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Sample records for hax-1 overexpression splicing

  1. UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion

    International Nuclear Information System (INIS)

    Hirasaka, Katsuya; Mills, Edward M.; Haruna, Marie; Bando, Aki; Ikeda, Chika; Abe, Tomoki; Kohno, Shohei; Nowinski, Sara M.; Lago, Cory U.; Akagi, Ken-ichi; Tochio, Hidehito; Ohno, Ayako; Teshima-Kondo, Shigetada; Okumura, Yuushi; Nikawa, Takeshi

    2016-01-01

    Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca"2"+. The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca"2"+, suggesting that the C-terminal domain of Hax-1 underwent a Ca"2"+-induced conformational change. In the Ca"2"+-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca"2"+ binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca"2"+. - Highlights: • UCP3 interacts with Hax-1. • The interaction of UCP3 and Hax-1 occurs in a calcium-dependent manner. • The C-terminal domain of Hax-1 undergoes a calcium-induced conformational change.

  2. The calcium binding properties and structure prediction of the Hax-1 protein.

    Science.gov (United States)

    Balcerak, Anna; Rowinski, Sebastian; Szafron, Lukasz M; Grzybowska, Ewa A

    2017-01-01

    Hax-1 is a protein involved in regulation of different cellular processes, but its properties and exact mechanisms of action remain unknown. In this work, using purified, recombinant Hax-1 and by applying an in vitro autoradiography assay we have shown that this protein binds Ca 2+ . Additionally, we performed structure prediction analysis which shows that Hax-1 displays definitive structural features, such as two α-helices, short β-strands and four disordered segments.

  3. UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion

    Energy Technology Data Exchange (ETDEWEB)

    Hirasaka, Katsuya, E-mail: hirasaka@nagasaki-u.ac.jp [Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki (Japan); Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Mills, Edward M. [Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX (United States); Haruna, Marie; Bando, Aki; Ikeda, Chika; Abe, Tomoki [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Kohno, Shohei; Nowinski, Sara M. [Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX (United States); Lago, Cory U. [Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Akagi, Ken-ichi [Section of Laboratory Equipment, National Institute of Biomedical Innovation, Osaka (Japan); Tochio, Hidehito [Graduate School of Engineering, Kyoto University, Kyoto (Japan); Ohno, Ayako; Teshima-Kondo, Shigetada [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Okumura, Yuushi [Department of Nutrition and Health, Sagami Woman' s University, Kanagawa (Japan); Nikawa, Takeshi [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan)

    2016-03-25

    Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca{sup 2+}. The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca{sup 2+}, suggesting that the C-terminal domain of Hax-1 underwent a Ca{sup 2+}-induced conformational change. In the Ca{sup 2+}-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca{sup 2+} binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca{sup 2+}. - Highlights: • UCP3 interacts with Hax-1. • The interaction of UCP3 and Hax-1 occurs in a calcium-dependent manner. • The C-terminal domain of Hax-1 undergoes a calcium-induced conformational change.

  4. HAX-1 Protects Glioblastoma Cells from Apoptosis through the Akt1 Pathway

    Directory of Open Access Journals (Sweden)

    Xin Deng

    2017-12-01

    Full Text Available Glioblastoma is the most common malignant tumor in central nervous system (CNS, and it is still insurmountable and has a poor prognosis. The proliferation and survival mechanism of glioma cells needs to be explored further for the development of glioma treatment. Hematopoietic-substrate-1 associated protein X-1 (HAX-1 has been reported as an anti-apoptosis protein that plays an important role in several malignant tumors. However, the effect and mechanism of HAX-1 in glioblastomas remains unknown. This study aimed to investigate the effect of HAX-1 in glioblastoma cells and explore the mechanism. The results of clone formation and Edu proliferation assay showed slower multiplication in HAX-1 knock-out cells. Flow cytometry showed cell cycle arrest mainly in G0/G1 phase. Apoptosis due to oxidative stress was increased after HAX-1 was knocked out. Western-blot assay exhibited that the levels of p21, Bax, and p53 proteins were significantly raised, and that the activation of the caspase cascade was enhanced in the absence of HAX-1. The degradation rate and ubiquitination of p53 declined because of the decrease in phosphorylation of proteins MDM2 and Akt1. Co-immunoprecipitation (Co-IP and immunefluorescent co-localization assays were performed to test the influence of HAX-1 on the interaction between Akt1 and Hsp90, which is crucial for the activity of Akt1. In conclusion, this novel study suggested that HAX-1 could affect the Akt1 pathway through Hsp90. The knock-out of HAX-1 leads to the inactivity of the Ak1t/MDM2 axis, which leads to increased levels of p53, and finally generates cell cycle arrest and results in the apoptosis of glioblastoma cells.

  5. Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

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    Hoyer-Fender Sigrid

    2010-04-01

    Full Text Available Abstract Background Pelota (PELO is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation. Results We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC, we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments. Conclusion We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.

  6. Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

    Science.gov (United States)

    Pittermann, Erik; Lachmann, Nico; MacLean, Glenn; Emmrich, Stephan; Ackermann, Mania; Göhring, Gudrun; Schlegelberger, Brigitte; Welte, Karl; Schambach, Axel; Heckl, Dirk; Orkin, Stuart H; Cantz, Tobias; Klusmann, Jan-Henning

    2017-06-13

    Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 ( HAX1 ) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1 W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1 -centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1 W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

  7. FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

    Science.gov (United States)

    Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

    2012-01-01

    Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

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

    Science.gov (United States)

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

    2015-01-01

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

  9. H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells.

    Science.gov (United States)

    Mazel-Sanchez, B; Boal-Carvalho, I; Silva, F; Dijkman, R; Schmolke, M

    2018-06-01

    Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans. IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction

  10. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

    Science.gov (United States)

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-09-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

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

    Science.gov (United States)

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

    2016-08-12

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  13. Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation

    OpenAIRE

    Siamakpour-Reihani, Sharareh; Peterson, Tabitha A.; Bradford, Andrew M.; Argiros, Haroula J.; Haas, Laura Lowell; Lor, Siamee N.; Haulsee, Zachary M.; Spuches, Anne M.; Johnson, Dennis L.; Rohrschneider, Larry R.; Shuster, Charles Brad; Lyons, Barbara A.

    2011-01-01

    Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been full...

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Abhishek Kumar

    2017-10-01

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

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

    KAUST Repository

    Cui, Peng; Zhang, ShouDong; Ding, Feng; Ali, Shahjahan; Xiong, Liming

    2014-01-01

    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

  17. Targeting Splicing in Prostate Cancer

    OpenAIRE

    Effrosyni Antonopoulou; Michael Ladomery

    2018-01-01

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

  18. Mechanical rebar splicing

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    Milosavljević Branko

    2014-01-01

    Full Text Available Different mechanical rebar splicing systems are presented, and design situations where mechanical splicing has advantage over reinforcement splicing by overlapping and welding are defined in this paper. New international standards for testing and proof of systems for mechanical rebar splicing quality are considered. Mechanical splicing system for rebar and bolt connection, usable in steel and reinforced concrete structural elements connections, is presented in this paper. There are only few examples of mechanical rebar splicing in our country. The most significant one - the pylon and beam connection at Ada Bridge in Belgrade is presented in the paper. Intensive development of production and use of mechanical rebar splicing systems, research in this area, as well as the publication of international standards prescribing requirements for quality and procedures for proof of quality, represent very good base for development of the corresponding technical norms in Serbia. The legislation in this area would quicken proof of quality procedures, attest and approval issuing for individual products, leading to wider use of this system in all situations where it is in advantage over the classical reinforcement splicing.

  19. 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...

  20. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

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

  1. 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

    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...

  2. The neurogenetics of alternative splicing.

    Science.gov (United States)

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

    2016-05-01

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

  3. 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

    -dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

  4. Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

    International Nuclear Information System (INIS)

    O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.; O'Brien, Kathryn M.; Reitter, Julie N.; Mills, Kenneth V.

    2010-01-01

    Research highlights: → The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. → Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. → The intein splices with Lys in place of the highly conserved penultimate His. → The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

  5. 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.

  6. 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...

  7. Alternative RNA splicing and cancer

    Science.gov (United States)

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  8. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Enrique Álvarez

    Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

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

    OpenAIRE

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

    NARCIS (Netherlands)

    Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

  17. 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.

  18. Spliced RNA of woodchuck hepatitis virus.

    Science.gov (United States)

    Ogston, C W; Razman, D G

    1992-07-01

    Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

  19. Aberrant alternative splicing is another hallmark of cancer.

    Science.gov (United States)

    Ladomery, Michael

    2013-01-01

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

  20. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    OpenAIRE

    Ladomery, Michael

    2013-01-01

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

  1. SplicePlot: a utility for visualizing splicing quantitative trait loci.

    Science.gov (United States)

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B

    2014-04-01

    RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

  2. Hematopoietic Substrate-1-Associated Protein X-1 Regulates the Proliferation and Apoptosis of Endothelial Progenitor Cells Through Akt Pathway Modulation.

    Science.gov (United States)

    Guo, Xin-Bin; Deng, Xin; Wei, Ying

    2018-03-01

    Endothelial precursor cells (EPCs) are involved in vasculogenesis of various physiological and pathological processes. The proliferation and survival mechanism of EPCs needs to be explored further for the purpose of developing an effective glioma treatment. Hematopoietic substrate-1-associated protein X-1 (HAX-1) has been reported as an anti-apoptotic protein that plays an important role in several malignant tumors. However, the effect and mechanism of HAX-1 on EPCs remains unknown. This study aims to investigate the effect of HAX-1 on the proliferation and apoptosis of EPCs and explore its mechanism. According to our results, HAX-1 was overexpressed in EPCs. The results of clone formation and 5-ethynyl-2'-deoxyuridine proliferation assay showed that HAX-1 promoted multiplication of EPCs. Flow cytometry showed HAX-1 knockout cell cycle arrest mainly in G0/G1 phase. Apoptosis analysis showed that HAX-1 could protect EPCs from apoptosis in oxidative stress. Western blot assay indicated that HAX-1 could inhibit the activation of caspase cascade and reduce the expression of p21, Bcl-2-associated X protein, and p53. HAX-1 also enhanced the degradation rate and ubiquitination of p53 through the promotion of phosphorylation of proteins MDM-2 and Akt1. Co-immunoprecipitation and immunofluorescent colocalization assays were performed to test the influence of HAX-1 on the interaction between Akt1 and heat shock protein 90 (Hsp90), which is crucial for the activity of Akt1. In conclusion, this novel study suggests that HAX-1 could facilitate the Akt1 pathway through Hsp90, which led to a decline in the levels of p53, and finally promoted the proliferation and inhibited the apoptosis of EPCs. Stem Cells 2018;36:406-419. © 2017 AlphaMed Press.

  3. 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

  4. 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...

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  6. Alternative Splicing in Neurogenesis and Brain Development.

    Science.gov (United States)

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

    2018-01-01

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

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  10. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    Science.gov (United States)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

  11. Quantitative nature of overexpression experiments

    Science.gov (United States)

    Moriya, Hisao

    2015-01-01

    Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation associated with the degree of overexpression. PMID:26543202

  12. Alternative Splicing Control of Abiotic Stress Responses.

    Science.gov (United States)

    Laloum, Tom; Martín, Guiomar; Duque, Paula

    2018-02-01

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

  13. CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

    Science.gov (United States)

    Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

    2009-12-01

    Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

  14. 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...

  15. Position dependence of the rous sarcoma virus negative regulator of splicing element reflects proximity to a 5' splice site

    International Nuclear Information System (INIS)

    Wang Yuedi; McNally, Mark T.

    2003-01-01

    Rous sarcoma virus (RSV) requires incomplete splicing of its viral transcripts to maintain efficient replication. A splicing inhibitor element, the negative regulator of splicing (NRS), is located near the 5' end of the RNA but the significance of this positioning is not known. In a heterologous intron the NRS functions optimally when positioned close to the authentic 5' splice site. This observation led us to investigate the basis of the position dependence. Four explanations were put forth and stressed the role of three major elements involved in splicing, the 3' splice site, the 5' splice site, and the 5' end cap structure. NRS function was unrelated to its position relative to the 3' splice site or the cap structure and appeared to depend on its position relative to the authentic 5' splice site. We conclude that position dependence may reflect distance constraints necessary for competition of the NRS with the authentic 5' splice site for pairing with the 3' splice sites

  16. 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.

  17. LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding

    Directory of Open Access Journals (Sweden)

    Ji Won Um

    2016-02-01

    Full Text Available The four members of the LRRTM family (LRRTM1-4 are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.

  18. 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.

  19. 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.

  20. 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_patt...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

  1. DEK protein overexpression predicts poor prognosis in pancreatic ductal adenocarcinoma.

    Science.gov (United States)

    Sun, Jie; Bi, Fangfang; Yang, Yang; Zhang, Yuan; Jin, Aihua; Li, Jinzi; Lin, Zhenhua

    2017-02-01

    DEK, a transcription factor, is involved in mRNA splicing, transcriptional control, cell division and differentiation. Recent studies suggest that DEK overexpression can promote tumorigenesis in a wide range of cancer cell types. However, little is known concerning the status of DEK in pancreatic ductal adenocarcinoma (PDAC). Based on the microarray data from Gene Expression Omnibus (GEO), the expression levels of DEK mRNA in PDAC tissues were significantly higher than levels in the adjacent non-tumor tissues. To explore the clinical features of DEK overexpression in PDAC, 87 PDAC and 52 normal pancreas tissues were selected for immunoenzyme staining of the DEK protein. Localization of the DEK protein was detected in PANC-1 pancreatic cancer cells using immunofluorescence (IF) staining. The correlations between DEK overexpression and the clinical features of PDAC were evaluated using the Chi-squared (χ2) and Fisher's exact tests. The survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazard models. The expression levels of DEK mRNA in PDAC tissues were significantly higher than that in the adjacent non‑tumor tissues. The DEK protein showed a primarily nuclear staining pattern in PDAC. The positive rate of the DEK protein was 52.9% (46/87) in PDAC, which was significantly higher than that in the adjacent normal pancreatic tissues (7.7%, 4/52). DEK overexpression in PDAC was correlated with tumor size, histological grade, tumor‑node‑metastasis (TNM) stage and overall survival (OS) rates. In addition, multivariate analysis demonstrated that DEK overexpression was an independent prognostic factor along with histological grade and TNM stage in patients with PDAC. In conclusion, DEK overexpression is associated with PDAC progression and may be a potential biomarker for poor prognostic evaluation in PDAC.

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

    Science.gov (United States)

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

    2009-05-01

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

  3. 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.

  4. 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.

  5. Variation in alternative splicing across human tissues

    OpenAIRE

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

    2004-01-01

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

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Ye E Zhou

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

  8. Genetics of alternative splicing evolution during sunflower domestication.

    Science.gov (United States)

    Smith, Chris C R; Tittes, Silas; Mendieta, J Paul; Collier-Zans, Erin; Rowe, Heather C; Rieseberg, Loren H; Kane, Nolan C

    2018-06-11

    Alternative splicing enables organisms to produce the diversity of proteins necessary for multicellular life by using relatively few protein-coding genes. Although differences in splicing have been identified among divergent taxa, the shorter-term evolution of splicing is understudied. The origins of novel splice forms, and the contributions of alternative splicing to major evolutionary transitions, are largely unknown. This study used transcriptomes of wild and domesticated sunflowers to examine splice differentiation and regulation during domestication. We identified substantial splicing divergence between wild and domesticated sunflowers, mainly in the form of intron retention. Transcripts with divergent splicing were enriched for seed-development functions, suggesting that artificial selection impacted splicing patterns. Mapping of quantitative trait loci (QTLs) associated with 144 differential splicing cases revealed primarily trans -acting variation affecting splicing patterns. A large proportion of identified QTLs contain known spliceosome proteins and are associated with splicing variation in multiple genes. Examining a broader set of wild and domesticated sunflower genotypes revealed that most differential splicing patterns in domesticated sunflowers likely arose from standing variation in wild Helianthus annuus and gained frequency during the domestication process. However, several domesticate-associated splicing patterns appear to be introgressed from other Helianthus species. These results suggest that sunflower domestication involved selection on pleiotropic regulatory alleles. More generally, our findings indicate that substantial differences in isoform abundances arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.

  9. Alternative RNA splicing and gastric cancer.

    Science.gov (United States)

    Li, Ying; Yuan, Yuan

    2017-07-01

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

  10. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

    NARCIS (Netherlands)

    Mueller, Nancy; Berkhout, Ben; Das, Atze T.

    2015-01-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

  11. Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

    Science.gov (United States)

    Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

    2014-01-01

    Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

  12. Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

    Science.gov (United States)

    Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

    2014-02-01

    Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

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

  15. 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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  17. Modelling reveals kinetic advantages of co-transcriptional splicing.

    Directory of Open Access Journals (Sweden)

    Stuart Aitken

    2011-10-01

    Full Text Available Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

  18. Modelling reveals kinetic advantages of co-transcriptional splicing.

    Science.gov (United States)

    Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

    2011-10-01

    Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

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

    Science.gov (United States)

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

    2017-07-07

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

  20. The 20S proteasome splicing activity discovered by SpliceMet.

    Directory of Open Access Journals (Sweden)

    Juliane Liepe

    2010-06-01

    Full Text Available The identification of proteasome-generated spliced peptides (PSP revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS. For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

  1. Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

    Science.gov (United States)

    Boroni, Mariana; Sammeth, Michael; Gava, Sandra Grossi; Jorge, Natasha Andressa Nogueira; Macedo, Andréa Mara; Machado, Carlos Renato; Mourão, Marina Moraes; Franco, Glória Regina

    2018-03-01

    Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.

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

    Directory of Open Access Journals (Sweden)

    Penny David

    2007-10-01

    Full Text Available Abstract Background Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. Results For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. Conclusion Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

  3. Alternative splicing in cancers: From aberrant regulation to new therapeutics.

    Science.gov (United States)

    Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

    2018-03-01

    Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. 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

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

    Science.gov (United States)

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

    2017-07-01

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

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

    Science.gov (United States)

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

    2017-05-30

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

  7. Loss of Endocan tumorigenic properties after alternative splicing of exon 2

    International Nuclear Information System (INIS)

    Depontieu, Florence; Grigoriu, Bogdan-Dragos; Scherpereel, Arnaud; Adam, Estelle; Delehedde, Maryse; Gosset, Philippe; Lassalle, Philippe

    2008-01-01

    Endocan was originally described as a dermatan sulfate proteoglycan found freely circulating in the blood. Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells. This molecule is the product of a single gene composed of 3 exons. Previous data showed that endocan mRNA is subject to alternative splicing with possible generation of two protein products. In the present study we identified, and functionally characterized, the alternative spliced product of the endocan gene: the exon 2-deleted endocan, called endocanΔ2. Stable, endocanΔ2-overexpressing cell lines were generated to investigate the biological activities of this new alternatively spliced product of endocan gene. Tumorigenesis was studied by inoculating endocan and endocanΔ2 expressing cell lines subcutaneously in SCID mice. Biochemical properties of endocan and endocanΔ2 were studied after production of recombinant proteins in various cell lines of human and murine origin. Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan. EndocanΔ2 did not promote tumor formation by 293 cells implanted in the skin of severe combined immunodeficient (SCID) mice. Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer

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

    Science.gov (United States)

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

    2017-01-01

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

  9. Hereditary cancer genes are highly susceptible to splicing mutations

    Science.gov (United States)

    Soemedi, Rachel; Maguire, Samantha; Murray, Michael F.; Monaghan, Sean F.

    2018-01-01

    Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing. PMID:29505604

  10. Hereditary cancer genes are highly susceptible to splicing mutations.

    Directory of Open Access Journals (Sweden)

    Christy L Rhine

    2018-03-01

    Full Text Available Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77% of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36% of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

  11. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    OpenAIRE

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted fro...

  12. Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

    Science.gov (United States)

    Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

    2016-08-01

    Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ferenc Jeanplong

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

  14. 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.

  15. 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...

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

    Science.gov (United States)

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

    2017-10-30

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

  17. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-05

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

  19. 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.

  20. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

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

    2015-01-01

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

  1. Language study on Spliced Semigraph using Folding techniques

    Science.gov (United States)

    Thiagarajan, K.; Padmashree, J.

    2018-04-01

    In this paper, we proposed algorithm to identify cut vertices and cut edges for n-Cut Spliced Semigraph and splicing the n-Cut Spliced Semigraph using cut vertices else cut edges or combination of cut vertex and cut edge and applying sequence of folding to the spliced semigraph to obtain the semigraph quadruple η(S)=(2, 1, 1, 1). We observed that the splicing and folding using both cut vertices and cut edges is applicable only for n-Cut Spliced Semigraph where n > 2. Also, we transformed the spliced semigraph into tree structure and studied the language for the semigraph with n+2 vertices and n+1 semivertices using Depth First Edge Sequence algorithm and obtain the language structure with sequence of alphabet ‘a’ and ‘b’.

  2. Multiple splicing defects in an intronic false exon.

    Science.gov (United States)

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  3. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    Science.gov (United States)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  4. SNEV overexpression extends the life span of human endothelial cells

    International Nuclear Information System (INIS)

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes

    2006-01-01

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells

  5. Entropic contributions to the splicing process

    International Nuclear Information System (INIS)

    Osella, Matteo; Caselle, Michele

    2009-01-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model

  6. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  7. 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.

  8. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

    Science.gov (United States)

    Seth, Puneet; Yeowell, Heather N

    2010-04-01

    Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease

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

    Science.gov (United States)

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

    2016-12-01

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

  10. 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

    and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  11. A study of alternative splicing in the pig

    Directory of Open Access Journals (Sweden)

    Jørgensen Claus B

    2010-05-01

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

  12. 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.

  13. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    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 by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......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...

  14. 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

  15. 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.

  16. Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis.

    Directory of Open Access Journals (Sweden)

    Marta Vicente-Crespo

    2008-02-01

    Full Text Available Muscleblind-like proteins (MBNL have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D are coded by the unique Drosophila muscleblind gene.We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3 minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a

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

    Science.gov (United States)

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  19. Alternative Splicing as a Target for Cancer Treatment.

    Science.gov (United States)

    Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

    2018-02-11

    Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

  20. 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......, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. CONCLUSION: Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form...... of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process....

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

    Directory of Open Access Journals (Sweden)

    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.

  2. The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis.

    NARCIS (Netherlands)

    Sugliani, M.; Brambilla, V.; Clerkx, E.J.M.; Koornneef, M.; Soppe, W.J.J.

    2010-01-01

    ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3- and ABI3-ß, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-ß transcript

  3. Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

    Directory of Open Access Journals (Sweden)

    Geetha Melangath

    Full Text Available Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional

  4. Revolutionizing membrane protein overexpression in bacteria

    NARCIS (Netherlands)

    Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

    The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory,

  5. Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells

    Science.gov (United States)

    Lu, Yu; Loh, Yuin-Han; Li, Hu; Cesana, Marcella; Ficarro, Scott B.; Parikh, Jignesh R.; Salomonis, Nathan; Toh, Cheng-Xu Delon; Andreadis, Stelios T.; Luckey, C. John; Collins, James J.; Daley, George Q.; Marto, Jarrod A.

    2014-01-01

    Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming. PMID:24813856

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

    Science.gov (United States)

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

    2015-11-15

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

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

    Indian Academy of Sciences (India)

    in this family revealed skipping of exon 22 in three members of this family. In one proband, a ... This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an ... of life) and presence of multiple tumors (multifocal). The ..... spliced RNA have been linked to parent of origin as well as.

  8. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2017-10-01

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

  9. 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.

  10. Antitumorigenic potential of STAT3 alternative splicing modulation.

    Science.gov (United States)

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

    2011-10-25

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

  11. 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...

  12. Connecting the dots: chromatin and alternative splicing in EMT.

    Science.gov (United States)

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  13. Conservation and sex-specific splicing of the doublesex gene

    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 ...

  14. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam

    2007-01-01

    suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has...

  15. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

  16. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

    Directory of Open Access Journals (Sweden)

    Yesenia Ríos

    2011-01-01

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

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

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

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

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

    Science.gov (United States)

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

    2017-01-01

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

  19. Alternative splicing of mutually exclusive exons--a review.

    Science.gov (United States)

    Pohl, Martin; Bortfeldt, Ralf H; Grützmann, Konrad; Schuster, Stefan

    2013-10-01

    Alternative splicing (AS) of pre-mRNAs in higher eukaryotes and several viruses is one major source of protein diversity. Usually, the following major subtypes of AS are distinguished: exon skipping, intron retention, and alternative 3' and 5' splice sites. Moreover, mutually exclusive exons (MXEs) represent a rare subtype. In the splicing of MXEs, two (or more) splicing events are not independent anymore, but are executed or disabled in a coordinated manner. In this review, several bioinformatics approaches for analyzing MXEs are presented and discussed. In particular, we revisit suitable definitions and nomenclatures, and bioinformatics tools for finding MXEs, adjacent and non-adjacent MXEs, clustered and grouped MXEs. Moreover, the molecular mechanisms for splicing MXEs proposed in the literature are reviewed and discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. 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...

  1. Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Bang, Jeanne Mv; Christensen, Lise L

    2018-01-01

    designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific...

  2. Gene Overexpression: Uses, Mechanisms, and Interpretation

    Science.gov (United States)

    2012-01-01

    The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease. PMID:22419077

  3. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

    DEFF Research Database (Denmark)

    Vorwerk, P; Christoffersen, C T; Müller, J

    1999-01-01

    to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase......The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found...... domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing...

  4. Low resistance splices for HTS devices and applications

    Science.gov (United States)

    Lalitha, S. L.

    2017-09-01

    This paper discusses the preparation methodology and performance evaluation of low resistance splices made of the second generation (2G) high-temperature superconductor (HTS). These splices are required in a broad spectrum of HTS devices including a large aperture, high-field solenoid built in the laboratory to demonstrate a superconducting magnetic energy storage (SMES) device. Several pancake coils are assembled in the form of a nested solenoid, and each coil requires a hundred meters or more of 2G (RE)BCO tape. However, commercial availability of this superconductor with a very uniform physical properties is currently limited to shorter piece lengths. This necessitates us having splices to inter-connect the tape pieces within a pancake coil, between adjacent pancake coils, and to attach HTS current leads to the magnet assembly. As a part of the optimization and qualification of splicing process, a systematic study was undertaken to analyze the electrical performance of splices in two different configurations suitable for this magnet assembly: lap joint and spiral joint. The electrical performance is quantified in terms of the resistance of splices estimated from the current-voltage characteristics. It has been demonstrated that a careful application of this splicing technique can generate lap joints with resistance less than 1 nΩ at 77 K.

  5. 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.

  6. 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.

  7. Diversification of the muscle proteome through alternative splicing.

    Science.gov (United States)

    Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

    2018-03-06

    Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

  8. 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.

  9. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Hou, Lichao [Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Chai, Yubo; Song, Qinghe; Chen, Sumin [Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Luo, Wenjing, E-mail: luowenj@fmmu.edu.cn [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Chen, Jingyuan, E-mail: jy_chen@fmmu.edu.cn [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China)

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  10. 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

    RNAs, 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...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly....

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

    Science.gov (United States)

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

    2012-09-15

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

  12. Conditional Toxin Splicing Using a Split Intein System.

    Science.gov (United States)

    Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

    2017-01-01

    Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

  13. Research on Splicing Method of Digital Relic Fragment Model

    Science.gov (United States)

    Yan, X.; Hu, Y.; Hou, M.

    2018-04-01

    In the course of archaeological excavation, a large number of pieces of cultural relics were unearthed, and the restoration of these fragments was done manually by traditional arts and crafts experts. In this process, cultural relics experts often try to splice the existing cultural relics, and then use adhesive to stick together the fragments of correct location, which will cause irreversible secondary damage to cultural relics. In order to minimize such damage, the surveyors combine 3D laser scanning with computer technology, and use the method of establishing digital cultural relics fragments model to make virtual splicing of cultural relics. The 3D software on the common market can basically achieve the model translation and rotation, using this two functions can be achieved manually splicing between models, mosaic records after the completion of the specific location of each piece of fragments, so as to effectively reduce the damage to the relics had tried splicing process.

  14. Seismic retrofit of spliced sleeve connections for precast bridge piers.

    Science.gov (United States)

    2017-03-01

    Grouted Splice Sleeve (GSS) connectors are being considered for connecting bridge columns, footings, and pier caps in Accelerated Bridge Construction (ABC). A repair technique for precast reinforced concrete bridge column-to-footing and column-to-pie...

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

    KAUST Repository

    Alshareef, Sahar; Ling, Yu; Butt, Haroon; Mariappan, Kiruthiga G.; Benhamed, Moussa; Mahfouz, Magdy M.

    2017-01-01

    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

  16. NUCKS overexpression in breast cancer

    Directory of Open Access Journals (Sweden)

    Kittas Christos

    2009-08-01

    Full Text Available Abstract Background NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR, real-time PCR (qRT-PCR and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. Results The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS. It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. Conclusion The results show overexpression of the NUCKS protein in a number of non

  17. The peculiarities of large intron splicing in animals.

    Directory of Open Access Journals (Sweden)

    Samuel Shepard

    Full Text Available In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These "large introns" must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5' and 3' acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing-a consecutive splicing from the 5'-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.

  18. Splicing regulatory factors, ageing and age-related disease.

    Science.gov (United States)

    Latorre, Eva; Harries, Lorna W

    2017-07-01

    Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Reflections on protein splicing: structures, functions and mechanisms

    Science.gov (United States)

    Anraku, Yasuhiro; Satow, Yoshinori

    2009-01-01

    Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. PMID:19907126

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Jayasinghe, Reyka G; Cao, Song; Gao, Qingsong; Wendl, Michael C; Vo, Nam Sy; Reynolds, Sheila M; Zhao, Yanyan; Climente-González, Héctor; Chai, Shengjie; Wang, Fang; Varghese, Rajees; Huang, Mo; Liang, Wen-Wei; Wyczalkowski, Matthew A; Sengupta, Sohini; Li, Zhi; Payne, Samuel H; Fenyö, David; Miner, Jeffrey H; Walter, Matthew J; Vincent, Benjamin; Eyras, Eduardo; Chen, Ken; Shmulevich, Ilya; Chen, Feng; Ding, Li

    2018-04-03

    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. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  2. The Functional Impact of Alternative Splicing in Cancer.

    Science.gov (United States)

    Climente-González, Héctor; Porta-Pardo, Eduard; Godzik, Adam; Eyras, Eduardo

    2017-08-29

    Alternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remain mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein-protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer, and some of them could potentially be considered alternative splicing drivers (AS drivers). Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. 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.

  4. CircSMARCA5 Inhibits Migration of Glioblastoma Multiforme Cells by Regulating a Molecular Axis Involving Splicing Factors SRSF1/SRSF3/PTB

    Directory of Open Access Journals (Sweden)

    Davide Barbagallo

    2018-02-01

    Full Text Available Circular RNAs (circRNAs have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5: it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student’s t-test, contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student’s t-test. Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma’s histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs, specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1, a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE. Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3 RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD substrate. Interestingly, SRSF3 is known to interplay

  5. The Spliced Leader Trans-Splicing Mechanism in Different Organisms: Molecular Details and Possible Biological Roles

    Directory of Open Access Journals (Sweden)

    Mainá eBitar

    2013-10-01

    Full Text Available The spliced leader (SL is a gene that generates a functional ncRNA that is composed of two regions: an intronic region of unknown function (SLi and an exonic region (SLe, which is transferred to the 5’ end of independent transcripts yielding mature mRNAs, in a process known as spliced leader trans-splicing (SLTS. The best described function for SLTS is to solve polycistronic transcripts into monocistronic units, specifically in Trypanosomatids. In other metazoans, it is speculated that the SLe addition could lead to increased mRNA stability, differential recruitment of the translational machinery, modification of the 5' region or a combination of these effects. Although important aspects of this mechanism have been revealed, several features remain to be elucidated. We have analyzed 157 SLe sequences from 148 species from 7 phyla and found a high degree of conservation among the sequences of species from the same phylum, although no considerable similarity seems to exist between sequences of species from different phyla. When analyzing case studies, we found evidence that a given SLe will always be related to a given set of transcripts in different species from the same phylum, and therefore, different SLe sequences from the same species would regulate different sets of transcripts. In addition, we have observed distinct transcript categories to be preferential targets for the SLe addition in different phyla. This work sheds light into crucial and controversial aspects of the SLTS mechanism. It represents a comprehensive study concerning various species and different characteristics of this important post-transcriptional regulatory mechanism.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2008-06-01

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

  8. 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.

  9. Statistical analysis of LHC main interconnection splices room temperature resistance (R-8) results

    CERN Document Server

    Heck, S

    2012-01-01

    During the 2008/2009 shutdown the so-called R-8/R-16 room temperature resistance test has been introduced for the quality control of the LHC main interconnection splices. It has been found that at present two groups of LHC main interconnection splices can be distinguished, so-called “old” splices produced during LHC installation, and so-called “new” splices produced during 2009. 2009 production splices are considered as the state-of-the art, which is reflected by a much smaller R-8 distribution as compared to that of splices produced during first LHC installation.

  10. Human Splice-Site Prediction with Deep Neural Networks.

    Science.gov (United States)

    Naito, Tatsuhiko

    2018-04-18

    Accurate splice-site prediction is essential to delineate gene structures from sequence data. Several computational techniques have been applied to create a system to predict canonical splice sites. For classification tasks, deep neural networks (DNNs) have achieved record-breaking results and often outperformed other supervised learning techniques. In this study, a new method of splice-site prediction using DNNs was proposed. The proposed system receives an input sequence data and returns an answer as to whether it is splice site. The length of input is 140 nucleotides, with the consensus sequence (i.e., "GT" and "AG" for the donor and acceptor sites, respectively) in the middle. Each input sequence model is applied to the pretrained DNN model that determines the probability that an input is a splice site. The model consists of convolutional layers and bidirectional long short-term memory network layers. The pretraining and validation were conducted using the data set tested in previously reported methods. The performance evaluation results showed that the proposed method can outperform the previous methods. In addition, the pattern learned by the DNNs was visualized as position frequency matrices (PFMs). Some of PFMs were very similar to the consensus sequence. The trained DNN model and the brief source code for the prediction system are uploaded. Further improvement will be achieved following the further development of DNNs.

  11. RNA splicing in a new rhabdovirus from Culex mosquitoes.

    Science.gov (United States)

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-07-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

  12. A Challenging Pie to Splice: Drugging the Spliceosome.

    Science.gov (United States)

    León, Brian; Kashyap, Manoj K; Chan, Warren C; Krug, Kelsey A; Castro, Januario E; La Clair, James J; Burkart, Michael D

    2017-09-25

    Since its discovery in 1977, the study of alternative RNA splicing has revealed a plethora of mechanisms that had never before been documented in nature. Understanding these transitions and their outcome at the level of the cell and organism has become one of the great frontiers of modern chemical biology. Until 2007, this field remained in the hands of RNA biologists. However, the recent identification of natural product and synthetic modulators of RNA splicing has opened new access to this field, allowing for the first time a chemical-based interrogation of RNA splicing processes. Simultaneously, we have begun to understand the vital importance of splicing in disease, which offers a new platform for molecular discovery and therapy. As with many natural systems, gaining clear mechanistic detail at the molecular level is key towards understanding the operation of any biological machine. This minireview presents recent lessons learned in this emerging field of RNA splicing chemistry and chemical biology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 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.

  14. The determinants of alternative RNA splicing in human cells.

    Science.gov (United States)

    Ramanouskaya, Tatsiana V; Grinev, Vasily V

    2017-12-01

    Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

  15. L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

    Science.gov (United States)

    Arévalo, Miguel; Núñez-Gómez, Elena; Pérez-Roque, Lucía; Pericacho, Miguel; González-Núñez, María; Langa, Carmen; Martínez-Salgado, Carlos; Perez-Barriocanal, Fernando; Bernabeu, Carmelo; Lopez-Novoa, José M.

    2014-01-01

    Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development. PMID:25313562

  16. Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

    Science.gov (United States)

    Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

    2017-08-01

    Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Alternative splicing: the pledge, the turn, and the prestige : The key role of alternative splicing in human biological systems.

    Science.gov (United States)

    Gallego-Paez, L M; Bordone, M C; Leote, A C; Saraiva-Agostinho, N; Ascensão-Ferreira, M; Barbosa-Morais, N L

    2017-09-01

    Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.

  18. 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 Caenorhabditis nematodes-more than 92% of cassette exons from Caenorhabditis elegans are conserved in Caenorhabditis briggsae and/or Caenorhabditis remanei. High levels of conservation extend to minor-form exons (present in a minority of transcripts) and are particularly pronounced for exons showing complex...... patterns of splicing. The functionality of the vast majority of cassette exons is underscored by various other features. We suggest that differences in conservation between lineages reflect differences in levels of functionality and further suggest that these differences are due to differences in intron...

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

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

  1. 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%.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-08-29

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

  4. 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...

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

    Science.gov (United States)

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

    2016-10-15

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

  6. DEDB: a database of Drosophila melanogaster exons in splicing graph form

    Directory of Open Access Journals (Sweden)

    Tan Tin

    2004-12-01

    Full Text Available Abstract Background A wealth of quality genomic and mRNA/EST sequences in recent years has provided the data required for large-scale genome-wide analysis of alternative splicing. We have capitalized on this by constructing a database that contains alternative splicing information organized as splicing graphs, where all transcripts arising from a single gene are collected, organized and classified. The splicing graph then serves as the basis for the classification of the various types of alternative splicing events. Description DEDB http://proline.bic.nus.edu.sg/dedb/index.html is a database of Drosophila melanogaster exons obtained from FlyBase arranged in a splicing graph form that permits the creation of simple rules allowing for the classification of alternative splicing events. Pfam domains were also mapped onto the protein sequences allowing users to access the impact of alternative splicing events on domain organization. Conclusions DEDB's catalogue of splicing graphs facilitates genome-wide classification of alternative splicing events for genome analysis. The splicing graph viewer brings together genome, transcript, protein and domain information to facilitate biologists in understanding the implications of alternative splicing.

  7. 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.

  8. 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...

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

    Science.gov (United States)

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

    2017-06-28

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

  10. [Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

    Science.gov (United States)

    Niimi, Hideki; Ogawa, Tomomi; Note, Rhougou; Hayashi, Shirou; Ueno, Tomohiro; Harada, Kenu; Uji, Yoshinori; Kitajima, Isao

    2010-12-01

    In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

  11. Systematic profiling of alternative splicing signature reveals prognostic predictor for ovarian cancer.

    Science.gov (United States)

    Zhu, Junyong; Chen, Zuhua; Yong, Lei

    2018-02-01

    The majority of genes are alternatively spliced and growing evidence suggests that alternative splicing is modified in cancer and is associated with cancer progression. Systematic analysis of alternative splicing signature in ovarian cancer is lacking and greatly needed. We profiled genome-wide alternative splicing events in 408 ovarian serous cystadenocarcinoma (OV) patients in TCGA. Seven types of alternative splicing events were curated and prognostic analyses were performed with predictive models and splicing network built for OV patients. Among 48,049 mRNA splicing events in 10,582 genes, we detected 2,611 alternative splicing events in 2,036 genes which were significant associated with overall survival of OV patients. Exon skip events were the most powerful prognostic factors among the seven types. The area under the curve of the receiver-operator characteristic curve for prognostic predictor, which was built with top significant alternative splicing events, was 0.937 at 2,000 days of overall survival, indicating powerful efficiency in distinguishing patient outcome. Interestingly, splicing correlation network suggested obvious trends in the role of splicing factors in OV. In summary, we built powerful prognostic predictors for OV patients and uncovered interesting splicing networks which could be underlying mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. A novel CARD containing splice-isoform of CIITA regulates nitric oxide synthesis in dendritic cells.

    Science.gov (United States)

    Huang, Dachuan; Lim, Sylvia; Chua, Rong Yuan Ray; Shi, Hong; Ng, Mah Lee; Wong, Siew Heng

    2010-03-01

    MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.

  13. KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

    Science.gov (United States)

    Fujita, Yuji; Masuda, Kiyoshi; Hamada, Junichi; Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

    2017-11-24

    KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

  14. Large exon size does not limit splicing in vivo.

    Science.gov (United States)

    Chen, I T; Chasin, L A

    1994-03-01

    Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

  15. Insights into alternative splicing of sarcomeric genes in the heart

    NARCIS (Netherlands)

    Weeland, Cornelis J.; van den Hoogenhof, Maarten M.; Beqqali, Abdelaziz; Creemers, Esther E.

    2015-01-01

    Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical

  16. Minor class splicing shapes the zebrafish transcriptome during development

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  17. The Database Management Module of the Splice System.

    Science.gov (United States)

    1983-06-01

    standardization is the only wise chocs . E. FUNCTIONS OF THE EATABASE MkNAGEMENT MODULE As a result of onqoing research in thmc impl1msntaticn of SPLICE, thns...u an e-v Offset by one or mc--l orders of ma#-inuIs inorcvesnnt --L tue execution time cf user transacdrioas. Purthermore, ’is s-toraqe requlrement

  18. A novel CDX2 isoform regulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Matthew E Witek

    Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

  19. Stiff, Strong Splice For A Composite Sandwich Structure

    Science.gov (United States)

    Schmaling, D.

    1991-01-01

    New type of splice for composite sandwich structure reduces peak shear stress in structure. Layers of alternating fiber orientation interposed between thin ears in adhesive joint. Developed for structural joint in spar of helicopter rotor blade, increases precision of control over thickness of adhesive at joint. Joint easy to make, requires no additional pieces, and adds little weight.

  20. 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.

  1. 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 ...

  2. Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

    1995-01-16

    We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

  3. Chinmo prevents transformer alternative splicing to maintain male sex identity.

    Directory of Open Access Journals (Sweden)

    Lydia Grmai

    2018-02-01

    Full Text Available Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx. Female-specific expression of Sex-lethal (Sxl causes alternative splicing of transformer (tra to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir and Female lethal (2d (Fl(2d. traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2d. Consistent with this, we show that both Vir and Fl(2d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

  4. 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.

  5. Chinmo prevents transformer alternative splicing to maintain male sex identity.

    Science.gov (United States)

    Grmai, Lydia; Hudry, Bruno; Miguel-Aliaga, Irene; Bach, Erika A

    2018-02-01

    Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

  6. NMR studies of two spliced leader RNAs using isotope labeling

    Energy Technology Data Exchange (ETDEWEB)

    Lapham, J.; Crothers, D.M. [Yale Univ., New Haven, CT (United States)

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

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

    International Nuclear Information System (INIS)

    Sun Chang; Southard, Catherine; Di Rienzo, Anna

    2009-01-01

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

  8. Flexural behavior of concrete beam with mechanical splices of reinforcement subjected to cyclic loading

    International Nuclear Information System (INIS)

    Nab, H. S.; Kim, W. B.

    2008-01-01

    In nuclear power plant structures, the mechanical rebar splices are designated and constructed on the basis of ACI and ASME code. Regardless of good performance on mechanical rebar splices, these splicing methods that did not be registered on ASME code have not restricted to apply to construction site. In this study, the main candidate splice is cold roll formed parallel threaded splice. This was registered newly in ASME Section III division 2 CC 4333 'Mechanical Splices' in 2004. To compare the traditional rebar splice with mechanical rebar splices, concrete beams were made to evaluate the ductility of spliced reinforcing bars. Based on Experimental results, it was identified that the mechanical rebar splices by parallel threaded coupler had better accumulated dissipation energy capacity to resist seismic behavior than the traditional lapping splices. It showed that concrete specimens with D36 reinforcing bar coupler are 1.8 times better performance and that concrete specimens with D22 reinforcing bar coupler are 2.8 times better performance. (authors)

  9. 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...

  10. Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

    Science.gov (United States)

    Rexiati, Maimaiti; Sun, Mingming; Guo, Wei

    2018-01-05

    Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

  11. PathwaySplice: An R package for unbiased pathway analysis of alternative splicing in RNA-Seq data.

    Science.gov (United States)

    Yan, Aimin; Ban, Yuguang; Gao, Zhen; Chen, Xi; Wang, Lily

    2018-04-24

    Pathway analysis of alternative splicing would be biased without accounting for the different number of exons or junctions associated with each gene, because genes with higher number of exons or junctions are more likely to be included in the "significant" gene list in alternative splicing. We present PathwaySplice, an R package that (1) Performs pathway analysis that explicitly adjusts for the number of exons or junctions associated with each gene; (2) Visualizes selection bias due to different number of exons or junctions for each gene and formally tests for presence of bias using logistic regression; (3) Supports gene sets based on the Gene Ontology terms, as well as more broadly defined gene sets (e.g. MSigDB) or user defined gene sets; (4) Identifies the significant genes driving pathway significance and (5) Organizes significant pathways with an enrichment map, where pathways with large number of overlapping genes are grouped together in a network graph. https://bioconductor.org/packages/release/bioc/html/PathwaySplice.html. lily.wangg@gmail.com, xi.steven.chen@gmail.com.

  12. Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

    Science.gov (United States)

    Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

    2017-01-01

    Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

  13. Significance of Aurora B overexpression in hepatocellular carcinoma. Aurora B Overexpression in HCC

    International Nuclear Information System (INIS)

    Lin, Zhong-Zhe; Jeng, Yung-Ming; Hu, Fu-Chang; Pan, Hung-Wei; Tsao, Hsin-Wei; Lai, Po-Lin; Lee, Po-Huang; Cheng, Ann-Lii; Hsu, Hey-Chi

    2010-01-01

    To investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC). The Aurora B and Aurora A mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the p53 and β-catenin genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of p53 and exon 3 of β-catenin. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines. Aurora B was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of Aurora B was associated with Aurora A overexpression (P = 0.0003) and p53 mutation (P = 0.002) and was inversely associated with β-catenin mutation (P = 0.002). Aurora B overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that Aurora B overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of p53 and β-catenin. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis. Aurora B overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment

  14. U2AF1 mutations alter splice site recognition in hematological malignancies.

    Science.gov (United States)

    Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E; Zebari, Ahmad S; Bradley, Philip; Bradley, Robert K

    2015-01-01

    Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains. © 2015 Ilagan et al.; Published by Cold Spring Harbor Laboratory Press.

  15. Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

    Science.gov (United States)

    Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

    2018-04-30

    Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. 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.

  17. Transgenic overexpression of 14-3-3 zeta protects hippocampus against endoplasmic reticulum stress and status epilepticus in vivo.

    Directory of Open Access Journals (Sweden)

    Gary P Brennan

    Full Text Available 14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ isoform has been linked to endoplasmic reticulum (ER function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress.

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

    KAUST Repository

    Lee, Keh Chien; Jang, Yun Hee; Kim, SoonKap; Park, Hyo-Young; Thu, May Phyo; Lee, Jeong Hwan; Kim, Jeong-Kook

    2017-01-01

    , 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

  19. 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.

  20. 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.

  1. Optical fabrication of large area photonic microstructures by spliced lens

    Science.gov (United States)

    Jin, Wentao; Song, Meng; Zhang, Xuehua; Yin, Li; Li, Hong; Li, Lin

    2018-05-01

    We experimentally demonstrate a convenient approach to fabricate large area photorefractive photonic microstructures by a spliced lens device. Large area two-dimensional photonic microstructures are optically induced inside an iron-doped lithium niobate crystal. The experimental setups of our method are relatively compact and stable without complex alignment devices. It can be operated in almost any optical laboratories. We analyze the induced triangular lattice microstructures by plane wave guiding, far-field diffraction pattern imaging and Brillouin-zone spectroscopy. By designing the spliced lens appropriately, the method can be easily extended to fabricate other complex large area photonic microstructures, such as quasicrystal microstructures. Induced photonic microstructures can be fixed or erased and re-recorded in the photorefractive crystal.

  2. Hand1 overexpression inhibits medulloblastoma metastasis

    Energy Technology Data Exchange (ETDEWEB)

    Asuthkar, Swapna; Guda, Maheedhara R. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Martin, Sarah E. [Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Antony, Reuben; Fernandez, Karen [Department of Pediatrics, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Lin, Julian [Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Tsung, Andrew J. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Illinois Neurological Institute, Peoria, IL 61656 (United States); Velpula, Kiran K., E-mail: velpula@uic.edu [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States)

    2016-08-19

    Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

  3. Hand1 overexpression inhibits medulloblastoma metastasis

    International Nuclear Information System (INIS)

    Asuthkar, Swapna; Guda, Maheedhara R.; Martin, Sarah E.; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J.; Velpula, Kiran K.

    2016-01-01

    Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

  4. Intergenic mRNA molecules resulting from trans-splicing.

    Science.gov (United States)

    Finta, Csaba; Zaphiropoulos, Peter G

    2002-02-22

    Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 or CYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 and CYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules, i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be approximately 0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2-13 of CYP3A4 revealed catalytic activity toward testosterone.

  5. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    Directory of Open Access Journals (Sweden)

    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

  6. 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.

  7. 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.

  8. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

    Directory of Open Access Journals (Sweden)

    Xuexia Zhou

    2014-12-01

    Full Text Available Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  9. 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.

  10. 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

  11. 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....

  12. Quantifying alternative splicing from paired-end RNA-sequencing data

    OpenAIRE

    Rossell, David; Stephan-Otto Attolini, Camille; Kroiss, Manuel; Stöcker, Almond

    2014-01-01

    RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely...

  13. 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.

  14. 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

  15. Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells

    International Nuclear Information System (INIS)

    Wei, Xing; Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin; Xiao, Xianzhong

    2010-01-01

    WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.

  16. Analysis of Few-Mode Multi-Core Fiber Splice Behavior Using an Optical Vector Network Analyzer

    DEFF Research Database (Denmark)

    Rommel, Simon; Mendinueta, Jose Manuel Delgado; Klaus, Werner

    2017-01-01

    The behavior of splices in a 3-mode 36-core fiber is analyzed using optical vector network analysis. Time-domain response analysis confirms splices may cause significant mode-mixing, while frequency-domain analysis shows splices may affect system level mode-dependent loss both positively and negativ......The behavior of splices in a 3-mode 36-core fiber is analyzed using optical vector network analysis. Time-domain response analysis confirms splices may cause significant mode-mixing, while frequency-domain analysis shows splices may affect system level mode-dependent loss both positively...

  17. Frequent Nek1 overexpression in human gliomas

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Jun [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Cai, Yu, E-mail: aihaozuqiu22@163.com [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Pin [Med-X Research Institute, Shanghai Jiao Tong University, Shanghai (China); Zhao, Weiguo [Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

    2016-08-05

    Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

  18. Frequent Nek1 overexpression in human gliomas

    International Nuclear Information System (INIS)

    Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

    2016-01-01

    Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

  19. HBV X Protein induces overexpression of HERV-W env through NF-κB in HepG2 cells.

    Science.gov (United States)

    Liu, Cong; Liu, Lijuan; Wang, Xiuling; Liu, Youyi; Wang, Miao; Zhu, Fan

    2017-12-01

    Human endogenous retrovirus W family (HERV-W) envelope (env) at chromosome 7 is highly expressed in the placenta and possesses fusogenic activity in trophoblast development. HERV-W env has been found to be overexpressed in some cancers and immune diseases. Viral transactivators can induce the overexpression of HERV-W env in human cell lines. Hepatitis B virus X protein (HBx) is believed to be a multifunctional oncogenic protein. Here, we reported that HBx could increase the promoter activity of HERV-W env and upregulate the mRNA levels of non-spliced and spliced HERV-W env and also its protein in human hepatoma HepG2 cells. Interestingly, we found that the inhibition of nuclear factor κB (NF-κB) using shRNA targeting NF-κB/p65 or PDTC (an inhibitor of NF-κB) could attenuate the upregulation of HERV-W env induced by HBx. These suggested that HBx might upregulate the expression of HERV-W env through NF-κB in HepG2 cells. This study might provide a new insight in HBV-associated liver diseases including HCC.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

    Science.gov (United States)

    Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki; Fukuoka, Junji; Gan, Wenjian; Hikasa, Takuya; Kaga, Yoshiaki; Takemoto, Masayuki; Magari, Masaki; Li, Xialu; Manley, James L; Ohmori, Hitoshi; Kanayama, Naoki

    2012-01-24

    Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

  5. Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

    Directory of Open Access Journals (Sweden)

    Kazumi Nakabayashi

    2015-12-01

    Full Text Available The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1 is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1...... and flPD-1 upon activation suggests an important interplay between the putative soluble PD-1 and flPD-1 possibly involved in maintenance of peripheral self-tolerance and prevention of autoimmunity....

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  8. 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.

  9. 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.

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

    NARCIS (Netherlands)

    Ishunina, Tatjana A.; Swaab, Dick F.

    2012-01-01

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

  11. 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...

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Nixon Tamara J

    2008-05-01

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

  14. Functional characterisation of an intron retaining K+ transporter of barley reveals intron-mediated alternate splicing

    KAUST Repository

    Shahzad, K.; Rauf, M.; Ahmed, M.; Malik, Z. A.; Habib, I.; Ahmed, Z.; Mahmood, K.; Ali, R.; Masmoudi, K.; Lemtiri-Chlieh, Fouad; Gehring, Christoph A; Berkowitz, G. A.; Saeed, N. A.

    2015-01-01

    Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K+ transporter, HvHKT2;1, with stably

  15. 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.

  16. 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...

  17. 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...

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

    Science.gov (United States)

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

    2017-07-07

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

  19. Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

    Directory of Open Access Journals (Sweden)

    Reut Shalgi

    2014-06-01

    Full Text Available During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ∼1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Reich Jens G

    2006-06-01

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

  2. Nd:YAG-laser-based time-domain reflectometry measurements of the intrinsic reflection signature from PMMA fiber splices

    Science.gov (United States)

    Lawson, Christopher M.; Michael, Robert R., Jr.; Dressel, Earl M.; Harmony, David W.

    1991-12-01

    Optical time domain reflectometry (OTDR) measurements have been performed on polished polymethylmethacrylate (PMMA) plastic fiber splices. After the dominant splice reflection sources due to surface roughness, inexact index matching, and fiber core misalignment were eliminated, an intrinsic OTDR signature 3 - 8 dB above the Rayleigh backscatter floor remained with all tested fibers. This minimum splice reflectivity exhibits characteristics that are consistent with sub-surface polymer damage and can be used for detection of PMMA fiber splices.

  3. Overexpressed TP73 induces apoptosis in medulloblastoma

    International Nuclear Information System (INIS)

    Castellino, Robert C; De Bortoli, Massimiliano; Lin, Linda L; Skapura, Darlene G; Rajan, Jessen A; Adesina, Adekunle M; Perlaky, Laszlo; Irwin, Meredith S; Kim, John YH

    2007-01-01

    Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to

  4. Overexpressed TP73 induces apoptosis in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Perlaky Laszlo

    2007-07-01

    Full Text Available Abstract Background Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. Methods We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Results Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and

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

    Science.gov (United States)

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

    2015-10-01

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

  6. 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.

  7. 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.

  8. 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.

  9. SOAPsplice: genome-wide ab initio detection of splice junctions from RNA-Seq data

    Directory of Open Access Journals (Sweden)

    Songbo eHuang

    2011-07-01

    Full Text Available RNA-Seq, a method using next generation sequencing technologies to sequence the transcriptome, facilitates genome-wide analysis of splice junction sites. In this paper, we introduce SOAPsplice, a robust tool to detect splice junctions using RNA-Seq data without using any information of known splice junctions. SOAPsplice uses a novel two-step approach consisting of first identifying as many reasonable splice junction candidates as possible, and then, filtering the false positives with two effective filtering strategies. In both simulated and real datasets, SOAPsplice is able to detect many reliable splice junctions with low false positive rate. The improvement gained by SOAPsplice, when compared to other existing tools, becomes more obvious when the depth of sequencing is low. SOAPsplice is freely available at http://soap.genomics.org.cn/soapsplice.html.

  10. On splice site prediction using weight array models: a comparison of smoothing techniques

    International Nuclear Information System (INIS)

    Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

    2007-01-01

    In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called 'splicing'. The positions where introns are cut and exons are spliced together are called 'splice sites'. Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed

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

    Science.gov (United States)

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

    2009-01-01

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

  12. 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.

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

    KAUST Repository

    Leoni, Guido; Le Pera, Loredana; Ferrè , Fabrizio; Raimondo, Domenico; Tramontano, Anna

    2011-01-01

    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.

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

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    Lorena Suarez-Artiles

    2018-01-01

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

  15. Interleukin-6 overexpression induces pulmonary hypertension.

    Science.gov (United States)

    Steiner, M Kathryn; Syrkina, Olga L; Kolliputi, Narasaish; Mark, Eugene J; Hales, Charles A; Waxman, Aaron B

    2009-01-30

    Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6-overexpressing transgenic mice (Tg(+)) and compared to wild-type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-beta and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

  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 and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

    Science.gov (United States)

    2014-01-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  19. 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

  20. IE Information No. 86-104: Unqualified butt splice connectors identified in qualified penetrations

    International Nuclear Information System (INIS)

    Jordan, E.L.

    1992-01-01

    During an NRC equipment qualification (EQ) inspection at Dresden Nuclear Power Station, May 19--23, 1986, a deficiency was discovered concerning a lack of similarity between tested and installed nylon insulated butt splices in EQ qualified GE electrical penetrations. commonwealth Edison sent four sample splices removed from Quad Cities Nuclear Power Station to Wyle Laboratory to further substantiate their qualification for use in a harsh environment. These splices were identical to those installed at Dresden. During the testing performed at Wyle Laboratory December 4--5, 1986, all four samples exhibited excessive leakage currents to ground when exposed to a steam environment. Commonwealth Edison consequently declared the splices unqualified and shut down its Quad Cities Unit 1 to rework the splices by wrapping them with previously qualified tape. Dresden Unit 2 has similarly reworked the splices by wrapping them with tape. Duane Arnold Energy Center also has commenced a shutdown in order to make repairs. The short circuits that occurred appeared to start by condensation entering the splice between the wire insulation and the nylon tubing. The arcing caused insulation degradation that then allowed arcs to pass through the insulation to the enclosure

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

    KAUST Repository

    Alshareef, Sahar; Ling, Yu; Butt, Haroon; Mariappan, Kiruthiga; Benhamed, Moussa; Mahfouz, Magdy

    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.

  2. 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.

  3. 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.

  4. A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer.

    Science.gov (United States)

    El-Athman, Rukeia; Fuhr, Luise; Relógio, Angela

    2018-06-20

    Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Genome wide identification of aberrant alternative splicing events in myotonic dystrophy type 2.

    Science.gov (United States)

    Perfetti, Alessandra; Greco, Simona; Fasanaro, Pasquale; Bugiardini, Enrico; Cardani, Rosanna; Garcia-Manteiga, Jose M; Manteiga, Jose M Garcia; Riba, Michela; Cittaro, Davide; Stupka, Elia; Meola, Giovanni; Martelli, Fabio

    2014-01-01

    Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.

  7. Judging the similarity of soundscapes does not require categorization: evidence from spliced stimuli.

    Science.gov (United States)

    Aucouturier, Jean-Julien; Defreville, Boris

    2009-04-01

    This study uses an audio signal transformation, splicing, to create an experimental situation where human listeners judge the similarity of audio signals, which they cannot easily categorize. Splicing works by segmenting audio signals into 50-ms frames, then shuffling and concatenating these frames back in random order. Splicing a signal masks the identification of the categories that it normally elicits: For instance, human participants cannot easily identify the sound of cars in a spliced recording of a city street. This study compares human performance on both normal and spliced recordings of soundscapes and music. Splicing is found to degrade human similarity performance significantly less for soundscapes than for music: When two spliced soundscapes are judged similar to one another, the original recordings also tend to sound similar. This establishes that humans are capable of reconstructing consistent similarity relations between soundscapes without relying much on the identification of the natural categories associated with such signals, such as their constituent sound sources. This finding contradicts previous literature and points to new ways to conceptualize the different ways in which humans perceive soundscapes and music.

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

    Science.gov (United States)

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

    2010-01-01

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

  9. The role of polypyrimidine tract-binding proteins and other hnRNP proteins in plant splicing regulation

    Directory of Open Access Journals (Sweden)

    Andreas eWachter

    2012-05-01

    Full Text Available Alternative precursor mRNA splicing is a widespread phenomenon in multicellular eukaryotes and represents a major means for functional expansion of the transcriptome. While several recent studies have revealed an important link between splicing regulation and fundamental biological processes in plants, many important aspects, such as the underlying splicing regulatory mechanisms, are so far not well understood. Splicing decisions are in general based on a splicing code that is determined by the dynamic interplay of splicing-controlling factors and cis-regulatory elements. Several members of the group of heterogeneous nuclear ribonucleoprotein (hnRNP proteins are well-known regulators of splicing in animals and the comparatively few reports on some of their plant homologues revealed similar functions. This also applies to polypyrimidine tract-binding proteins (PTBs, a thoroughly investigated class of hnRNP proteins with splicing regulatory functions in both animals and plants. Further examples from plants are auto- and cross-regulatory splicing circuits of glycine-rich RNA-binding proteins (GRPs and splicing enhancement by oligouridylatebinding proteins. Besides their role in defining splice site choice, hnRNP proteins are also involved in multiple other steps of nucleic acid metabolism, highlighting the functional versatility of this group of proteins in higher eukaryotes.

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

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

    2018-02-28

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

  13. 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)

  14. 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].

  15. Splice-Switching Therapy for Spinal Muscular Atrophy

    Directory of Open Access Journals (Sweden)

    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.

  16. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

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

  17. Characterization of fibroblast growth factor receptor 2 overexpression in the human breast cancer cell line SUM-52PE

    International Nuclear Information System (INIS)

    Tannheimer, Stacey L; Rehemtulla, Alnawaz; Ethier, Stephen P

    2000-01-01

    The fibroblast growth factor receptor (FGFR)2 gene has been shown to be amplified in 5-10% of breast cancer patients. A breast cancer cell line developed in our laboratory, SUM-52PE, was shown to have a 12-fold amplification of the FGFR2 gene, and FGFR2 message was found to be overexpressed 40-fold in SUM-52PE cells as compared with normal human mammary epithelial (HME) cells. Both human breast cancer (HBC) cell lines and HME cells expressed two FGFR2 isoforms, whereas SUM-52PE cells overexpressed those two isoforms, as well as several unique FGFR2 polypeptides. SUM-52PE cells expressed exclusively FGFR2-IIIb isoforms, which are high-affinity receptors for fibroblast growth factor (FGF)-1 and FGF-7. Differences were identified in the expression of the extracellular Ig-like domains, acid box and carboxyl termini, and several variants not previously reported were isolated from these cells. The FGFR family of receptor tyrosine kinases includes four members, all of which are highly alternatively spliced and glycosylated. For FGFR2, alternative splicing of the second half of the third Ig-like domain, involving exons IIIb and IIIc, is a mutually exclusive choice that affects ligand binding specificity and affinity [1,2,3]. It appears that the second half of the third Ig-like domain can dictate high affinity for FGF-2 or keratinocyte growth factor (KGF), whereas affinity for FGF-1 appears to remain the same [3]. Alternative splicing of the carboxyl terminus has been shown to involve at least two different exons that can produce at least three different variants. The C1-type and C2-type carboxyl termini are encoded by the same exon, and have two different splice acceptor sites, whereas the C3-type carboxyl terminus is encoded by a separate exon [4]. The biologic significance of the C1 carboxyl terminus, as compared with the shorter C3 variant found primarily in tumorigenic samples, has been studied in NIH3T3 transfection assays, in which C3 variants were able to produce

  18. 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.

  19. Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

    Science.gov (United States)

    Fu, Xiang-Dong

    2017-04-01

    EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

  20. 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...

  1. Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

    International Nuclear Information System (INIS)

    Asai, Kengo; Platt, Craig; Cochrane, Alan

    2003-01-01

    The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export

  2. Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

    NARCIS (Netherlands)

    Yu, Hongyou; de Vos, Paul; Ren, Yijin

    OBJECTIVE: The hypothesis of the present study is that overexpression of osteoprotegerin (OPG) promotes preosteoblast maturation. MATERIALS AND METHODS: The preosteoblast cell line MC3T3-E1 was transfected with OPG overexpression. OPG expression was confirmed by enzyme-linked immunosorbent assay

  3. Sex determination in insects: a binary decision based on alternative splicing.

    Science.gov (United States)

    Salz, Helen K

    2011-08-01

    The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c......Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently...... needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined...

  5. 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.

  6. Analysis and prediction of gene splice sites in four Aspergillus genomes

    DEFF Research Database (Denmark)

    Wang, Kai; Ussery, David; Brunak, Søren

    2009-01-01

    Several Aspergillus fungal genomic sequences have been published, with many more in progress. Obviously, it is essential to have high-quality, consistently annotated sets of proteins from each of the genomes, in order to make meaningful comparisons. We have developed a dedicated, publicly available......, splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window...... better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene....

  7. DORT and TORT workshop -- Outline for presentation for splicing with TORSED and TORSET

    International Nuclear Information System (INIS)

    Barnett, A.

    1998-04-01

    This paper addresses the problem of solving a problem which is larger than can be accommodated by the computer system at your disposal. This can result from two constrains: (1) The available memory of the machine is too small to contain the problem. (2) Individual files may be too large to store on-line. It also addresses the problem of what to do when you want to alter only a subset of a solution space of a larger problem and don't want to rerun the entire problem. These problems can be solved by splicing with TORSED AND TORSET. If the basic shape of your problem is cylindrical and azimuthally uniform, with only a small region of three-dimensionality, then the best splicing method is the TORSED -- DORT to TORT splice. However, if there is no part of the problem which is azimuthally constant, then one might want to consider a TORT to TORT splice. Both methods are discussed here

  8. 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...

  9. The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

    Science.gov (United States)

    Zhao, Chen; Pyle, Anna Marie

    2017-12-01

    The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. 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

    patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary...... or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events...

  11. Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

    Directory of Open Access Journals (Sweden)

    Ana Rivera-Barahona

    Full Text Available The spf/ash mouse model of ornithine transcarbamylase (OTC deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations.

  12. 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...

  13. 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.

  14. 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

  15. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Horner David S

    2010-10-01

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

  18. Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat

    OpenAIRE

    1991-01-01

    Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the...

  19. 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.

  20. Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Yuanyuan Wang

    2015-06-01

    Full Text Available The regulation of expression of X-box-binding protein-1 (XBP1, a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5′-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

  1. Dimethylarginine Dimethylaminohydrolase Overexpression enhances Insulin Sensitivity

    Science.gov (United States)

    Sydow, Karsten; Mondon, Carl E.; Schrader, Joerg; Konishi, Hakuoh; Cooke, John P.

    2011-01-01

    Objective Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. Methods and Results To test this hypothesis we employed a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22–70 weeks) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistence index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2μM) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. Conclusions These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity. PMID:18239148

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

    Science.gov (United States)

    Liu, Jing; Hu, Jiaxin; Corey, David R.

    2012-01-01

    Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

  7. Fast rate of evolution in alternatively spliced coding regions of mammalian genes

    Directory of Open Access Journals (Sweden)

    Nurtdinov Ramil N

    2006-04-01

    Full Text Available Abstract Background At least half of mammalian genes are alternatively spliced. Alternative isoforms are often genome-specific and it has been suggested that alternative splicing is one of the major mechanisms for generating protein diversity in the course of evolution. Another way of looking at alternative splicing is to consider sequence evolution of constitutive and alternative regions of protein-coding genes. Indeed, it turns out that constitutive and alternative regions evolve in different ways. Results A set of 3029 orthologous pairs of human and mouse alternatively spliced genes was considered. The rate of nonsynonymous substitutions (dN, the rate of synonymous substitutions (dS, and their ratio (ω = dN/dS appear to be significantly higher in alternatively spliced coding regions compared to constitutive regions. When N-terminal, internal and C-terminal alternatives are analysed separately, C-terminal alternatives appear to make the main contribution to the observed difference. The effects become even more pronounced in a subset of fast evolving genes. Conclusion These results provide evidence of weaker purifying selection and/or stronger positive selection in alternative regions and thus one more confirmation of accelerated evolution in alternative regions. This study corroborates the theory that alternative splicing serves as a testing ground for molecular evolution.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2017-10-05

    Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. C-MET overexpression and amplification in gliomas.

