TROPONIN T ALTERNATIVE SPLICING IN EMBRYONIC HEART

Summary

Principal Investigator: THOMAS ALEXANDER COOPER
Affiliation: Baylor College of Medicine
Country: USA
Abstract: DESCRIPTION: The long term goal of this proposal is to understand the m o lecular basis for the regulation of pre-mRNA alternative splicing. Alternative splicing is a common and key regulatory step for the expression of diverse protein isoforms according to cell-specific or developmental programs. Despite significant advances in the understanding of the biochemistry of pre-mRNA splicing, little is known about the molecular components or mechanisms that regulate splice site selection. The PI will continue his investigation of developmentally regulated alternative splicing using the chicken cardiac troponin T (cTNT) gene in which a single alternative exon (exon 5) is included in embryonic striated muscle and skipped in the adult. During the last funding period we have made significant progress with regard to both constitutive and alternative splicing mechanisms. The investigator's accomplishments include: (I) identification and characterization of a previously unknown constitutive splicing element (referred to as a splicing enhancer) located within cTNT exon 5 and other alternative and constitutive exons; (ii) demonstration that a subset of the SR protein family of essential splicing factors binds to the enhancer and activates splicing; (iii) identification of an intronic element that is necessary and sufficient to activate muscle-specific exon inclusion in vivo; and (iv) reconstitution of a muscle-specific splicing in a cell free complementation system. A major focus of this proposal is to identify, isolate, and characterize factors that activate exon inclusion in embryonic striated muscle. A muscle-specific activator sequence has been localized within a 142 nucleotide segment immediately downstream of the alternative exon. The critical determinants within this region will be defined. A prime target is a conserved sequence found in two similarly regulated mammalian genes. The established in vitro complementation system will be used to characterize muscle-specific splicing activity. Functionally significant RNA-binding proteins will be identified using an approach that directly correlates in vitro RNA-binding with in vivo splicing activity. In vitro splicing and RNA-binding will be used as complementary assays to identify, characterize and, ultimately, isolate by cDNA cloning the factors that regulate a cell-specific splicing event. An understanding of cTNT alternative splicing could provide a paradigm for regulatory mechanisms in vertebrates. Regulation of alternative splicing is a fundamental process required for normal development and cellular function. Alterations of alternative splicing pathways are associated with pathologic changes in a number of diseases. Therefore, insights gained from these studies will be directly applicable to basic molecular mechanisms that affect human health.
Funding Period: 1991-07-01 - 2000-06-30
more information: NIH RePORT

Top Publications

  1. pmc Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development
    Jimena Giudice
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA
    Nat Commun 5:3603. 2014
  2. pmc Myotonic dystrophy mouse models: towards rational therapy development
    Mário Gomes-Pereira
    INSERM U781, Universite Paris Descartes, Faculte de Medicine Necker Enfants Malades, Paris, France
    Trends Mol Med 17:506-17. 2011
  3. pmc Functional consequences of developmentally regulated alternative splicing
    Auinash Kalsotra
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA
    Nat Rev Genet 12:715-29. 2011
  4. pmc Pre-mRNA splicing in disease and therapeutics
    Ravi K Singh
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Trends Mol Med 18:472-82. 2012
  5. pmc Antisense oligonucleotides: rising stars in eliminating RNA toxicity in myotonic dystrophy
    Zhihua Gao
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Hum Gene Ther 24:499-507. 2013
  6. pmc Reexpression of pyruvate kinase M2 in type 1 myofibers correlates with altered glucose metabolism in myotonic dystrophy
    Zhihua Gao
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 110:13570-5. 2013
  7. pmc Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron
    Gloria V Echeverria
    Integrative Molecular and Biomedical Sciences Program, Baylor College of Medicine, Houston, TX 77030, USA, Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA and Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 42:1893-903. 2014
  8. pmc The Mef2 Transcription Network Is Disrupted in Myotonic Dystrophy Heart Tissue, Dramatically Altering miRNA and mRNA Expression
    Auinash Kalsotra
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Cell Rep 6:336-45. 2014
  9. pmc Identification of MBNL1 and MBNL3 domains required for splicing activation and repression
    Ioannis Grammatikakis
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 39:2769-80. 2011
  10. pmc RNA and disease
    Thomas A Cooper
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Cell 136:777-93. 2009

Scientific Experts

  • T A Cooper
  • Auinash Kalsotra
  • Amanda J Ward
  • Zhihua Gao
  • Gloria V Echeverria
  • Ravi K Singh
  • Young Hwa Goo
  • Jimena Giudice
  • Christopher B Burge
  • Ioannis Grammatikakis
  • Mário Gomes-Pereira
  • N Muge Kuyumcu-Martinez
  • James P Orengo
  • Jin Han
  • Nuno André Faustino
  • Xander H T Wehrens
  • Wei Wang
  • Wei Li
  • Chad J Creighton
  • Priyatansh Gurha
  • Marissa A Scavuzzo
  • Zheng Xia
  • Eric T Wang
  • Genevieve Gourdon
  • Xinshu Xiao
  • Jason M Johnson
  • John C Castle
  • Guey Shin Wang
  • Donnie Bundman

Detail Information

Publications20

  1. pmc Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development
    Jimena Giudice
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA
    Nat Commun 5:3603. 2014
    ..These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics. ..
  2. pmc Myotonic dystrophy mouse models: towards rational therapy development
    Mário Gomes-Pereira
    INSERM U781, Universite Paris Descartes, Faculte de Medicine Necker Enfants Malades, Paris, France
    Trends Mol Med 17:506-17. 2011
    ..More importantly, it provides critical assessment of their suitability and limitations for preclinical testing of emerging therapeutic strategies...
  3. pmc Functional consequences of developmentally regulated alternative splicing
    Auinash Kalsotra
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA
    Nat Rev Genet 12:715-29. 2011
    ..Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is used by other regulatory mechanisms...
  4. pmc Pre-mRNA splicing in disease and therapeutics
    Ravi K Singh
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Trends Mol Med 18:472-82. 2012
    ..In addition to discussing the disorders caused by these mutations, this review summarizes therapeutic approaches that have emerged to correct splicing of individual genes or target the splicing machinery...
  5. pmc Antisense oligonucleotides: rising stars in eliminating RNA toxicity in myotonic dystrophy
    Zhihua Gao
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Hum Gene Ther 24:499-507. 2013
    ..ASO targeting approaches will also provide avenues for the treatment of other repeat RNA-mediated diseases...
  6. pmc Reexpression of pyruvate kinase M2 in type 1 myofibers correlates with altered glucose metabolism in myotonic dystrophy
    Zhihua Gao
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 110:13570-5. 2013
    ..We propose that PKM2 expression induces changes in type 1 fibers associated with muscle atrophy and muscle weakness in DM1. ..
  7. pmc Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron
    Gloria V Echeverria
    Integrative Molecular and Biomedical Sciences Program, Baylor College of Medicine, Houston, TX 77030, USA, Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA and Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 42:1893-903. 2014
    ..These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion. ..
  8. pmc The Mef2 Transcription Network Is Disrupted in Myotonic Dystrophy Heart Tissue, Dramatically Altering miRNA and mRNA Expression
    Auinash Kalsotra
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Cell Rep 6:336-45. 2014
    ..We conclude that loss of MEF2 in DM1 heart causes pathogenic features through aberrant expression of both miRNA and mRNA targets. ..
  9. pmc Identification of MBNL1 and MBNL3 domains required for splicing activation and repression
    Ioannis Grammatikakis
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 39:2769-80. 2011
    ..These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins...
  10. pmc RNA and disease
    Thomas A Cooper
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Cell 136:777-93. 2009
    ..The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry is providing new RNA-based tools for developing therapeutics...
  11. pmc The pathobiology of splicing
    Amanda J Ward
    Departments of Molecular and Cellular Biology and Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    J Pathol 220:152-63. 2010
    ..An understanding of the role of splicing in disease expands potential opportunities for therapeutic intervention by either directly addressing the cause or by providing novel approaches to circumvent disease processes...
  12. pmc Identification of CELF splicing activation and repression domains in vivo
    Jin Han
    Department of Pathology, Baylor College of Medicine Houston, TX 77030, USA
    Nucleic Acids Res 33:2769-80. 2005
    ..These results provide a foundation for identifying CELF-interacting proteins involved in activated and/or repressed splicing...
  13. pmc A bichromatic fluorescent reporter for cell-based screens of alternative splicing
    James P Orengo
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 34:e148. 2006
    ....
  14. pmc Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation
    N Muge Kuyumcu-Martinez
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Mol Cell 28:68-78. 2007
    ..These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a noncoding RNA...
  15. pmc A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart
    Auinash Kalsotra
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Proc Natl Acad Sci U S A 105:20333-8. 2008
    ..These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development...
  16. pmc CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA
    Young Hwa Goo
    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
    Nucleic Acids Res 37:4275-86. 2009
    ..We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP...
  17. pmc Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment
    Nuno André Faustino
    Department of Pathology, Baylor College of Medicine, One Baylor Place, Houston, TX 77030, USA
    Mol Cell Biol 25:879-87. 2005
    ..For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins...