to transcripts that cannot encode a normal protein.

  3. The Alternative Splicing Regulator Tra2b Is Required for Somitogenesis and Regulates Splicing of an Inhibitory Wnt11b Isoform

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    Darwin S. Dichmann

    2015-02-01

    Full Text Available Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping, 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short. We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism.

  4. Zinc-induced modulation of SRSF6 activity alters Bim splicing to promote generation of the most potent apoptotic isoform BimS.

    Science.gov (United States)

    Hara, Hirokazu; Takeda, Tatsuya; Yamamoto, Nozomi; Furuya, Keisuke; Hirose, Kazuya; Kamiya, Tetsuro; Adachi, Tetsuo

    2013-07-01

    Bim is a member of the pro-apoptotic BH3-only Bcl-2 family of proteins. Bim gene undergoes alternative splicing to produce three predominant splicing variants (BimEL, BimL and BimS). The smallest variant BimS is the most potent inducer of apoptosis. Zinc (Zn(2+)) has been reported to stimulate apoptosis in various cell types. In this study, we examined whether Zn(2+) affects the expression of Bim in human neuroblastoma SH-SY5Y cells. Zn(2+) triggered alterations in Bim splicing and induced preferential generation of BimS, but not BimEL and BimL, in a dose- and time-dependent manner. Other metals (cadmium, cobalt and copper) and stresses (oxidative, endoplasmic reticulum and genotoxic stresses) had little or no effect on the expression of BimS. To address the mechanism of Zn(2+)-induced preferential generation of BimS, which lacks exon 4, we developed a Bim mini-gene construct. Deletion analysis using the Bim mini-gene revealed that predicted binding sites of the SR protein SRSF6, also known as SRp55, are located in the intronic region adjacent to exon 4. We also found that mutations in the predicted SRSF6-binding sites abolished generation of BimS mRNA from the mutated Bim mini-gene. In addition, a UV cross-linking assay followed by Western blotting showed that SRSF6 directly bound to the predicted binding site and Zn(2+) suppressed this binding. Moreover, Zn(2+) stimulated SRSF6 hyper-phosphorylation. TG003, a cdc2-like kinase inhibitor, partially prevented Zn(2+)-induced generation of BimS and SRSF6 hyper-phosphorylation. Taken together, our findings suggest that Zn(2+) inhibits the activity of SRSF6 and promotes elimination of exon 4, leading to preferential generation of BimS. © 2013 FEBS.

  5. Multivariate Analysis and Visualization of Splicing Correlations in Single-Gene Transcriptomes

    Directory of Open Access Journals (Sweden)

    Agnew William S

    2007-01-01

    Full Text Available Abstract Background RNA metabolism, through 'combinatorial splicing', can generate enormous structural diversity in the proteome. Alternative domains may interact, however, with unpredictable phenotypic consequences, necessitating integrated RNA-level regulation of molecular composition. Splicing correlations within transcripts of single genes provide valuable clues to functional relationships among molecular domains as well as genomic targets for higher-order splicing regulation. Results We present tools to visualize complex splicing patterns in full-length cDNA libraries. Developmental changes in pair-wise correlations are presented vectorially in 'clock plots' and linkage grids. Higher-order correlations are assessed statistically through Monte Carlo analysis of a log-linear model with an empirical-Bayes estimate of the true probabilities of observed and unobserved splice forms. Log-linear coefficients are visualized in a 'spliceprint,' a signature of splice correlations in the transcriptome. We present two novel metrics: the linkage change index, which measures the directional change in pair-wise correlation with tissue differentiation, and the accuracy index, a very simple goodness-of-fit metric that is more sensitive than the integrated squared error when applied to sparsely populated tables, and unlike chi-square, does not diverge at low variance. Considerable attention is given to sparse contingency tables, which are inherent to single-gene libraries. Conclusion Patterns of splicing correlations are revealed, which span a broad range of interaction order and change in development. The methods have a broad scope of applicability, beyond the single gene – including, for example, multiple gene interactions in the complete transcriptome.

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

    Directory of Open Access Journals (Sweden)

    Haroon Kalam

    2017-03-01

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

  7. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  8. ZmbZIP60 mRNA is spliced in maize in response to ER stress

    Directory of Open Access Journals (Sweden)

    Li Yanjie

    2012-03-01

    Full Text Available Abstract Background Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA. Results A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT or tunicamycin (TM or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb. Conclusions The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

  9. RNA-Seq Analysis of Differential Splice Junction Usage and Intron Retentions by DEXSeq.

    Directory of Open Access Journals (Sweden)

    Yafang Li

    Full Text Available Alternative splicing is an important biological process in the generation of multiple functional transcripts from the same genomic sequences. Differential analysis of splice junctions (SJs and intron retentions (IRs is helpful in the detection of alternative splicing events. In this study, we conducted differential analysis of SJs and IRs by use of DEXSeq, a Bioconductor package originally designed for differential exon usage analysis in RNA-seq data analysis. We set up an analysis pipeline including mapping of RNA-seq reads, the preparation of count tables of SJs and IRs as the input files, and the differential analysis in DEXSeq. We analyzed the public RNA-seq datasets generated from RNAi experiments on Drosophila melanogaster S2-DRSC cells to deplete RNA-binding proteins (GSE18508. The analysis confirmed previous findings on the alternative splicing of the trol and Ant2 (sesB genes in the CG8144 (ps-depletion experiment and identified some new alternative splicing events in other RNAi experiments. We also identified IRs that were confirmed in our SJ analysis. The proposed method used in our study can output the genomic coordinates of differentially used SJs and thus enable sequence motif search. Sequence motif search and gene function annotation analysis helped us infer the underlying mechanism in alternative splicing events. To further evaluate this method, we also applied the method to public RNA-seq data from human breast cancer (GSE45419 and the plant Arabidopsis (SRP008262. In conclusion, our study showed that DEXSeq can be adapted to differential analysis of SJs and IRs, which will facilitate the identification of alternative splicing events and provide insights into the molecular mechanisms of transcription processes and disease development.

  10. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    Science.gov (United States)

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. Copyright © 2015 Elsevier Ltd. All rights

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

    Directory of Open Access Journals (Sweden)

    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

  12. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  13. Methods to detect faulty splices in the superconducting magnet system of the LHC

    International Nuclear Information System (INIS)

    Bailey, R.; Bellesia, B.; Lasheras, N.Catalan; Dahlerup-Petersen, K.; Denz, R.; Robles, C.; Koratzinos, M.; Pojer, M.; Ponce, L.; Saban, R.; Schmidt, R.

    2009-01-01

    The incident of 19 September 2008 at the LHC was caused by a faulty inter-magnet splice of about 200 n(Omega) resistance. Cryogenic and electrical techniques have been developed to detect other abnormal splices, either between or inside the magnets. The existing quench protection system can be used to detect internal splices with R > 20 n(Omega). Since this system does not cover the bus between magnets, the cryogenic system is used to measure the rate of temperature rise due to ohmic heating. Accuracy of a few mK/h, corresponding to a few Watts, has been achieved, allowing detection of excess resistance, if it is more than 40 n(Omega) in a cryogenic subsector (two optical cells). Follow-up electrical measurements are made in regions identified by the cryogenic system. These techniques have detected two abnormal internal magnet splices of 100 n(Omega) and 50 n(Omega) respectively. In 2009, this ad hoc system will be replaced with a permanent one to monitor all splices at the n(Omega) level

  14. A View of Pre-mRNA Splicing from RNase R Resistant RNAs

    Directory of Open Access Journals (Sweden)

    Hitoshi Suzuki

    2014-05-01

    Full Text Available During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

  15. Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing.

    Science.gov (United States)

    Li, Sanshu; Breaker, Ronald R

    2013-03-01

    Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5' splice site, which greatly increases use of this 5' splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

  16. Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

    Directory of Open Access Journals (Sweden)

    Ulrich F. Müller

    2017-01-01

    Full Text Available Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

  17. Predicting human splicing branchpoints by combining sequence-derived features and multi-label learning methods.

    Science.gov (United States)

    Zhang, Wen; Zhu, Xiaopeng; Fu, Yu; Tsuji, Junko; Weng, Zhiping

    2017-12-01

    Alternative splicing is the critical process in a single gene coding, which removes introns and joins exons, and splicing branchpoints are indicators for the alternative splicing. Wet experiments have identified a great number of human splicing branchpoints, but many branchpoints are still unknown. In order to guide wet experiments, we develop computational methods to predict human splicing branchpoints. Considering the fact that an intron may have multiple branchpoints, we transform the branchpoint prediction as the multi-label learning problem, and attempt to predict branchpoint sites from intron sequences. First, we investigate a variety of intron sequence-derived features, such as sparse profile, dinucleotide profile, position weight matrix profile, Markov motif profile and polypyrimidine tract profile. Second, we consider several multi-label learning methods: partial least squares regression, canonical correlation analysis and regularized canonical correlation analysis, and use them as the basic classification engines. Third, we propose two ensemble learning schemes which integrate different features and different classifiers to build ensemble learning systems for the branchpoint prediction. One is the genetic algorithm-based weighted average ensemble method; the other is the logistic regression-based ensemble method. In the computational experiments, two ensemble learning methods outperform benchmark branchpoint prediction methods, and can produce high-accuracy results on the benchmark dataset.

  18. Conformations of JNK3α splice variants analyzed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Park, Ji Young; Yun, Youngjoo; Chung, Ka Young

    2017-03-01

    c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family that regulate apoptosis, inflammation, cytokine production, and metabolism. MAPKs undergo various splicing within their kinase domains. Unlike other MAPKs, JNKs have alternative splicing at the C-terminus, resulting in long and short variants. Functional or conformational effects due to the elongated C-terminal tail in the long splice variants have not been investigated nor has the conformation of the C-terminal tail been analyzed. Here, we analyzed the conformation of the elongated C-terminal tail and investigated conformational differences between long and short splice variants of JNKs using JNK3α2 and JNK3α1 as models. We adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to analyze the conformation. HDX-MS revealed that the C-terminal tail is mostly intrinsically disordered, and that the conformation of the kinase domain of JNK3α2 is more dynamic than that of JNK3α1. The different conformation dynamics between long and short splice variants of JNK3α might affect the cellular functions of JNK3. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

    Directory of Open Access Journals (Sweden)

    Junyu Zhang

    Full Text Available Autoimmune polyendocrine syndrome type 1 (APS-1 is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203 containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1.

  20. Consolidation of the 13 kA Splices in the Electrical Feedboxes of the LHC

    CERN Document Server

    Perin, A; Duarte Ramos, D; Pirotte, O; Principe, R; Savary, F; Scheuerlein, C; Tock, J Ph; Verweij, A

    2012-01-01

    In 2008 a defective connection in one of the 13 kA dipole circuits of the LHC caused an electric breakdown that resulted in extensive damage in a sector of the accelerator. The investigation performed after the accident showed the necessity to consolidate the electrical splices of the 13 kA dipole and quadrupole circuits in order to operate the LHC at its nominal energy of 7 TeV. These circuits are powered through electrical feedboxes located at each end of the 8 sectors of the LHC. In the feedboxes the current is routed from room temperature to the superconducting magnets busbars along current leads and superconducting busbars and flows through at least two internal splices. These splices are based on the same technology as the magnet-to-magnet ones but they are significantly different in terms of environment and configuration. As for the magnet to magnet splices, a consolidation will be necessary to operate them at nominal current. This paper presents an analysis of the properties of these splices and the t...

  1. Effect of 5-fluorouracil incorporation into pre-mRNA on RNA splicing in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Doong, S.L.

    1988-01-01

    5-Fluorouracil(FUra) has been proven useful in the chemotherapy of a number of cancers. The mechanism underlying its cytotoxicity is controversial. We are interested in studying the FUra effect on the fidelity of the pre-mRNA splicing process. ({sup 32}P)-labeled human {beta}-globin pre-mRNA containing the first two exons and the first intervening sequence was synthesized in the presence of UTP, FUTP, or both. The appearance of a new minor spliced product was dependent on both the pH of the splicing reaction and the extent of FUra incorporation into pre-mRNA. At least 84% substitution of U by FUra was required to observe the presence of the abnormal splicing pathway. The new spliced product was sequenced and found to contain an additional 20 bases derived from the 3{prime} end of the intervening sequence. Nearest neighbor analysis, RNase T{sub 1} fingerprinting, and short primer extension experiments were carried out to assess the extent of transcription infidelity induced by FUra. Site directed mutagenesis was performed to determine the sequence(s) of FUra substitution which contribute to missplicing in vitro.

  2. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

    Science.gov (United States)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M; Mohammad, Dara K; Saleh, Amer F; Sutlu, Tolga; Nordin, Joel Z; Guterstam, Peter; Gustafsson, Manuela O; Kharazi, Shabnam; Piątosa, Barbara; Roberts, Thomas C; Behlke, Mark A; Wood, Matthew J A; Gait, Michael J; Lundin, Karin E; El Andaloussi, Samir; Månsson, Robert; Berglöf, Anna; Wengel, Jesper; Smith, C I Edvard

    2014-09-01

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

  3. Splice-correction strategies for treatment of X-linked agammaglobulinemia.

    Science.gov (United States)

    Bestas, Burcu; Turunen, Janne J; Blomberg, K Emelie M; Wang, Qing; Månsson, Robert; El Andaloussi, Samir; Berglöf, Anna; Smith, C I Edvard

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.

  4. LEMONS - A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes.

    Directory of Open Access Journals (Sweden)

    Liron Levin

    Full Text Available RNA-seq is becoming a preferred tool for genomics studies of model and non-model organisms. However, DNA-based analysis of organisms lacking sequenced genomes cannot rely on RNA-seq data alone to isolate most genes of interest, as DNA codes both exons and introns. With this in mind, we designed a novel tool, LEMONS, that exploits the evolutionary conservation of both exon/intron boundary positions and splice junction recognition signals to produce high throughput splice-junction predictions in the absence of a reference genome. When tested on multiple annotated vertebrate mRNA data, LEMONS accurately identified 87% (average of the splice-junctions. LEMONS was then applied to our updated Mediterranean chameleon transcriptome, which lacks a reference genome, and predicted a total of 90,820 exon-exon junctions. We experimentally verified these splice-junction predictions by amplifying and sequencing twenty randomly selected genes from chameleon DNA templates. Exons and introns were detected in 19 of 20 of the positions predicted by LEMONS. To the best of our knowledge, LEMONS is currently the only experimentally verified tool that can accurately predict splice-junctions in organisms that lack a reference genome.

  5. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    Energy Technology Data Exchange (ETDEWEB)

    Tennyson, C.N.; Worton, R.G. [Univ. of Toronto and the Hospital for Sick Children, Ontario (Canada)

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  6. Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F.

    Science.gov (United States)

    Wang, Erming; Aslanzadeh, Vahid; Papa, Filomena; Zhu, Haiyan; de la Grange, Pierre; Cambi, Franca

    2012-01-01

    In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5' splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.

  7. How to find the real one (at the level of pre-mRNA splicing).

    Science.gov (United States)

    Rauch, T; Kiss, Ibolya

    2003-01-01

    The mature mRNA always carries nucleotide sequences that faithfully mirror the protein product according to the niles of the genetic code. However, in the chromosome, the nucleotide sequence that represents a certain protein is interrupted by additional sequences. Therefore, most eukaryotic genes are longer than their final mRNA products. The human genome project revealed that only a tiny portion of sequences serves as protein-coding region and almost one quarter of the genome is occupied by non-coding intervening sequences. The elimination of these non-coding regions from the precursor RNA in a process termed splicing must be extremely precise, because even a single nucleotide mistake may cause a fatal error. At present, two types of intervening sequences have been identified in protein-coding genes. One of them, the U2-dependent or major-class is prevalent and represents 99% of known sequences. The other one, the so-called U12-dependent or minor-class of introns, occurs in much lesser amounts in the genome. The basic problem of nuclear splicing concerns i/ the molecular mechanisms, which ensure that the coding regions are correctly recognized and spliced together: ii/ the principles and mechanisms that guarantee the high fidelity of the splicing system; iii/ the differences in the excision mechanisms of the two classes of introns. We are going to present models explaining how intervening sequences are accurately removed and the coding regions correctly juxtaposed. The two splicing mechanisms will also be compared.

  8. On the path to genetic novelties: insights from programmed DNA elimination and RNA splicing.

    Science.gov (United States)

    Catania, Francesco; Schmitz, Jürgen

    2015-01-01

    Understanding how genetic novelties arise is a central goal of evolutionary biology. To this end, programmed DNA elimination and RNA splicing deserve special consideration. While programmed DNA elimination reshapes genomes by eliminating chromatin during organismal development, RNA splicing rearranges genetic messages by removing intronic regions during transcription. Small RNAs help to mediate this class of sequence reorganization, which is not error-free. It is this imperfection that makes programmed DNA elimination and RNA splicing excellent candidates for generating evolutionary novelties. Leveraging a number of these two processes' mechanistic and evolutionary properties, which have been uncovered over the past years, we present recently proposed models and empirical evidence for how splicing can shape the structure of protein-coding genes in eukaryotes. We also chronicle a number of intriguing similarities between the processes of programmed DNA elimination and RNA splicing, and highlight the role that the variation in the population-genetic environment may play in shaping their target sequences. © 2015 Wiley Periodicals, Inc.

  9. The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis

    Science.gov (United States)

    Zhang, Cui-Jun; Zhou, Jin-Xing; Liu, Jun; Ma, Ze-Yang; Zhang, Su-Wei; Dou, Kun; Huang, Huan-Wei; Cai, Tao; Liu, Renyi; Zhu, Jian-Kang; He, Xin-Jian

    2013-01-01

    DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing. PMID:23524848

  10. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    Science.gov (United States)

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  11. Integrative analysis revealed the molecular mechanism underlying RBM10-mediated splicing regulation.

    Science.gov (United States)

    Wang, Yongbo; Gogol-Döring, Andreas; Hu, Hao; Fröhler, Sebastian; Ma, Yunxia; Jens, Marvin; Maaskola, Jonas; Murakawa, Yasuhiro; Quedenau, Claudia; Landthaler, Markus; Kalscheuer, Vera; Wieczorek, Dagmar; Wang, Yang; Hu, Yuhui; Chen, Wei

    2013-09-01

    RBM10 encodes an RNA binding protein. Mutations in RBM10 are known to cause multiple congenital anomaly syndrome in male humans, the TARP syndrome. However, the molecular function of RBM10 is unknown. Here we used PAR-CLIP to identify thousands of binding sites of RBM10 and observed significant RBM10-RNA interactions in the vicinity of splice sites. Computational analyses of binding sites as well as loss-of-function and gain-of-function experiments provided evidence for the function of RBM10 in regulating exon skipping and suggested an underlying mechanistic model, which could be subsequently validated by minigene experiments. Furthermore, we demonstrated the splicing defects in a patient carrying an RBM10 mutation, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10-mediated splicing regulation and provided a molecular link to understanding a human congenital disorder. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

  12. TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity

    International Nuclear Information System (INIS)

    Na, Lei; Tang, Yan-Dong; Liu, Jian-Dong; Yu, Chang-Qing; Sun, Liu-Ke; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun; Zhou, Jian-Hua

    2014-01-01

    Highlights: • TRIMe7-CypA expresses in rhesus and pig-tailed, but not long-tailed macaques. • TRIMe7-CypA does not show the restriction to a HIV-GFP report virus in vitro. • It acts as a negative modulator to TRIM5α likely by competitive inhibition. - Abstract: The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition

  13. TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity

    Energy Technology Data Exchange (ETDEWEB)

    Na, Lei [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Tang, Yan-Dong [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Biotechnology Institute of Southern Medical University, Guangzhou 510515 (China); Liu, Jian-Dong; Yu, Chang-Qing; Sun, Liu-Ke; Lin, Yue-Zhi; Wang, Xue-Feng [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Wang, Xiaojun, E-mail: xjw@hvri.ac.cn [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Zhou, Jian-Hua, E-mail: jianhua_uc@126.com [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Harbin Pharmaceutical Group Biovaccine Company, Harbin 150069 (China)

    2014-04-04

    Highlights: • TRIMe7-CypA expresses in rhesus and pig-tailed, but not long-tailed macaques. • TRIMe7-CypA does not show the restriction to a HIV-GFP report virus in vitro. • It acts as a negative modulator to TRIM5α likely by competitive inhibition. - Abstract: The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition.

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

    Science.gov (United States)

    Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

    2008-10-02

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

  15. High qualitative and quantitative conservation of alternative splicing in Caenorhabditis elegans and Caenorhabditis briggsae

    DEFF Research Database (Denmark)

    Rukov, Jakob Lewin; Irimia, Manuel; Mørk, Søren

    2007-01-01

    the qualitative and quantitative expression of 21 orthologous alternative splice events through the development of 2 nematode species separated by 85-110 Myr of evolutionary time. We demonstrate that most of these alternative splice events present in Caenorhabditis elegans are conserved in Caenorhabditis briggsae...... mechanisms controlling AS are to a large extent conserved during the evolution of Caenorhabditis. This strong conservation indicates that both major and minor splice forms have important functional roles and that the relative quantities in which they are expressed are crucial. Our results therefore suggest...... that the quantitative regulation of isoform expression levels is an intrinsic part of most AS events. Moreover, our results indicate that AS contributes little to transcript variation in Caenorhabditis genes and that gene duplication may be the major evolutionary mechanism for the origin of novel transcripts in these 2...

  16. Proposed Method for the Verification of the LHC Bus Bar Splices during Commissioning at Cryogenic Conditions

    CERN Document Server

    Calvi, M; Rodríguez-Mateos, F

    2007-01-01

    The commissioning of the Large Hadron Collider at CERN includes the powering of about 1600 superconducting electrical circuits to currents ranging from 55 A to 11.8 kA. A large number of splices (over 70'000) are present at the magnet interconnects, which can only be validated with current at cryogenic conditions. This paper discusses the thermal effects related to possible faulty splices during the powering of the circuits. The calculations of the quench and detection currents, as well as the hot spot temperatures, are described. The heat transfer model with the surrounding coolant and the current profiles inside the splices are presented. This study is completed with a sensitivity analysis on the hot spot temperature with respect to the model parameters. Finally, the implications with respect to the powering ramps and parameters to be applied during the first powering are discussed.

  17. 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.

  18. 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

  19. Changes in Alternative Splicing as Pharmacodynamic Markers for Sudemycin D6

    Directory of Open Access Journals (Sweden)

    Morgan Thurman

    2017-09-01

    Full Text Available Objective: The aim of the study was to define pharmacodynamic markers for sudemycin D6, an experimental cancer drug that changes alternative splicing in human blood. Methods: Blood samples from 12 donors were incubated with sudemycin D6 for up to 24 hours, and at several time points total RNA from lymphocytes was prepared and the pre-messenger RNA (mRNA splicing patterns were analyzed with reverse transcription-polymerase chain reaction. Results: Similar to immortalized cells, blood lymphocytes change alternative splicing due to sudemycin D6 treatment. However, lymphocytes in blood respond slower than immortalized cultured cells. Conclusions: Exon skipping in the DUSP11 and SRRM1 pre-mRNAs are pharmacodynamic markers for sudemycin D6 treatment and show effects beginning at 9 hours after treatment.

  20. A SMN-Dependent U12 Splicing Event Essential for Motor Circuit Function

    Science.gov (United States)

    Lotti, Francesco; Imlach, Wendy L.; Saieva, Luciano; Beck, Erin S.; Hao, Le T.; Li, Darrick K.; Jiao, Wei; Mentis, George Z.; Beattie, Christine E.; McCabe, Brian D.; Pellizzoni, Livio

    2012-01-01

    SUMMARY Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a novel protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA. PMID:23063131

  1. Production and Quality Assurance of Main Busbar Interconnection Splices during the LHC 2008-2009 Shutdown.

    CERN Document Server

    Bertinelli, F; Dalin, J-M; Fessia, P; Flora, R H; Heck, S; Pfeffer, H; Prin, H; Scheuerlein, C; Thonet, P; Tock, J-P; Williams, L

    2011-01-01

    The main busbar interconnection splices of the Large Hadron Collider are assembled by inductive soldering of the Rutherford type cables and the copper profiles of the stabilizer. Following the September 2008 incident, the assembly process and the quality assurance have been improved, with new measurement and diagnostics methods introduced. In the 2008-2009 shutdown the resistance both in the superconducting and in the normal conducting states have been the focus for improvements. The introduction of gamma radiography has allowed the visualization of voids between cable and stabilizer. It is now known that during the standard soldering heating cycle solder is lost from the busbar extremities adjacent to the splice profiles, leaving parts of the cable in poor contact with the stabilizer. A room temperature resistance measurement has been introduced as a simple, non-destructive test to measure the electrical continuity of the splice in its normal conducting state. An ultrasonic test has been performed systematic...

  2. 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...

  3. 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.

  4. Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

    Science.gov (United States)

    Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

    2013-08-01

    Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  5. A biophysical model for identifying splicing regulatory elements and their interactions.

    Directory of Open Access Journals (Sweden)

    Ji Wen

    Full Text Available Alternative splicing (AS of precursor mRNA (pre-mRNA is a crucial step in the expression of most eukaryotic genes. Splicing factors (SFs play an important role in AS regulation by binding to the cis-regulatory elements on the pre-mRNA. Although many splicing factors (SFs and their binding sites have been identified, their combinatorial regulatory effects remain to be elucidated. In this paper, we derive a biophysical model for AS regulation that integrates combinatorial signals of cis-acting splicing regulatory elements (SREs and their interactions. We also develop a systematic framework for model inference. Applying the biophysical model to a human RNA-Seq data set, we demonstrate that our model can explain 49.1%-66.5% variance of the data, which is comparable to the best result achieved by biophysical models for transcription. In total, we identified 119 SRE pairs between different regions of cassette exons that may regulate exon or intron definition in splicing, and 77 SRE pairs from the same region that may arise from a long motif or two different SREs bound by different SFs. Particularly, putative binding sites of polypyrimidine tract-binding protein (PTB, heterogeneous nuclear ribonucleoprotein (hnRNP F/H and E/K are identified as interacting SRE pairs, and have been shown to be consistent with the interaction models proposed in previous experimental results. These results show that our biophysical model and inference method provide a means of quantitative modeling of splicing regulation and is a useful tool for identifying SREs and their interactions. The software package for model inference is available under an open source license.

  6. Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

    Directory of Open Access Journals (Sweden)

    Xinping Wang

    Full Text Available Amelogenin, the major enamel matrix protein in tooth development, has been demonstrated to play a significant role in tooth enamel formation. Previous studies have identified the alternative splicing of amelogenin in many mammalian vertebrates as one mechanism for amelogenin heterogeneous expression in teeth. While amelogenin and its splicing forms in mammalian vertebrates have been cloned and sequenced, the amelogenin gene, especially its splicing forms in non-mammalian species, remains largely unknown. To better understand the mechanism underlying amelogenin evolution, we previously cloned and characterized an amelogenin gene sequence from a squamate, the green iguana. In this study, we employed RT-PCR to amplify the amelogenin gene from the black spiny-tailed iguana Ctenosaura similis teeth, and discovered a novel splicing form of the amelogenin gene. The transcript of the newly identified iguana amelogenin gene (named C. Similis-T2L is 873 nucleotides long encoding an expected polypeptide of 206 amino acids. The C. Similis-T2L contains a unique exon denominated exon X, which is located between exon 5 and exon 6. The C. Similis-T2L contains 7 exons including exon 1, 2, 3, 5, X, 6, and 7. Analysis of the secondary and tertiary structures of T2L amelogenin protein demonstrated that exon X has a dramatic effect on the amelogenin structures. This is the first report to provide definitive evidence for the amelogenin alternative splicing in non-mammalian vertebrates, revealing a unique exon X and the splicing form of the amelogenin gene transcript in Ctenosaura similis.

  7. Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

    Science.gov (United States)

    Shi, Lisong; Luo, Chunyan; Ahmed, Mairaj K; Attaie, Ali B; Ye, Xiaoqian

    2016-04-01

    Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

  8. 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

  9. Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2004-09-01

    Full Text Available Abstract Background Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. Methods We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. Results Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. Conclusions We propose to combine the computational prediction of alternative splice isoforms with experimental validation for

  10. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins.

    Directory of Open Access Journals (Sweden)

    Ying Lin

    Full Text Available Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa N-intein fragment and S11 split inteins having a very small (6 aa C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100% of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4 s(-1 to ~3.8 × 10(-4 s(-1, which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.

  11. Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

    Science.gov (United States)

    Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

    2016-06-01

    Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  12. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S.Tiong; Roca, Xavier

    2017-01-01

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers. PMID:29100409

  13. Heat-shrinkable splicing materials for Class 1E wire and cable systems in nuclear power generating stations

    International Nuclear Information System (INIS)

    Handa, Katsue; Maruyama, Masahiro; Kanno, Mikio; Ohya, Shingo; Nagakawa, Seiji; Sugimori, Mikihiro

    1987-01-01

    This report describes the shapes of heat-shrinkable splicing materials (cable sleeve and breakout, and round end cap) made of polyolefine resin, their application to cable splicing, and the properties of the materials as well as of the splice using them. Particularly, the report features introduction of their properties as determined by tests under the same conditions as used in Japan in qualifying tests on wires and cables for nuclear power generating stations. The heat-shrinkable splicing materials proved to be equal in properties to flame-retardant cables for nuclear power plants when tested for oxygen index and subjected to a vertical flame test on ''insulated wire'' and a vertical tray flame test on the cable splice. It was also confirmed that Class 1E cable using these splicing materials could stand the most rigorous environmental test in Japan. Therefore they can be used for splicing Class 1E wires and cables and the splice formed with them can be regarded as Class 1E specified in IEEE Std. 383. (author)

  14. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kvissel, Anne-Katrine; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-01-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  16. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S Tiong; Roca, Xavier

    2017-09-29

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis -acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM -polymorphism-associated TKI-resistant CML and other cancers.

  17. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Jin Shang

    2015-01-01

    Full Text Available Low back pain (LBP is a very prevalent disease and degenerative disc diseases (DDDs usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0 to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale.

  18. The Effective Lifetime of ACSR Full Tension Splice Connector Operated at Higher Temperature

    International Nuclear Information System (INIS)

    Wang, Jy-An John; Lara-Curzio, Edgar; King Jr, Thomas J.; Graziano, Joe; Chan, John; Goodwin, Tip

    2009-01-01

    This paper is to address the issues related to integrity of ACSR full tension splice connectors operated at high temperatures. A protocol of integrating analytical and experimental approaches to evaluate the integrity of a full tension single-stage splice connector (SSC) assembly during service at high operating temperature was developed. Based on the developed protocol the effective lifetime evaluation was demonstrated with ACSR Drake conductor SSC systems. The investigation indicates that thermal cycling temperature and frequency, conductor cable tension loading, and the compressive residual stress field within a SSC system have significant impact on the SSC integrity and the associated effective lifetime

  19. 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...

  20. Split and Splice Approach for Highly Selective Targeting of Human NSCLC Tumors

    Science.gov (United States)

    2014-10-01

    for Stx2A-specific intracellular delivery mechanism by replacing four lysine residues in its C-terminal part with arginines and confirming that the...Fig. 3. Substitution of all 4 lysines in the C-terminal intein part by arginines preserves its trans-splicing activity in vivo as revealed by using...not directly applicable for in vivo trans-splicing of Stx2A split toxin due to the presence of four lysines in the C-terminal part of the intein

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

    Directory of Open Access Journals (Sweden)

    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

  2. Unique splicing pattern of the TCF7L2 gene in human pancreatic islets

    DEFF Research Database (Denmark)

    Osmark, P; Hansson, O; Jonsson, Anna Elisabet

    2009-01-01

    Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex s...... splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored....

  3. Effect of confinement on bond strength of hot-dip galvanized lap splices in concrete structures

    International Nuclear Information System (INIS)

    Fakhran, Mazen

    2004-01-01

    Galvanizing the reinforcing steel is one of the methods used to protect bars against corrosion. Galvanizing is a hot dip process where the reinforcing bars are immersed in an aqueous pre flux solution of zinc ammonium chloride at a controlled temperature between 840 and 850 degrees F. In 2001, a research program was started at AUB to evaluate experimentally the effect of hot dip galvanizing on the bond capacity of tension lap splices anchored in full-scale beam specimens designed to fail in bond splitting mode. The test results indicated that the use of galvanized bars had a negligible effect on bond strength of reinforcement in normal strength. However, galvanizing caused an average of 20 percent decrease in bond strength of reinforcement in high strength concrete. The primary objective of research reported in this thesis, is the need to find a solution to eliminate the bond reduction of galvanized bars in high strength concrete. It is significant to evaluate the positive effect of the addition of transverse reinforcement in the splice region. The hypothesis to be tested is that such transverse reinforcement will insure uniform bond stress distribution over the entire splice region, thus mobilizing all bar lugs along the splice in the stress transfer mechanism between the bar and the surrounding concrete. Such mechanism might reduce the significant decrease in bond strength in high strength concrete due to galvanizing. To achieve this objective, eighteen full-scale beam specimens were tested in positive bending. Each beam was reinforced with bars spliced in a constant moment region at midspam. The splice length was chosen in such a way that the beams failed in bond splitting of the concrete cover in the splice region. The main variables were type of coating (black or galvanized bars), bar size (20, 25 and 32 mm), and amount of transverse reinforcement in the splice region (0, 2 or 4 stirrups). The test results indicated that confinement did not have a significant

  4. Angular interpolations and splice options for three-dimensional transport computations

    International Nuclear Information System (INIS)

    Abu-Shumays, I.K.; Yehnert, C.E.

    1996-01-01

    New, accurate and mathematically rigorous angular Interpolation strategies are presented. These strategies preserve flow and directionality separately over each octant of the unit sphere, and are based on a combination of spherical harmonics expansions and least squares algorithms. Details of a three-dimensional to three-dimensional (3-D to 3-D) splice method which utilizes the new angular interpolations are summarized. The method has been implemented in a multidimensional discrete ordinates transport computer program. Various features of the splice option are illustrated by several applications to a benchmark Dog-Legged Void Neutron (DLVN) streaming and transport experimental assembly

  5. Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6

    DEFF Research Database (Denmark)

    Mantri, M.; Webby, C.J.; Loik, N.D.

    2012-01-01

    The lysyl 5S-hydroxylase, JMJD6 acts on proteins involved in RNA splicing. We find that in the absence of substrate JMJD6 catalyses turnover of 2OG to succinate. H-NMR analyses demonstrate that consumption of 2OG is coupled to succinate formation. MS analyses reveal that JMJD6 undergoes self......-hydroxylation in the presence of Fe(ii) and 2OG resulting in production of 5S-hydroxylysine residues. JMJD6 in human cells is also found to be hydroxylated. Self-hydroxylation of JMJD6 may play a regulatory role in modulating the hydroxylation status of proteins involved in RNA splicing. This journal is...

  6. 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

  7. Analysis of Histone Deacetylase 7 (HDAC7) Alternative Splicing and Its Role in Embryonic Stem Cell Differentiation Toward Smooth Muscle Lineage.

    Science.gov (United States)

    Yang, Junyao; Margariti, Andriana; Zeng, Lingfang

    2016-01-01

    Histone deacetylases (HDACs) have a central role in the regulation of gene expression, which undergoes alternative splicing during embryonic stem cell (ES) cell differentiation. Alternative splicing gives rise to vast diversity over gene information, arousing public concerns in the last decade. In this chapter, we describe a strategy to detect HDAC7 alternative splicing and analyze its function on ES cell differentiation.

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

    Directory of Open Access Journals (Sweden)

    Barbara Wappenschmidt

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

  9. Expression microarray analysis reveals alternative splicing of LAMA3 and DST genes in head and neck squamous cell carcinoma.

    Science.gov (United States)

    Li, Ryan; Ochs, Michael F; Ahn, Sun Mi; Hennessey, Patrick; Tan, Marietta; Soudry, Ethan; Gaykalova, Daria A; Uemura, Mamoru; Brait, Mariana; Shao, Chunbo; Westra, William; Bishop, Justin; Fertig, Elana J; Califano, Joseph A

    2014-01-01

    Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors. We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score-the Maximum-Minimum Exon Score (MMES)--designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort. After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001). Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.

  10. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated IG{epsilon} gene transcription by recruitment of HDAC1

    DEFF Research Database (Denmark)

    Dong, Lijie; Zhang, Xinyu; Fu, Xiao

    2010-01-01

    of phosphorylation, and IL-4 stimulation increased tyrosine phosphorylation of PSF and STAT6. Functional analysis demonstrated that ectopic expression of PSF resulted in inhibition of STAT6-mediated gene transcriptional activation and mRNA expression of Ig heavy chain germline Ig ε, while knockdown of PSF increased......Regulation of transcription requires cooperation between sequence specific transcription factors and numerous coregulatory proteins. In IL-4/IL-13 signaling several coactivators for STAT6 have been identified, but the molecular mechanisms of STAT6-mediated gene transcription are still not fully...... understood. Here we identified by proteomic approach that PTB-associated splicing factor (PSF) interacts with STAT6. In cells the interaction required IL-4 stimulation and was observed both with endogenous and ectopically expressed proteins. The ligand dependency of the interaction suggested involvement...

  11. Silencing the Snail-dependent RNA splice regulator ESRP1 drives malignant transformation of human pulmonary epithelial cells.

    Science.gov (United States)

    Walser, Tonya C; Jing, Zhe; Tran, Linh M; Lin, Ying Q; Yakobian, Natalie; Wang, Gerald; Krysan, Kostyantyn; Zhu, Li X; Sharma, Sherven; Lee, Mi-Heon; Belperio, John A; Ooi, Aik T; Gomperts, Brigitte N; Shay, Jerry W; Larsen, Jill E; Minna, John D; Hong, Long-Sheng; Fishbein, Michael C; Dubinett, Steven M

    2018-02-05

    Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated including in non-small cell lung cancer (NSCLC). Here we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo. Snail-mediated transformation relied upon silencing of the tumor suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC. Copyright ©2018, American Association for Cancer Research.

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

    Directory of Open Access Journals (Sweden)

    Susan N Bolch

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

  13. A novel RNA-splicing mutation in TRAPPC2 gene causing X-linked ...

    Indian Academy of Sciences (India)

    large Chinese family with SEDT and identified a novel RNA- splicing mutation in the TRAPPC2 gene. Materials and methods. Patients. The proband of this family is 38-year-old man who sought medical attention because of chronic pain in weight-bearing joints. The proband's height is 135 cm and his arm span is 155 cm.

  14. Differential dynamics of splicing factor SC35 during the cell cycle

    Indian Academy of Sciences (India)

    Srinivas

    Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear ... and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during ... mitotic cells was determined with respect to markers for.

  15. Computational Recognition of RNA Splice Sites by Exact Algorithms for the Quadratic Traveling Salesman Problem

    Directory of Open Access Journals (Sweden)

    Anja Fischer

    2015-06-01

    Full Text Available One fundamental problem of bioinformatics is the computational recognition of DNA and RNA binding sites. Given a set of short DNA or RNA sequences of equal length such as transcription factor binding sites or RNA splice sites, the task is to learn a pattern from this set that allows the recognition of similar sites in another set of DNA or RNA sequences. Permuted Markov (PM models and permuted variable length Markov (PVLM models are two powerful models for this task, but the problem of finding an optimal PM model or PVLM model is NP-hard. While the problem of finding an optimal PM model or PVLM model of order one is equivalent to the traveling salesman problem (TSP, the problem of finding an optimal PM model or PVLM model of order two is equivalent to the quadratic TSP (QTSP. Several exact algorithms exist for solving the QTSP, but it is unclear if these algorithms are capable of solving QTSP instances resulting from RNA splice sites of at least 150 base pairs in a reasonable time frame. Here, we investigate the performance of three exact algorithms for solving the QTSP for ten datasets of splice acceptor sites and splice donor sites of five different species and find that one of these algorithms is capable of solving QTSP instances of up to 200 base pairs with a running time of less than two days.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    BACKGROUND: BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patte...

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

    Science.gov (United States)

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

    2010-01-01

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

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

    African Journals Online (AJOL)

    The Na+-K+-ATPase is an essential transport enzyme expressed in all animal tissues, where it generates ion gradients to maintain membrane potential and drive the transport of other solutes. It also balances metabolism and body temperature. In this study, the characterization of three novel bovine ATP1B2 splice variants, ...

  19. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

    KAUST Repository

    Zhang, Runxuan

    2017-04-05

    Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

  20. Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ...

    Indian Academy of Sciences (India)

    Unknown

    1 Handayama,. Hamamatsu ... structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in ... structural change in an mRNA transcript, leading to the production of a ...

  1. 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

  2. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  3. Splicing transitions of the anchoring protein ENH during striated muscle development.

    Science.gov (United States)

    Ito, Jumpei; Hashimoto, Taiki; Nakamura, Sho; Aita, Yusuke; Yamazaki, Tomoko; Schlegel, Werner; Takimoto, Koichi; Maturana, Andrés D

    2012-05-04

    The ENH (PDLIM5) protein acts as a scaffold to tether various functional proteins at subcellular sites via PDZ and three LIM domains. Splicing of the ENH primary transcript generates various products with different repertories of protein interaction modules. Three LIM-containing ENH predominates in neonatal cardiac tissue, whereas LIM-less ENHs are abundant in adult hearts, as well as skeletal muscles. Here we examine the timing of splicing transitions of ENH gene products during postnatal heart development and C2C12 myoblast differentiation. Real-time PCR analysis shows that LIM-containing ENH1 mRNA is gradually decreased during postnatal heart development, whereas transcripts with the short exon 5 appear in the late postnatal period and continues to increase until at least one month after birth. The splicing transition from LIM-containing ENH1 to LIM-less ENHs is also observed during the early period of C2C12 differentiation. This transition correlates with the emergence of ENH transcripts with the short exon 5, as well as the expression of myogenin mRNA. In contrast, the shift from the short exon 5 to the exon 7 occurs in the late differentiation period. The timing of this late event corresponds to the appearance of mRNA for the skeletal myosin heavy chain MYH4. Thus, coordinated and stepwise splicing transitions result in the production of specific ENH transcripts in mature striated muscles. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Histone H3 lysine 36 methylation affects temperature-induced alternative splicing and flowering in plants

    NARCIS (Netherlands)

    Pajoro, A.; Severing, E.; Angenent, G.C.; Immink, R.G.H.

    2017-01-01

    Background: Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in

  5. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events.

    Science.gov (United States)

    Liu, Jinfeng; Lee, William; Jiang, Zhaoshi; Chen, Zhongqiang; Jhunjhunwala, Suchit; Haverty, Peter M; Gnad, Florian; Guan, Yinghui; Gilbert, Houston N; Stinson, Jeremy; Klijn, Christiaan; Guillory, Joseph; Bhatt, Deepali; Vartanian, Steffan; Walter, Kimberly; Chan, Jocelyn; Holcomb, Thomas; Dijkgraaf, Peter; Johnson, Stephanie; Koeman, Julie; Minna, John D; Gazdar, Adi F; Stern, Howard M; Hoeflich, Klaus P; Wu, Thomas D; Settleman, Jeff; de Sauvage, Frederic J; Gentleman, Robert C; Neve, Richard M; Stokoe, David; Modrusan, Zora; Seshagiri, Somasekar; Shames, David S; Zhang, Zemin

    2012-12-01

    Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.

  6. Splitting the spectral flow and the SU(3) Casson invariant for spliced sums

    DEFF Research Database (Denmark)

    Boden, Hans U.; Himpel, Benjamin

    2009-01-01

    We show that the SU(3) Casson invariant for spliced sums along certain torus knots equals 16 times the product of their SU(2) Casson knot invariants. The key step is a splitting formula for su(n) spectral flow for closed 3–manifolds split along a torus....

  7. Alternative Splicing of P/Q-Type Ca2+ Channels Shapes Presynaptic Plasticity

    Directory of Open Access Journals (Sweden)

    Agnes Thalhammer

    2017-07-01

    Full Text Available Alternative splicing of pre-mRNAs is prominent in the mammalian brain, where it is thought to expand proteome diversity. For example, alternative splicing of voltage-gated Ca2+ channel (VGCC α1 subunits can generate thousands of isoforms with differential properties and expression patterns. However, the impact of this molecular diversity on brain function, particularly on synaptic transmission, which crucially depends on VGCCs, is unclear. Here, we investigate how two major splice isoforms of P/Q-type VGCCs (Cav2.1[EFa/b] regulate presynaptic plasticity in hippocampal neurons. We find that the efficacy of P/Q-type VGCC isoforms in supporting synaptic transmission is markedly different, with Cav2.1[EFa] promoting synaptic depression and Cav2.1[EFb] synaptic facilitation. Following a reduction in network activity, hippocampal neurons upregulate selectively Cav2.1[EFa], the isoform exhibiting the higher synaptic efficacy, thus effectively supporting presynaptic homeostatic plasticity. Therefore, the balance between VGCC splice variants at the synapse is a key factor in controlling neurotransmitter release and presynaptic plasticity.

  8. Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells.

    Directory of Open Access Journals (Sweden)

    Winston Koh

    Full Text Available Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.

  9. CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

    Czech Academy of Sciences Publication Activity Database

    Dušková, Eva; Hnilicová, Jarmila; Staněk, David

    2014-01-01

    Roč. 11, č. 7 (2014), s. 865-874 ISSN 1547-6286 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68378050 Keywords : alternative splicing * fibronectin * p300 * histone acetylation * promoter Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 4.974, year: 2014

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

  12. Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

    2008-11-01

    Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

  13. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    International Nuclear Information System (INIS)

    Hu, Yi; Ericsson, Ida; Doseth, Berit; Liabakk, Nina B.; Krokan, Hans E.; Kavli, Bodil

    2014-01-01

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation

  14. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

    DEFF Research Database (Denmark)

    Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas

    2011-01-01

    There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a p...

  15. Defective splicing, disease and therapy: searching for master checkpoints in exon definition.

    Science.gov (United States)

    Buratti, Emanuele; Baralle, Marco; Baralle, Francisco E

    2006-01-01

    The number of aberrant splicing processes causing human disease is growing exponentially and many recent studies have uncovered some aspects of the unexpectedly complex network of interactions involved in these dysfunctions. As a consequence, our knowledge of the various cis- and trans-acting factors playing a role on both normal and aberrant splicing pathways has been enhanced greatly. However, the resulting information explosion has also uncovered the fact that many splicing systems are not easy to model. In fact we are still unable, with certainty, to predict the outcome of a given genomic variation. Nonetheless, in the midst of all this complexity some hard won lessons have been learned and in this survey we will focus on the importance of the wide sequence context when trying to understand why apparently similar mutations can give rise to different effects. The examples discussed in this summary will highlight the fine 'balance of power' that is often present between all the various regulatory elements that define exon boundaries. In the final part, we shall then discuss possible therapeutic targets and strategies to rescue genetic defects of complex splicing systems.

  16. CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

    Czech Academy of Sciences Publication Activity Database

    Dušková, E.; Hnilicová, Jarmila; Staněk, D.

    2014-01-01

    Roč. 11, č. 7 (2014), s. 1-10 ISSN 1547-6286 R&D Projects: GA ČR(CZ) GBP305/12/G034 Grant - others:Charles University Prague(CZ) 274111 Institutional support: RVO:61388971 Keywords : alternative splicing * fibronectin * p300 Subject RIV: EE - Microbiology, Virology Impact factor: 4.974, year: 2014

  17. CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

    Czech Academy of Sciences Publication Activity Database

    Dušková, Eva; Hnilicová, Jarmila; Staněk, David

    2014-01-01

    Roč. 11, č. 7 (2014), s. 865-874 ISSN 1547-6286 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68378050 Keywords : alternative splicing * fibronectin * p300 * histone acetylation * promoter Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.974, year: 2014

  18. Spliced DNA sequences in the Paramecium germline: their properties and evolutionary potential.

    Science.gov (United States)

    Catania, Francesco; McGrath, Casey L; Doak, Thomas G; Lynch, Michael

    2013-01-01

    Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic--often coding--DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation.

  19. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  20. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

    DEFF Research Database (Denmark)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

    2014-01-01

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense...

  1. Human aldehyde dehydrogenase genes: alternatively spliced transcriptional variants and their suggested nomenclature.

    Science.gov (United States)

    Black, William J; Stagos, Dimitrios; Marchitti, Satori A; Nebert, Daniel W; Tipton, Keith F; Bairoch, Amos; Vasiliou, Vasilis

    2009-11-01

    The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer's disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. Data-mining methods were used to retrieve all human ALDH sequences. Alternatively spliced transcriptional variants were determined based on (i) criteria for sequence integrity and genomic alignment; (ii) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and (iii) if available, empirical evidence of variants from the literature. Alternatively spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. To avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily.

  2. Differential dynamics of splicing factor SC35 during the cell cycle

    Indian Academy of Sciences (India)

    We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in ...

  3. Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

    Czech Academy of Sciences Publication Activity Database

    Divina, Petr; Kvitkovicova, Andrea; Buratti, E.; Vorechovsky, I.

    2009-01-01

    Roč. 17, č. 6 (2009), s. 759-765 ISSN 1018-4813 Institutional research plan: CEZ:AV0Z50520514 Keywords : mutation * cryptic splice site * exon skipping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.564, year: 2009

  4. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  5. Characterization of viral RNA splicing using whole-transcriptome datasets from host species.

    Science.gov (United States)

    Zhou, Chengran; Liu, Shanlin; Song, Wenhui; Luo, Shiqi; Meng, Guanliang; Yang, Chentao; Yang, Hua; Ma, Jinmin; Wang, Liang; Gao, Shan; Wang, Jian; Yang, Huanming; Zhao, Yun; Wang, Hui; Zhou, Xin

    2018-02-19

    RNA alternative splicing (AS) is an important post-transcriptional mechanism enabling single genes to produce multiple proteins. It has been well demonstrated that viruses deploy host AS machinery for viral protein productions. However, knowledge on viral AS is limited to a few disease-causing viruses in model species. Here we report a novel approach to characterizing viral AS using whole transcriptome dataset from host species. Two insect transcriptomes (Acheta domesticus and Planococcus citri) generated in the 1,000 Insect Transcriptome Evolution (1KITE) project were used as a proof of concept using the new pipeline. Two closely related densoviruses (Acheta domesticus densovirus, AdDNV, and Planococcus citri densovirus, PcDNV, Ambidensovirus, Densovirinae, Parvoviridae) were detected and analyzed for AS patterns. The results suggested that although the two viruses shared major AS features, dramatic AS divergences were observed. Detailed analysis of the splicing junctions showed clusters of AS events occurred in two regions of the virus genome, demonstrating that transcriptome analysis could gain valuable insights into viral splicing. When applied to large-scale transcriptomics projects with diverse taxonomic sampling, our new method is expected to rapidly expand our knowledge on RNA splicing mechanisms for a wide range of viruses.

  6. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Science.gov (United States)

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

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

    Science.gov (United States)

    2008-09-01

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

  8. Population genetics of duplicated alternatively spliced exons of the Dscam gene in Daphnia and Drosophila

    NARCIS (Netherlands)

    Brites, Daniela; Encinas-Viso, Francisco; Ebert, Dieter; Du Pasquier, Louis; Haag, Christoph R.

    2011-01-01

    In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This

  9. Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, M M

    2010-01-01

    In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthe...

  10. Phylogenetic analyses suggest reverse splicing spread of group I introns in fungal ribosomal DNA

    Directory of Open Access Journals (Sweden)

    Simon Dawn M

    2005-11-01

    Full Text Available Abstract Background Group I introns have spread into over 90 different sites in nuclear ribosomal DNA (rDNA with greater than 1700 introns reported in these genes. These ribozymes generally spread through endonuclease-mediated intron homing. Another putative pathway is reverse splicing whereby a free group I intron inserts into a homologous or heterologous RNA through complementary base-pairing between the intron and exon RNA. Reverse-transcription of the RNA followed by general recombination results in intron spread. Here we used phylogenetics to test for reverse splicing spread in a taxonomically broadly sampled data set of fungal group I introns including 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont Symbiotaphrina buchneri. Results Our analyses reveal a complex evolutionary history of the fungal introns with many cases of vertical inheritance (putatively for the 9 introns in S. buchneri and intron lateral transfer. There are several examples in which introns, many of which are still present in S. buchneri, may have spread through reverse splicing into heterologous rDNA sites. If the S. buchneri introns are ancient as we postulate, then group I intron loss was widespread in fungal rDNA evolution. Conclusion On the basis of these results, we suggest that the extensive distribution of fungal group I introns is at least partially explained by the reverse splicing movement of existing introns into ectopic rDNA sites.

  11. 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 amo...

  12. G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

    Energy Technology Data Exchange (ETDEWEB)

    Willing, M.; Deschenes, S. [Univ. of Iowa, Iowa City, IA (United States)

    1994-09-01

    We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exon 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.

  13. Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

    Directory of Open Access Journals (Sweden)

    Stefan Walzik

    2011-04-01

    Full Text Available Alternative splicing creates several Na(v1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v1.5 splice variants have been discovered. Four of them, namely Na(v1.5a, Na(v1.5c, Na(v1.5d, and Na(v1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3 and isolated cardiac conduction disease (CCD. When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v1.5, hNa(v1.5a, and hNa(v1.5c. In contrast, the hNa(v1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v1.5e, the neonatal variant of hNa(v1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

  14. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

  15. Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Bolte Kathrin

    2010-04-01

    Full Text Available Abstract Background Alternative splicing (AS involving tandem acceptors that are separated by three nucleotides (NAGNAG is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human and Mus musculus (mouse. It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice. In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss. Results Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor. Our classifier achieved a balanced specificity and sensitivity of ≥ 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454, we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125 of the well-supported cases of AS being predicted correctly. Conclusion NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns, and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the

  16. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    Science.gov (United States)

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. PMID:20068555

  17. Depletion of somatic mutations in splicing-associated sequences in cancer genomes.

    Science.gov (United States)

    Hurst, Laurence D; Batada, Nizar N

    2017-11-07

    An important goal of cancer genomics is to identify systematically cancer-causing mutations. A common approach is to identify sites with high ratios of non-synonymous to synonymous mutations; however, if synonymous mutations are under purifying selection, this methodology leads to identification of false-positive mutations. Here, using synonymous somatic mutations (SSMs) identified in over 4000 tumours across 15 different cancer types, we sought to test this assumption by focusing on coding regions required for splicing. Exon flanks, which are enriched for sequences required for splicing fidelity, have ~ 17% lower SSM density compared to exonic cores, even after excluding canonical splice sites. While it is impossible to eliminate a mutation bias of unknown cause, multiple lines of evidence support a purifying selection model above a mutational bias explanation. The flank/core difference is not explained by skewed nucleotide content, replication timing, nucleosome occupancy or deficiency in mismatch repair. The depletion is not seen in tumour suppressors, consistent with their role in positive tumour selection, but is otherwise observed in cancer-associated and non-cancer genes, both essential and non-essential. Consistent with a role in splicing modulation, exonic splice enhancers have a lower SSM density before and after controlling for nucleotide composition; moreover, flanks at the 5' end of the exons have significantly lower SSM density than at the 3' end. These results suggest that the observable mutational spectrum of cancer genomes is not simply a product of various mutational processes and positive selection, but might also be shaped by negative selection.

  18. Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.

    Science.gov (United States)

    McNeil, Bonnie A; Zimmerly, Steven

    2014-06-01

    Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. © 2014 McNeil and Zimmerly; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Sample Size Estimation for Detection of Splicing Events in Transcriptome Sequencing Data.

    Science.gov (United States)

    Kaisers, Wolfgang; Schwender, Holger; Schaal, Heiner

    2017-09-05

    Merging data from multiple samples is required to detect low expressed transcripts or splicing events that might be present only in a subset of samples. However, the exact number of required replicates enabling the detection of such rare events often remains a mystery but can be approached through probability theory. Here, we describe a probabilistic model, relating the number of observed events in a batch of samples with observation probabilities. Therein, samples appear as a heterogeneous collection of events, which are observed with some probability. The model is evaluated in a batch of 54 transcriptomes of human dermal fibroblast samples. The majority of putative splice-sites (alignment gap-sites) are detected in (almost) all samples or only sporadically, resulting in an U-shaped pattern for observation probabilities. The probabilistic model systematically underestimates event numbers due to a bias resulting from finite sampling. However, using an additional assumption, the probabilistic model can predict observed event numbers within a events (mean 7122 in alignments from TopHat alignments and 86,215 in alignments from STAR). We conclude that the probabilistic model provides an adequate description for observation of gap-sites in transcriptome data. Thus, the calculation of required sample sizes can be done by application of a simple binomial model to sporadically observed random events. Due to the large number of uniquely observed putative splice-sites and the known stochastic noise in the splicing machinery, it appears advisable to include observation of rare splicing events into analysis objectives. Therefore, it is beneficial to take scores for the validation of gap-sites into account.

  20. Examining the intersection between splicing, nuclear export and small RNA pathways.

    Science.gov (United States)

    Nabih, Amena; Sobotka, Julia A; Wu, Monica Z; Wedeles, Christopher J; Claycomb, Julie M

    2017-11-01

    Nuclear Argonaute/small RNA pathways in a variety of eukaryotic species are generally known to regulate gene expression via chromatin modulation and transcription attenuation in a process known as transcriptional gene silencing (TGS). However, recent data, including genetic screens, phylogenetic profiling, and molecular mechanistic studies, also point to a novel and emerging intersection between the splicing and nuclear export machinery with nuclear Argonaute/small RNA pathways in many organisms. In this review, we summarize the field's current understanding regarding the relationship between splicing, export and small RNA pathways, and consider the biological implications for coordinated regulation of transcripts by these pathways. We also address the importance and available approaches for understanding the RNA regulatory logic generated by the intersection of these particular pathways in the context of synthetic biology. The interactions between various eukaryotic RNA regulatory pathways, particularly splicing, nuclear export and small RNA pathways provide a type of combinatorial code that informs the identity ("self" versus "non-self") and dictates the fate of each transcript in a cell. Although the molecular mechanisms for how splicing and nuclear export impact small RNA pathways are not entirely clear at this early stage, the links between these pathways are widespread across eukaryotic phyla. The link between splicing, nuclear export, and small RNA pathways is emerging and establishes a new frontier for understanding the combinatorial logic of gene regulation across species that could someday be harnessed for therapeutic, biotechnology and agricultural applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F.

    Directory of Open Access Journals (Sweden)

    Erming Wang

    Full Text Available In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5' splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.

  2. Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

    Science.gov (United States)

    Ortigão-Farias, João Ramalho; Di-Blasi, Tatiana; Telleria, Erich Loza; Andorinho, Ana Carolina; Lemos-Silva, Thais; Ramalho-Ortigão, Marcelo; Tempone, Antônio Jorge; Traub-Csekö, Yara Maria

    2018-02-01

    BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

  3. New approach for high reliability, low loss splicing between silica and ZBLAN fibers

    Science.gov (United States)

    Carbonnier, Robin; Zheng, Wenxin

    2018-02-01

    In the past decade, ZBLAN (ZrF4-BaF2-LaF3-NaF) fibers have drawn increasing interest for laser operations at wavelengths where Fused Silica-based (SiO2) fibers do not perform well. One limitation to the expansion of ZBLAN fiber lasers today is the difficulty to efficiently inject and extract light in/from the guiding medium using SiO2 fibers. Although free space and butt coupling have provided acceptable results, consistent and long lasting physical joints between SiO2 and ZBLAN fibers will allow smaller, cheaper, and more robust component manufacturing. While low loss splices have been reported using a traditional splicing approach, the very low mechanical strength of the joint makes it difficult to scale. Difficulties in achieving a strong bond are mainly due to the large difference of transition temperature between ZBLAN and SiO2 fibers ( 260°C vs 1175°C). This paper presents results obtained by using the high thermal expansion coefficient of the ZBLAN fiber to encapsulate a smaller SiO2 fiber. A CO2 laser glass processing system was used to control the expansion and contraction of the ZBLAN material during the splicing process for optimum reliability. This method produced splices between 125μm ZBLAN to 80μm SiO2 fibers with average transmission loss of 0.225dB (measured at 1550nm) and average ultimate tension strength of 121.4gf. The Resulting splices can be durably packaged without excessive care. Other combinations using 125μm SiO2 fibers tapered to 80μm are also discussed.

  4. Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Ann-Katrin Claudius

    2014-04-01

    Full Text Available The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1 strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

  5. Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

    Science.gov (United States)

    Treutlein, Barbara; Gokce, Ozgun; Quake, Stephen R; Südhof, Thomas C

    2014-04-01

    Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

  6. Alternative Splicing Regulation of Cancer-Related Pathways in Caenorhabditis elegans: An In Vivo Model System with a Powerful Reverse Genetics Toolbox

    Directory of Open Access Journals (Sweden)

    Sergio Barberán-Soler

    2013-01-01

    Full Text Available Alternative splicing allows for the generation of protein diversity and fine-tunes gene expression. Several model systems have been used for the in vivo study of alternative splicing. Here we review the use of the nematode Caenorhabditis elegans to study splicing regulation in vivo. Recent studies have shown that close to 25% of genes in the worm genome undergo alternative splicing. A big proportion of these events are functional, conserved, and under strict regulation either across development or other conditions. Several techniques like genome-wide RNAi screens and bichromatic reporters are available for the study of alternative splicing in worms. In this review, we focus, first, on the main studies that have been performed to dissect alternative splicing in this system and later on examples from genes that have human homologs that are implicated in cancer. The significant advancement towards understanding the regulation of alternative splicing and cancer that the C. elegans system has offered is discussed.

  7. A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein.

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    Georgios Valianatos

    Full Text Available MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX. Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing Mdm

  8. Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

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    Mario Gustavo Mayer

    2005-08-01

    Full Text Available Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.

  9. Some sufficient conditions for persistency and permanency of two stages DNA splicing languages via Yusof-Goode approach

    Science.gov (United States)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-07-01

    Splicing system that was first introduced by Head makes a connection between field of formal language theory and molecular biology. This system modeled the biological process of splitting and ligating on double stranded deoxyribonucleic acid (DNA) molecules under effect of restriction enzymes and appropriate ligase. In this paper, the concept of two stages DNA splicing languages is introduced. Some sufficient conditions for persistency and permanency of the above DNA splicing languages focusing on two rules and two initial strings will be investigated by usingYusof-Good (Y-G) approach.

  10. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    Science.gov (United States)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  11. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    Science.gov (United States)

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

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    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

  13. The Cancer Exome Generated by Alternative mRNA Splicing Dilutes Predicted HLA Class I Epitope Density

    DEFF Research Database (Denmark)

    Stranzl, Thomas; Larsen, Mette Voldby; Lund, Ole

    2012-01-01

    Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes...... is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based...... on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I...

  14. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

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    Jeong H Ahn

    2016-08-01

    Full Text Available The elongation phase of transcription by RNA Polymerase II (Pol II involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation.

  15. Performance of lap splices in large-scale column specimens affected by ASR and/or DEF-extension phase.

    Science.gov (United States)

    2015-03-01

    A large experimental program, consisting of the design, construction, curing, exposure, and structural load : testing of 16 large-scale column specimens with a critical lap splice region that were influenced by varying : stages of alkali-silica react...

  16. Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

    Science.gov (United States)

    Butty, Vincent L; Boutz, Paul L; Begum, Shahinoor; Kimble, Amy L; Sharp, Phillip A; Burge, Christopher B

    2018-01-01

    Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation. PMID:29293084

  17. Modeling of DNA single stage splicing language via Yusof-Goode approach: One string with two rules

    Science.gov (United States)

    Lim, Wen Li; Yusof, Yuhani; Mudaber, Mohammad Hassan

    2015-02-01

    Splicing system plays a pivotal role in attempts to recombine sets of double-stranded DNA molecules when acted by restriction enzymes and ligase. Traditional method of finding the result of DNA recombination through experiment is both time and money consuming. Hence, finding the number of patterns of DNA single stage splicing language through formalism of splicing system is a way to optimize the searching process. From the biological perspective, it predicts the number of types of molecules that will exist in the system under existence of restriction enzymes and ligase. In this paper, some theorems, corollaries and examples that lead to the predictions of single stage splicing languages involving one pattern string and two rules are presented via Yusof-Goode approach.

  18. Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART.

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    Jesse D Deere

    Full Text Available Highly active antiretroviral therapy (HAART can reduce levels of human immunodeficiency virus type 1 (HIV-1 to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA, leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load.

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

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    Omar Soukarieh

    2016-01-01

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

  20. The Cancer Exome Generated by Alternative mRNA Splicing Dilutes Predicted HLA Class I Epitope Density

    OpenAIRE

    Stranzl, Thomas; Larsen, Mette V.; Lund, Ole; Nielsen, Morten; Brunak, Søren

    2012-01-01

    Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the c...

  1. Interplay between exonic splicing enhancers, mRNA processing, and mRNA surveillance in the dystrophic Mdx mouse.

    Directory of Open Access Journals (Sweden)

    Massimo Buvoli

    2007-05-01

    Full Text Available Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5' and 3' splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD, lacks dystrophin by virtue of a premature termination codon (PTC in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described.Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3' splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS, but induces exclusively nonsense-mediated mRNA decay (NMD. Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24.Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon-exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites.

  2. New insights into the genomic organization and splicing of the doublesex gene, a terminal regulator of sexual differentiation in the silkworm Bombyx mori.

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    Jianping Duan

    Full Text Available Sex-determination mechanisms differ among organisms. The primary mechanism is diverse, whereas the terminal regulator is relatively-conserved. We analyzed the transcripts of the Bombyx mori doublesex gene (Bmdsx, and reported novel results concerning the genomic organization and expression of Bmdsx. Bmdsx consists of nine exons and eight introns, of which two exons are novel and have not been reported previously. Bmdsx transcripts are spliced to generate seventeen alternatively-spliced forms and eleven putative trans-spliced variants. Thirteen of the alternatively-spliced forms and five of the putative trans-spliced forms are reported here for the first time. Sequence analysis predicts that ten female-specific, six male-specific splice forms and one splice form found in males and females will result in four female-specific, two male-specific Dsx proteins and one Dsx protein common to males and females. The Dsx proteins are expected to be functional and regulate downstream target genes. Some of the predicted Dsx proteins are described here for the first time. Therefore the expression of the dsx gene in B. mori results in a variety of cis- and trans-spliced transcripts and multiple Dsx proteins. These findings show that in B. mori there is a complicated pattern of dsx splicing, and that the regulation of splicing and sex-specific functions of lepidopteran dsx have evolved complexity.

  3. NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.

    Science.gov (United States)

    Adamia, Sophia; Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Tenen, Daniel G; Stone, Richard M; Griffin, James D

    2014-05-01

    Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

  4. A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

    Science.gov (United States)

    Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

    2014-03-01

    Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

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

    Science.gov (United States)

    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.

  6. Splicing Regulation of Pro-Inflammatory Cytokines and Chemokines: At the Interface of the Neuroendocrine and Immune Systems.

    Science.gov (United States)

    Shakola, Felitsiya; Suri, Parul; Ruggiu, Matteo

    2015-09-07

    Alternative splicing plays a key role in posttranscriptional regulation of gene expression, allowing a single gene to encode multiple protein isoforms. As such, alternative splicing amplifies the coding capacity of the genome enormously, generates protein diversity, and alters protein function. More than 90% of human genes undergo alternative splicing, and alternative splicing is especially prevalent in the nervous and immune systems, tissues where cells need to react swiftly and adapt to changes in the environment through carefully regulated mechanisms of cell differentiation, migration, targeting, and activation. Given its prevalence and complexity, this highly regulated mode of gene expression is prone to be affected by disease. In the following review, we look at how alternative splicing of signaling molecules—cytokines and their receptors—changes in different pathological conditions, from chronic inflammation to neurologic disorders, providing means of functional interaction between the immune and neuroendocrine systems. Switches in alternative splicing patterns can be very dynamic and can produce signaling molecules with distinct or antagonistic functions and localization to different subcellular compartments. This newly discovered link expands our understanding of the biology of immune and neuroendocrine cells, and has the potential to open new windows of opportunity for treatment of neurodegenerative disorders.

  7. SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

    Science.gov (United States)

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  8. 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.

  9. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

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    Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

    2012-04-25

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  10. A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy.

    Science.gov (United States)

    Dwianingsih, Ery Kus; Malueka, Rusdy Ghazali; Nishida, Atsushi; Itoh, Kyoko; Lee, Tomoko; Yagi, Mariko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

    2014-08-01

    Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient's DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.

  11. Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

    Science.gov (United States)

    Armero, Victoria E S; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

    2017-01-01

    Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

  12. Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches

    Science.gov (United States)

    Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Griffith, Graziella; Philippi, Susanne; Précigout, Guillaume; Mamchaoui, Kamel; Jollet, Arnaud; Voit, Thomas; Garcia, Luis

    2013-01-01

    RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations. PMID:23861443

  13. Clk/STY (cdc2-like kinase 1 and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes.

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    Pengfei Li

    Full Text Available The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8-10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6 from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.