    Science.gov (United States)

    Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

    2015-01-01

    We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

  15. Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

    Science.gov (United States)

    Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

    2016-03-01

    Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

  16. 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.

  17. Cloning, overexpression, and characterization of cobrotoxin

    International Nuclear Information System (INIS)

    Hsieh, H.-C.; Kumar, Thallampuranam Krishnaswamy S.; Yu Chin

    2004-01-01

    Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12 mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo autocatalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled ( 15 N and 13 C) and all the resonances ( 1 H, 13 C, and 15 N) in the protein have been unambiguously assigned. 1 H- 15 N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1 H- 15 N chemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the α7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders

  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. Orphan receptor GPR110, an oncogene overexpressed in lung and prostate cancer

    International Nuclear Information System (INIS)

    Lum, Amy M; Wang, Bruce B; Beck-Engeser, Gabriele B; Li, Lauri; Channa, Namitha; Wabl, Matthias

    2010-01-01

    GPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues. Various epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry. We found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential

  20. 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 ...

  1. Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

    Directory of Open Access Journals (Sweden)

    Yacine Kharraz

    Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

  2. 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.

  3. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    International Nuclear Information System (INIS)

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M.M.

    2005-01-01

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF 35 . CIR was found to interact with U2AF 35 through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation

  4. 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

  5. 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.

  6. 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.

  7. Theory on the Coupled Stochastic Dynamics of Transcription and Splice-Site Recognition

    Science.gov (United States)

    Murugan, Rajamanickam; Kreiman, Gabriel

    2012-01-01

    Eukaryotic genes are typically split into exons that need to be spliced together to form the mature mRNA. The splicing process depends on the dynamics and interactions among transcription by the RNA polymerase II complex (RNAPII) and the spliceosomal complex consisting of multiple small nuclear ribonucleo proteins (snRNPs). Here we propose a biophysically plausible initial theory of splicing that aims to explain the effects of the stochastic dynamics of snRNPs on the splicing patterns of eukaryotic genes. We consider two different ways to model the dynamics of snRNPs: pure three-dimensional diffusion and a combination of three- and one-dimensional diffusion along the emerging pre-mRNA. Our theoretical analysis shows that there exists an optimum position of the splice sites on the growing pre-mRNA at which the time required for snRNPs to find the 5′ donor site is minimized. The minimization of the overall search time is achieved mainly via the increase in non-specific interactions between the snRNPs and the growing pre-mRNA. The theory further predicts that there exists an optimum transcript length that maximizes the probabilities for exons to interact with the snRNPs. We evaluate these theoretical predictions by considering human and mouse exon microarray data as well as RNAseq data from multiple different tissues. We observe that there is a broad optimum position of splice sites on the growing pre-mRNA and an optimum transcript length, which are roughly consistent with the theoretical predictions. The theoretical and experimental analyses suggest that there is a strong interaction between the dynamics of RNAPII and the stochastic nature of snRNP search for 5′ donor splicing sites. PMID:23133354

  8. Theory on the coupled stochastic dynamics of transcription and splice-site recognition.

    Directory of Open Access Journals (Sweden)

    Rajamanickam Murugan

    Full Text Available Eukaryotic genes are typically split into exons that need to be spliced together to form the mature mRNA. The splicing process depends on the dynamics and interactions among transcription by the RNA polymerase II complex (RNAPII and the spliceosomal complex consisting of multiple small nuclear ribonucleo proteins (snRNPs. Here we propose a biophysically plausible initial theory of splicing that aims to explain the effects of the stochastic dynamics of snRNPs on the splicing patterns of eukaryotic genes. We consider two different ways to model the dynamics of snRNPs: pure three-dimensional diffusion and a combination of three- and one-dimensional diffusion along the emerging pre-mRNA. Our theoretical analysis shows that there exists an optimum position of the splice sites on the growing pre-mRNA at which the time required for snRNPs to find the 5' donor site is minimized. The minimization of the overall search time is achieved mainly via the increase in non-specific interactions between the snRNPs and the growing pre-mRNA. The theory further predicts that there exists an optimum transcript length that maximizes the probabilities for exons to interact with the snRNPs. We evaluate these theoretical predictions by considering human and mouse exon microarray data as well as RNAseq data from multiple different tissues. We observe that there is a broad optimum position of splice sites on the growing pre-mRNA and an optimum transcript length, which are roughly consistent with the theoretical predictions. The theoretical and experimental analyses suggest that there is a strong interaction between the dynamics of RNAPII and the stochastic nature of snRNP search for 5' donor splicing sites.

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

    Directory of Open Access Journals (Sweden)

    Zhihua Ju

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

  10. [Overexpression of FKS1 to improve yeast autolysis-stress].

    Science.gov (United States)

    Li, Jia; Wang, Jinjing; Li, Qi

    2015-09-01

    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

  11. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    To investigate whether transgenic overexpression of the zebrafish BAFF leads to ... and BAFF proteins were expressed separately and confirmed in HeLa cells. ... body homogenate of zebrafish and demonstrated a significant increase in ...

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

    International Nuclear Information System (INIS)

    Chiu Yali; Ouyang Pin

    2006-01-01

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

  13. Prognostic implication of aquaporin 1 overexpression in resected lung adenocarcinoma.

    Science.gov (United States)

    Bellezza, Guido; Vannucci, Jacopo; Bianconi, Fortunato; Metro, Giulio; Del Sordo, Rachele; Andolfi, Marco; Ferri, Ivana; Siccu, Paola; Ludovini, Vienna; Puma, Francesco; Sidoni, Angelo; Cagini, Lucio

    2017-12-01

    Aquaporins (AQPs) are a group of transmembrane water-selective channel proteins thought to play a role in the regulation of water permeability for plasma membranes. Indeed, high AQP levels have been suggested to promote the progression, invasion and metastasis of tumours. Specifically, AQP1 and AQP5 overexpression in lung adenocarcinoma (AC) have been suggested to be involved in molecular mechanisms in lung cancer. The aim of this retrospective cohort single-centre study was to assess both the levels of expression and therein the prognostic significance, regarding outcome of AQP1 and AQP5 in resected AC patients. Patients with histological diagnoses of lung AC submitted to pulmonary resection were included in this cohort study. Tissue microarrays containing cores from 185 ACs were prepared. AQP1 and AQP5 expressions were assessed by immunohistochemistry. Results were scored as either low (Score 0-2) or high (Score 3-9). Clinical data, pathological tumour-node-metastasis staging and follow-up were recorded. Multivariate Cox survival analysis and Fisher's t-test were performed. AQP1 overexpression was detected in 85 (46%) patients, while AQP5 overexpression was observed in 45 (24%) patients. AQP1 did not result being significantly correlated with clinical and pathological parameters, while AQP5 resulted more expressed in AC with mucinous and papillary predominant patterns. Patients with AQP1 overexpression had shorter disease-free survival (P = 0.001) compared with patients without AQP1 overexpression. Multivariate analysis confirmed that AQP1 overexpression was significantly associated with shorter disease-free survival (P = 0.001). Our results evidenced that AQP1 overexpression resulted in a shorter disease-free survival in lung AC patients. Being so, AQP1 overexpression might be an important prognostic marker in lung AC. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights

  14. Tobacco, alcohol, and p53 overexpression in early colorectal neoplasia

    International Nuclear Information System (INIS)

    Terry, Mary Beth; Neugut, Alfred I; Mansukhani, Mahesh; Waye, Jerome; Harpaz, Noam; Hibshoosh, Hanina

    2003-01-01

    The p53 tumor suppressor gene is commonly mutated in colorectal cancer. While the effect of p53 mutations on colorectal cancer prognosis has been heavily studied, less is known about how epidemiologic risk factors relate to p53 status, particularly in early colorectal neoplasia prior to clinically invasive colorectal cancer (including adenomas, carcinoma in situ (CIS), and intramucosal carcinoma). We examined p53 status, as measured by protein overexpression, in 157 cases with early colorectal neoplasia selected from three New York City colonoscopy clinics. After collecting paraffin-embedded tissue blocks, immunohistochemistry was performed using an anti-p53 monoclonal mouse IgG 2 a [BP53-12-1] antibody. We analyzed whether p53 status was different for risk factors for colorectal neoplasia relative to a polyp-free control group (n = 508). p53 overexpression was found in 10.3%, 21.7%, and 34.9%, of adenomatous polyps, CIS, and intramucosal cases, respectively. Over 90% of the tumors with p53 overexpression were located in the distal colon and rectum. Heavy cigarette smoking (30+ years) was associated with cases not overexpressing p53 (OR = 1.8, 95% CI = 1.1–2.9) but not with those cases overexpressing p53 (OR = 1.0, 95% CI = 0.4–2.6). Heavy beer consumption (8+ bottles per week) was associated with cases overexpressing p53 (OR = 4.0, 95% CI = 1.3–12.0) but not with cases without p53 overexpression (OR = 1.6, 95% CI = 0.7–3.7). Our findings that p53 overexpression in early colorectal neoplasia may be positively associated with alcohol intake and inversely associated with cigarette smoking are consistent with those of several studies of p53 expression and invasive cancer, and suggest that there may be relationships of smoking and alcohol with p53 early in the adenoma to carcinoma sequence

  15. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-01

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

  18. 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

  19. Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.

    Science.gov (United States)

    Peter, Angela K; Miller, Gaynor; Capote, Joana; DiFranco, Marino; Solares-Pérez, Alhondra; Wang, Emily L; Heighway, Jim; Coral-Vázquez, Ramón M; Vergara, Julio; Crosbie-Watson, Rachelle H

    2017-06-06

    Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd -/- ) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. Alternative splicing of proteins from the SG

  20. Overexpression of Enterococcus faecalis elr operon protects from phagocytosis.

    Science.gov (United States)

    Cortes-Perez, Naima G; Dumoulin, Romain; Gaubert, Stéphane; Lacoux, Caroline; Bugli, Francesca; Martin, Rebeca; Chat, Sophie; Piquand, Kevin; Meylheuc, Thierry; Langella, Philippe; Sanguinetti, Maurizio; Posteraro, Brunella; Rigottier-Gois, Lionel; Serror, Pascale

    2015-05-25

    Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

  1. 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

  2. MKLN1 splicing defect in dogs with lethal acrodermatitis.

    Directory of Open Access Journals (Sweden)

    Anina Bauer

    2018-03-01

    Full Text Available Lethal acrodermatitis (LAD is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of ~1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN1:c.400+3A>C. This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.

  3. 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  5. 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.

  6. 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.

  7. 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

  8. RNA Splicing in a New Rhabdovirus from Culex Mosquitoes▿†

    Science.gov (United States)

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-01-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae. PMID:21507977

  9. 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.

  10. The splicing of tiny introns of Paramecium is controlled by MAGO.

    Science.gov (United States)

    Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

    2018-07-15

    The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    CERN Document Server

    Bailey, R; Catalan Lasheras, N; Dahlerup-Petersen, K; Denz, R; Robles, C; Koratzinos, M; Pojer, M; Ponce, L; Saban, R; Schmidt, R; Siemko, A; Solfaroli Camillocci, M; Thiesen, H; Vergara Fernandez, A; Flora, R H; Charifoulline, Z; Bednarek, M; Górnicki, E; Jurkiewicz, P; Kapusta, P; Strait, J

    2010-01-01

    The incident of 19 September 2008 at the LHC was caused by a faulty inter-magnet splice of about 200 nΩ 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 nΩ. 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 nΩ 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 nΩ and 50 nΩ respectively. In 2009, this ad hoc system will be replaced with a permanent one to monitor all splices at the nΩ level.

  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. 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.

  14. Alternative splicing at the intersection of biological timing, development, and stress responses.

    Science.gov (United States)

    Staiger, Dorothee; Brown, John W S

    2013-10-01

    High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

  15. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract

    Energy Technology Data Exchange (ETDEWEB)

    Ogg, S C; Anderson, P; Wickens, M P [Univ. of Wisconsin, Madison (USA)

    1990-01-11

    Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intro. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, the authors determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. They demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3{prime} and 5{prime} sites as are used in C. elegans. The branch point used lies in the inserted sequences. They conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

  16. HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

    Science.gov (United States)

    Hu, Yingchuan; Bandla, Santhoshi; Godfrey, Tony E.; Tan, Dongfeng; Luketich, James D.; Pennathur, Arjun; Qiu, Xing; Hicks, David G.; Peters, Jeffrey; Zhou, Zhongren

    2011-01-01

    The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of BE, 18 cases of low grade dysplasia and 15 cases of high grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and CISH methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by CISH and high density microarrays. We further confirm the similar frequency of HER2 amplification by CISH (18.10%; 21/116) and SNP 6.0 microarrays (16.4%, 19/116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12.1 % (14/116) of esophageal adenocarcinoma and 6.67% (1/15) of HGD. No HER2 amplification or overexpression was identified in BE or LGD. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by CISH than protein overexpression in esophageal adenocarcinoma (18.10% vs 12.9%). A modified two-step model for esophageal adenocarcinoma HER-2 testing is recommend for clinical esophageal adenocarcinoma HER-2 trial. PMID:21460800

  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. 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...

  20. 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 potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19...... effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue...

  1. 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.

  2. 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

    Alternative splicing (AS) is an important contributor to proteome diversity and is regarded as an explanatory factor for the relatively low number of human genes compared with less complex animals. To assess the evolutionary conservation of AS and its developmental regulation, we have investigated...... 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....... Moreover, we find that relative isoform expression levels vary significantly during development for 78% of the AS events and that this quantitative variation is highly conserved between the 2 species. Our results suggest that AS is generally tightly regulated through development and that the regulatory...

  3. 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...

  4. 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)

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

    International Nuclear Information System (INIS)

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

    2017-01-01

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

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  7. Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information

    DEFF Research Database (Denmark)

    Hebsgaard, Stefan M.; Korning, Peter G.; Tolstrup, Niels

    1996-01-01

    Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local predicition of splice sites, is refin...

  8. 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)

  9. Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

    DEFF Research Database (Denmark)

    Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick

    2007-01-01

    (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model...

  10. 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

  11. Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8(+) T Cells during Bacterial Infection

    NARCIS (Netherlands)

    Platteel, Anouk C M|info:eu-repo/dai/nl/375805613; Liepe, Juliane; Textoris-Taube, Kathrin; Keller, Christin; Henklein, Petra; Schalkwijk, Hanna H; Cardoso, Rebeca; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M|info:eu-repo/dai/nl/115553843

    2017-01-01

    Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to

  12. Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Anouk C.M. Platteel

    2017-08-01

    Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

  13. A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

    Directory of Open Access Journals (Sweden)

    Patricia Peking

    2016-01-01

    Full Text Available RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5′ trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5′ RNA trans-splicing molecule, capable of replacing Col7a1 exons 1–15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5′ RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

  14. Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans.

    Science.gov (United States)

    Awan, Ali R; Manfredo, Amanda; Pleiss, Jeffrey A

    2013-07-30

    Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multicellular eukaryotes and is associated with organismal complexity. Although alternative splicing is widespread in vertebrates, little is known about the evolutionary origins of this process, in part because of the absence of phylogenetically conserved events that cross major eukaryotic clades. Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Remarkably, an evolutionary analysis of four of the exons identified here as subject to skipping in S. pombe reveals high sequence conservation and perfect length conservation with their homologs in scores of plants, animals, and fungi. Moreover, alternative splicing of two of these exons have been documented in multiple vertebrate organisms, making these the first demonstrations of identical alternative-splicing patterns in species that are separated by over 1 billion y of evolution.

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

    Directory of Open Access Journals (Sweden)

    Chang Kung-Yen

    2011-07-01

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

  16. 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

  17. New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.

    Directory of Open Access Journals (Sweden)

    Mireia Mora

    Full Text Available Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300 is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA of intron 5 (c.653-1G>A in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

  18. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

    Directory of Open Access Journals (Sweden)

    Anne-Mette Hartung

    2016-05-01

    Full Text Available Costello syndrome (CS may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE and creation of an Exonic Splicing Silencer (ESS. We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

  19. 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

  20. 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.

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

    Science.gov (United States)

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

    2010-03-05

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

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Delerue-Audegond Audrey

    2008-12-01

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

  5. Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

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    Hitoshi Suzuki

    Full Text Available BACKGROUND: Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes. Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. CONCLUSIONS/SIGNIFICANCE: Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell

  6. 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.

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Kazushi Inoue

    2015-01-01

    Full Text Available Breast cancer (BC is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor and human epidermal growth factor receptor 2 (HER2, play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER , HER2 , and CD44 genes from the viewpoint of BC development. ERα36 , a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2 has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms ( CD44s , CD44v play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci

  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. 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...

  11. 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

  12. 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...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned...... and RCK2 (4 aa at SS1) and upstream of the calcium "bowl" (27 aa at SS4). Two other truncated variants, X2(92) and X2(188), lacking the intracellular C-terminal (stop downstream of S6), were cloned from cerebral vascular/meningeal tissue. They appear non-functional as no current expression was observed...

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

    Science.gov (United States)

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

    2002-10-01

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

  15. 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

  16. Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

    Science.gov (United States)

    Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

    2003-01-01

    Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

  17. 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

  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. Sexy splicing: regulatory interplays governing sex determination from Drosophila to mammals.

    Science.gov (United States)

    Lalli, Enzo; Ohe, Kenji; Latorre, Elisa; Bianchi, Marco E; Sassone-Corsi, Paolo

    2003-02-01

    A remarkable array of strategies is used to produce sexual differentiation in different species. Complex gene hierarchies govern sex determination pathways, as exemplified by the classic D. melanogaster paradigm, where an interplay of transcriptional, splicing and translational mechanisms operate. Molecular studies support the hypothesis that genetic sex determination pathways evolved in reverse order, from downstream to upstream genes, in the cascade. The recent identification of a role for the key regulatory factors SRY and WT1(+KTS) in pre-mRNA splicing indicates that important steps in the mammalian sex determination process are likely to operate at the post-transcriptional level.

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

    KAUST Repository

    Ling, Yu; Alshareef, Sahar; Butt, Haroon; Lozano-Juste, Jorge; Li, Lixin; Galal, Aya A.; Moustafa, Ahmed; Momin, Afaque Ahmad Imtiyaz; Tashkandi, Manal; Richardson, Dale N.; Fujii, Hiroaki; Arold, Stefan T.; Rodriguez, Pedro L.; Duque, Paula; Mahfouz, Magdy M.

    2016-01-01

    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

  1. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    Science.gov (United States)

    Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  2. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    Directory of Open Access Journals (Sweden)

    Rodrigo S Lacruz

    Full Text Available We have previously identified amelotin (AMTN as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL and ameloblastin (AMBN was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  3. 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

    The span of roof trusses with punched metal plate fasteners (nail plates) makes it often necessary to use splice joints in the top and bottom chords. In the finite element models used for design of the trusses these splice joints are normally assumed to be either rotationally stiff or pinned....... Timber-to-timber contact and non-linear elastic behaviour are included in the model. Results from tests with joints under fourpoint bending are compared with predictions given by TrussLab, and a good agreement is found. Splice joints in trusses with nail plates may be assumed to be rotationally stiff...... if their deformation has no significant effect upon the distribution of member forces according to Eurocode 5. Two simple guidelines for the design and location of splice joints are given in Eurocode 5 for treating the splice joints as rotationally stiff. The reasonability of these guidelines and the influence...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    BRCA1 and 176 BRCA2 unique variants, from 77 publications. At least six independent reviewers from research and/or clinical settings comprehensively examined splicing assay methods and data reported for 22 variant assays of 21 variants in four publications, and classified the variants using the 5-tier......Splicing assays are commonly undertaken in the clinical setting to assess the clinical relevance of sequence variants in disease predisposition genes. A 5-tier classification system incorporating both bioinformatic and splicing assay information was previously proposed as a method to provide...... of results, and the lack of quantitative data for the aberrant transcripts. We propose suggestions for minimum reporting guidelines for splicing assays, and improvements to the 5-tier splicing classification system to allow future evaluation of its performance as a clinical tool....

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

    NARCIS (Netherlands)

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

    1996-01-01

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

  6. PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor

    International Nuclear Information System (INIS)

    Maita, Hiroshi; Kitaura, Hirotake; Keen, T. Jeffrey; Inglehearn, Chris F.; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M.M.

    2004-01-01

    PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). However, RP9 is a rare disease and only two missense mutations have been described, so the report of a link between PAP-1 and RP9 was tentative. The precise cellular role of PAP-1 was also unknown at that time. We now report that PAP-1 localizes in nuclear speckles containing the splicing factor SC35 and interacts directly with another splicing factor, U2AF35. Furthermore, we used in vitro and in vivo splicing assays to show that PAP-1 has an activity, which alters the pattern of pre-mRNA splicing and that this activity is dependent on the phosphorylation state of PAP-1. We used the same splicing assay to examine the activities of two mutant forms of PAP-1 found in RP9 patients. The results showed that while one of the mutations, H137L, had no effect on splicing activity compared with that of wild-type PAP-1, the other, D170G, resulted in both a defect in splicing activity and a decreased proportion of phosphorylated PAP-1. The D170G mutation may therefore cause RP by altering splicing of retinal genes through a decrease in PAP-1 phosphorylation. These results demonstrate that PAP-1 has a role in pre-mRNA splicing and, given that three other splicing factors have been implicated in adRP, this finding provides compelling further evidence that PAP-1 is indeed the RP9 gene

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

    Directory of Open Access Journals (Sweden)

    Barbara Wappenschmidt

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

  8. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    International Nuclear Information System (INIS)

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-01-01

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

  9. Prognostic implication of NQO1 overexpression in hepatocellular carcinoma.

    Science.gov (United States)

    Lin, Lijuan; Sun, Jie; Tan, Yan; Li, Zhenling; Kong, Fanyong; Shen, Yue; Liu, Chao; Chen, Litian

    2017-11-01

    To explore the role of NQO1 overexpression for prognostic implication in hepatocellular carcinoma (HCC), NQO1 mRNA levels were detected in HCC fresh tissue samples of HCC and nontumor tissues, respectively. One hundred fifty-six cases of HCC meeting strict follow-up criteria were selected for immunohistochemical staining of NQO1 protein. Correlations between NQO1 overexpression and clinicopathological features of HCC were evaluated using χ 2 tests, survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient 5-year survival was analyzed using Cox proportional hazards analysis. In results, the levels of NQO1 mRNA were significantly up-regulated in 14 fresh tissue samples of HCC. Immunohistochemical analysis showed that the NQO1 expression and overexpression rates were significantly higher in HCC samples compared with either adjacent nontumor tissues or normal liver tissues. NQO1 overexpression correlated to tumor size, venous infiltration and late pTNM stage of HCC. NQO1 overexpression was also related to low disease-free survival and 5-year survival rates. In the late-stage group, disease-free and 5-year survival rates of patients with NQO1 overexpression were significantly lower than those of patients without NQO1 expression. Further analysis using a Cox proportional hazards regression model revealed that NQO1 expression emerged as a significant independent hazard factor for the 5-year survival rate of patients with HCC. Therefore, NQO1 plays an important role in the progression of HCC. NQO1 may potentially be used as an independent biomarker for prognostic evaluation of HCC. Copyright © 2017. Published by Elsevier Inc.

  10. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    Science.gov (United States)

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. © 2015 International Federation for Cell Biology.

  11. 49 CFR 236.74 - Protection of insulated wire; splice in underground wire.

    Science.gov (United States)

    2010-10-01

    ... underground wire. 236.74 Section 236.74 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RULES, STANDARDS, AND INSTRUCTIONS GOVERNING... wire; splice in underground wire. Insulated wire shall be protected from mechanical injury. The...

  12. 49 CFR 234.241 - Protection of insulated wire; splice in underground wire.

    Science.gov (United States)

    2010-10-01

    ... underground wire. 234.241 Section 234.241 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GRADE CROSSING SIGNAL SYSTEM SAFETY... of insulated wire; splice in underground wire. Insulated wire shall be protected from mechanical...

  13. 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.

  14. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

    Science.gov (United States)

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-05-28

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

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

    Science.gov (United States)

    2016-08-01

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

  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. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of electric... material and 21/2 inches of conductor insulation. The type, amperage, voltage rating, and construction of...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing...

  20. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

    Science.gov (United States)

    Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

    2010-10-01

    Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

  1. Role and convergent evolution of competing RNA secondary structures in mutually exclusive splicing.

    Science.gov (United States)

    Yue, Yuan; Hou, Shouqing; Wang, Xiu; Zhan, Leilei; Cao, Guozheng; Li, Guoli; Shi, Yang; Zhang, Peng; Hong, Weiling; Lin, Hao; Liu, Baoping; Shi, Feng; Yang, Yun; Jin, Yongfeng

    2017-10-03

    Exon or cassette duplication is an important means of expanding protein and functional diversity through mutually exclusive splicing. However, the mechanistic basis of this process in non-arthropod species remains poorly understood. Here, we demonstrate that MRP1 genes underwent tandem exon duplication in Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, Echinodermata, and early-diverging Chordata but not in late-diverging vertebrates. Interestingly, these events were of independent origin in different phyla, suggesting convergent evolution of alternative splicing. Furthermore, we showed that multiple sets of clade-conserved RNA pairings evolved to guide species-specific mutually exclusive splicing in Arthropoda. Importantly, we also identified a similar structural code in MRP exon clusters of the annelid, Capitella teleta, and chordate, Branchiostoma belcheri, suggesting an evolutionarily conserved competing pairing-guided mechanism in bilaterians. Taken together, these data reveal the molecular determinants and RNA pairing-guided evolution of species-specific mutually exclusive splicing spanning more than 600 million years of bilaterian evolution. These findings have a significant impact on our understanding of the evolution of and mechanism underpinning isoform diversity and complex gene structure.

  2. Alternative splicing of cyclooxygenase-1 mRNA in the human iris

    NARCIS (Netherlands)

    Dröge, M.J; van Sorge, A.A; van Haeringen, N.J; Quax, Wim; Zaagsma, Hans; Droge, MJ

    2003-01-01

    dIn homogenates of the human iris, the nonsteroidal antiinflammatory drug (NSAID) S(+)flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human

  3. 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

    , 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...

  4. 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....

  5. Regulation of Neurexin 1[beta] Tertiary Structure and Ligand Binding through Alternative Splicing

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Kaiser C.; Kuczynska, Dorota A.; Wu, Irene J.; Murray, Beverly H.; Sheckler, Lauren R.; Rudenko, Gabby (Michigan)

    2008-08-04

    Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a splice-insert signaling code. In particular, neurexin 1{beta} carrying an alternative splice insert at site SS{number_sign}4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1{beta}+SS{number_sign}4 reveals dramatic rearrangements to the 'hypervariable surface', the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop {beta}10-{beta}11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca{sup 2+}-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1{beta} isoforms acquire neuroligin splice isoform selectivity.

  6. 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.

  7. 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. PMID:23737328

  8. 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.

  9. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    DEFF Research Database (Denmark)

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E

    2016-01-01

    by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti......-cancer therapies based on SSO-mediated HRAS exon 2 skipping....

  10. regSNPs-splicing: a tool for prioritizing synonymous single-nucleotide substitution.

    Science.gov (United States)

    Zhang, Xinjun; Li, Meng; Lin, Hai; Rao, Xi; Feng, Weixing; Yang, Yuedong; Mort, Matthew; Cooper, David N; Wang, Yue; Wang, Yadong; Wells, Clark; Zhou, Yaoqi; Liu, Yunlong

    2017-09-01

    While synonymous single-nucleotide variants (sSNVs) have largely been unstudied, since they do not alter protein sequence, mounting evidence suggests that they may affect RNA conformation, splicing, and the stability of nascent-mRNAs to promote various diseases. Accurately prioritizing deleterious sSNVs from a pool of neutral ones can significantly improve our ability of selecting functional genetic variants identified from various genome-sequencing projects, and, therefore, advance our understanding of disease etiology. In this study, we develop a computational algorithm to prioritize sSNVs based on their impact on mRNA splicing and protein function. In addition to genomic features that potentially affect splicing regulation, our proposed algorithm also includes dozens structural features that characterize the functions of alternatively spliced exons on protein function. Our systematical evaluation on thousands of sSNVs suggests that several structural features, including intrinsic disorder protein scores, solvent accessible surface areas, protein secondary structures, and known and predicted protein family domains, show significant differences between disease-causing and neutral sSNVs. Our result suggests that the protein structure features offer an added dimension of information while distinguishing disease-causing and neutral synonymous variants. The inclusion of structural features increases the predictive accuracy for functional sSNV prioritization.

  11. 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.

  12. 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

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  14. Impact of Selected Parameters on the Fatigue Strength of Splices on Multiply Textile Conveyor Belts

    Science.gov (United States)

    Bajda, Mirosław; Błażej, Ryszard; Hardygóra, Monika

    2016-10-01

    Splices are the weakest points in the conveyor belt loop. The strength of these joints, and thus their design as well as the method and quality of splicing, determine the strength of the whole conveyor belt loop. A special zone in a splice exists, where the stresses in the adjacent plies or cables differ considerably from each other. This results in differences in the elongation of these elements and in additional shearing stresses in the rubber layer. The strength of the joints depends on several factors, among others on the parameters of the joined belt, on the connecting layer and the technology of joining, as well as on the materials used to make the joint. The strength of the joint constitutes a criterion for the selection of a belt suitable for the operating conditions, and therefore methods of testing such joints are of great importance. This paper presents the method of testing fatigue strength of splices made on multi-ply textile conveyor belts and the results of these studies.

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

    Science.gov (United States)

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

    2014-10-15

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

  16. 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

  17. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

    Directory of Open Access Journals (Sweden)

    Elvezia Maria Paraboschi

    2015-09-01

    Full Text Available Abnormalities in RNA metabolism and alternative splicing (AS are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls, followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p = 0.0015 by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  18. Triiodothyronine affects the alternative splicing of thyroid hormone receptor alpha mRNA

    NARCIS (Netherlands)

    Timmer, D. C.; Bakker, O.; Wiersinga, W. M.

    2003-01-01

    The c-erbAalpha gene encodes two thyroid hormone receptors, TRalpha1 and TRalpha2, that arise from alternative splicing of the TRalpha pre-mRNA. TRalpha2 is not able to bind triiodothyronine (T-3) and acts as a weak antagonist of TRs. It has been suggested that the balance of TRalpha1 to TRalpha2 is

  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. 30 CFR 77.601 - Trailing cables or portable cables; temporary splices.

    Science.gov (United States)

    2010-07-01

    ... LABOR COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS, SURFACE COAL MINES AND SURFACE WORK AREAS OF UNDERGROUND COAL MINES Trailing Cables § 77.601 Trailing cables or portable cables; temporary... or splices that heat or spark under load shall not be used. ...

  1. Chemotherapy induces alternative transcription and splicing: Facts and hopes for cancer treatment.

    Science.gov (United States)

    Lambert, Charles A; Garbacki, Nancy; Colige, Alain C

    2017-10-01

    Alternative promoter usage, alternative splicing and alternative cleavage/polyadenylation (referred here as to alternative transcription and splicing) are main instruments to diversify the transcriptome from a limited set of genes. There is a good deal of evidence that chemotherapeutic drugs affect these processes, but the therapeutic incidence of these effects is poorly documented. The scope of this study is to review the impact of chemotherapy on alternative transcription and splicing and to discuss potential implications in cancer therapy. A literature survey identified >2200 events induced by chemotherapeutic drugs. The molecular pathways involved in these regulations are briefly discussed. The GO terms associated with the alternative transcripts are mainly related to cell cycle/division, mRNA processing, DNA repair, macromolecules catabolism and chromatin. A large fraction (43%) of transcripts are also related to the new hallmarks of cancer, mostly genetic instability and replicative immortality. Finally, we ask the question of the impact of alternative transcription and splicing on drug efficacy and of the possible curative benefit of combining chemotherapy and pharmaceutical regulation of this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Mutant allele of rna14 in fission yeast affects pre-mRNA splicing

    Indian Academy of Sciences (India)

    transcript. Rna14 protein in budding yeast has been implicated in cleavage and ... Subsequently, genetic interaction of Rna14 with prp1 and physical .... molecular yeast techniques as described by Moreno et al. ..... To elucidate the role of Rna14 in splicing, RT-PCR analysis ..... design principles of a dynamic RNP machine.

  3. 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.

  4. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

    KAUST Repository

    Zhang, Runxuan; Calixto, Cristiane  P.  G.; Marquez, Yamile; Venhuizen, Peter; Tzioutziou, Nikoleta A.; Guo, Wenbin; Spensley, Mark; Entizne, Juan Carlos; Lewandowska, Dominika; ten  Have, Sara; Frei  dit  Frey, Nicolas; Hirt, Heribert; James, Allan B.; Nimmo, Hugh G.; Barta, Andrea; Kalyna, Maria; Brown, John  W.  S.

    2017-01-01

    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.

  5. 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.

  6. 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.

  7. Field gradient calculation of HTS double-pancake coils considering the slanted turns and the splice

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Geon Woo; Kim, Jin Sub; Song, Seung Hyun; Ko, Tae Kuk [Yonsei University, Seoul (Korea, Republic of); Lee, Woo Seung [JH ENGINEERING CO., LTD., Gunpo (Korea, Republic of); Lee, On You [Korea National University of Transportation, Chungju (Korea, Republic of)

    2017-03-15

    To obtain Nuclear Magnetic Resonance (NMR) measurement of membrane protein, an NMR magnet is required to generate high intensity, homogeneity, and stability of field. A High-Temperature Superconducting (HTS) magnet is a promising alternative to a conventional Low-Temperature Superconducting (LTS) NMR magnet for high field, current density, and stability margin. Conventionally, an HTS coil has been wound by several winding techniques such as Single-Pancake (SP), Double-Pancake (DP), and layer-wound. The DP winding technique has been frequently used for a large magnet because long HTS wire is generally difficult to manufacture, and maintenance of magnet is convenient. However, magnetic field generated by the slanted turns and the splice leads to field inhomogeneity in Diameter of Spherical Volume (DSV). The field inhomogeneity degrades performance of NMR spectrometer and thus effect of the slanted turns and the splice should be analyzed. In this paper, field gradient of HTS double-pancake coils considering the slanted turns and the splice was calculated using Biot-Savart law and numerical integration. The calculation results showed that magnetic field produced by the slanted turns and the splice caused significant inhomogeneity of field.

  8. 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

  9. Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa

    Science.gov (United States)

    Pan, Xinyuan; Chen, Xue; Liu, Xiaoxing; Gao, Xiang; Kang, Xiaoli; Xu, Qihua; Chen, Xuejuan; Zhao, Kanxing; Zhang, Xiumei; Chu, Qiaomei; Wang, Xiuying

    2014-01-01

    Purpose Seven genes involved in precursor mRNA (pre-mRNA) splicing have been implicated in autosomal dominant retinitis pigmentosa (adRP). We sought to detect mutations in all seven genes in Chinese families with RP, to characterize the relevant phenotypes, and to evaluate the prevalence of mutations in splicing genes in patients with adRP. Methods Six unrelated families from our adRP cohort (42 families) and two additional families with RP with uncertain inheritance mode were clinically characterized in the present study. Targeted sequence capture with next-generation massively parallel sequencing (NGS) was performed to screen mutations in 189 genes including all seven pre-mRNA splicing genes associated with adRP. Variants detected with NGS were filtered with bioinformatics analyses, validated with Sanger sequencing, and prioritized with pathogenicity analysis. Results Mutations in pre-mRNA splicing genes were identified in three individual families including one novel frameshift mutation in PRPF31 (p.Leu366fs*1) and two known mutations in SNRNP200 (p.Arg681His and p.Ser1087Leu). The patients carrying SNRNP200 p.R681H showed rapid disease progression, and the family carrying p.S1087L presented earlier onset ages and more severe phenotypes compared to another previously reported family with p.S1087L. In five other families, we identified mutations in other RP-related genes, including RP1 p. Ser781* (novel), RP2 p.Gln65* (novel) and p.Ile137del (novel), IMPDH1 p.Asp311Asn (recurrent), and RHO p.Pro347Leu (recurrent). Conclusions Mutations in splicing genes identified in the present and our previous study account for 9.5% in our adRP cohort, indicating the important role of pre-mRNA splicing deficiency in the etiology of adRP. Mutations in the same splicing gene, or even the same mutation, could correlate with different phenotypic severities, complicating the genotype–phenotype correlation and clinical prognosis. PMID:24940031

  10. 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

  11. PASSion: a pattern growth algorithm-based pipeline for splice junction detection in paired-end RNA-Seq data.

    Science.gov (United States)

    Zhang, Yanju; Lameijer, Eric-Wubbo; 't Hoen, Peter A C; Ning, Zemin; Slagboom, P Eline; Ye, Kai

    2012-02-15

    RNA-seq is a powerful technology for the study of transcriptome profiles that uses deep-sequencing technologies. Moreover, it may be used for cellular phenotyping and help establishing the etiology of diseases characterized by abnormal splicing patterns. In RNA-Seq, the exact nature of splicing events is buried in the reads that span exon-exon boundaries. The accurate and efficient mapping of these reads to the reference genome is a major challenge. We developed PASSion, a pattern growth algorithm-based pipeline for splice site detection in paired-end RNA-Seq reads. Comparing the performance of PASSion to three existing RNA-Seq analysis pipelines, TopHat, MapSplice and HMMSplicer, revealed that PASSion is competitive with these packages. Moreover, the performance of PASSion is not affected by read length and coverage. It performs better than the other three approaches when detecting junctions in highly abundant transcripts. PASSion has the ability to detect junctions that do not have known splicing motifs, which cannot be found by the other tools. Of the two public RNA-Seq datasets, PASSion predicted ≈ 137,000 and 173,000 splicing events, of which on average 82 are known junctions annotated in the Ensembl transcript database and 18% are novel. In addition, our package can discover differential and shared splicing patterns among multiple samples. The code and utilities can be freely downloaded from https://trac.nbic.nl/passion and ftp://ftp.sanger.ac.uk/pub/zn1/passion.

  12. 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

  13. Low-loss, robust fusion splicing of silica to chalcogenide fiber for integrated mid-infrared laser technology development.

    Science.gov (United States)

    Thapa, Rajesh; Gattass, Rafael R; Nguyen, Vinh; Chin, Geoff; Gibson, Dan; Kim, Woohong; Shaw, L Brandon; Sanghera, Jasbinder S

    2015-11-01

    We demonstrate a low-loss, repeatable, and robust splice between single-mode silica fiber and single-mode chalcogenide (CHG) fiber. These splices are particularly difficult to create because of the significant difference in the two fibers' glass transition temperatures (∼1000°C) as well as the large difference in the coefficients of thermal expansion between the fibers (∼20×10(-6)/°C). With 90% light coupled through the silica-CHG fiber splice, predominantly in the fundamental circular-symmetric mode, into the core of the CHG fiber and with 0.5 dB of splice loss measured around the wavelength of 2.5 μm, after correcting only for the Fresnel loss, the silica-CHG splice offers excellent beam quality and coupling efficiency. The tensile strength of the splice is greater than 12 kpsi, and the laser damage threshold is greater than 2 W (CW) and was limited by the available laser pump power. We also utilized this splicing technique to demonstrate 2 to 4.5 μm ultrabroadband supercontinuum generation in a monolithic all-fiber system comprising a CHG fiber and a high peak power 2 μm pulsed Raman-shifted thulium fiber laser. This is a major development toward compact form factor commercial applications of soft-glass mid-IR fibers.

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  16. 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.

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

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    Xiaoming Yi

    2000-09-01

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

  18. Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

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    Hulse, R P; Beazley-Long, N; Hua, J; Kennedy, H; Prager, J; Bevan, H; Qiu, Y; Fernandes, E S; Gammons, M V; Ballmer-Hofer, K; Gittenberger de Groot, A C; Churchill, A J; Harper, S J; Brain, S D; Bates, D O; Donaldson, L F

    2014-11-01

    Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. Copyright © 2014. Published by Elsevier Inc.

  19. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

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    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.

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

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

    2011-03-03

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

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

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    Rute Martins

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

  2. Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

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    Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

    2016-08-01

    Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

  3. Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin.

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    Krivokuca, Ana; Dragos, Vita Setrajcic; Stamatovic, Ljiljana; Blatnik, Ana; Boljevic, Ivana; Stegel, Vida; Rakobradovic, Jelena; Skerl, Petra; Jovandic, Stevo; Krajc, Mateja; Magic, Mirjana Brankovic; Novakovic, Srdjan

    2018-04-01

    Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel