Mechanisms Regulating Alternative pre-mRNA Splicing

Summary

Principal Investigator: James Patton
Abstract: Most eukaryotic genes are interrupted by non-coding introns that must be removed from pre-mRNA transcripts for the production of functional proteins. The splicing of introns is remarkably efficient and rapid, an amazing feat considering that the signals that delineate introns from exons are not conserved in higher eukaryotes. A corollary of the importance of splicing in normal cell function is that approximately 15% of characterized genetic diseases involve mutations that cause defects in splicing. Further, 60% or more of human genes are subject to alternative splicing requiring that cells not only be able to accurately recognize the difference between introns and exons, but also between alternatively spliced exons. We have been studying the regulation of alternative splicing focusing on the molecular mechanisms by which specific splice sites are activated or repressed. In the first part of this proposal, experiments are designed to understand the regulation of the mutually exclusive exons 2 and 3 of the alpha- tropomyosin gene. Dissection of the cis-acting sequences needed to control splicing have identified elements flanking exon 3 that are needed to repress splicing in smooth muscle cells. PTB (hnRNP I) binds to one of these elements but the identity of factors that bind to a directly adjacent, conserved UGC repeat remains unknown. Biochemical purification is proposed to identify this factor and determine how it participates with PTB to repress splicing. In contrast to repression of exon 3, activation of exon 2 is needed in smooth muscle cells mediated by four purine-rich enhancer elements and one or more members of the SR protein family of splicing factors. In vivo and in vitro splicing assays will be used to identify which SR proteins are essential for exon 2 activation. In the second part of this proposal, regulation of splicing by SR proteins will be expanded to include characterization of a new SR-related protein (SRrp86) that can activate or repress specific SR family members. The specific targets of SRrp86 are unknown but preliminary data suggests that it functions by protein-protein interaction. Experiments are proposed to determine how such interaction alters SR protein activity and to identify specific SR protein targets. Overall, the experiments in this proposal seek to understand the mechanistic basis underlying the repression and activation of splicing by SR proteins and hnRNP proteins.
Funding Period: 2002-08-01 - 2009-01-31
more information: NIH RePORT

Top Publications

  1. ncbi siRNA therapeutics: big potential from small RNAs
    R C C Ryther
    Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
    Gene Ther 12:5-11. 2005
  2. pmc Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F
    J Barrett Crawford
    Department of Biological Sciences, Box 1820 Station B, Vanderbilt University, Nashville, TN 37235, USA
    Mol Cell Biol 26:8791-802. 2006
  3. ncbi The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs
    Rui Peng
    Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA
    RNA Biol 3:69-76. 2006
  4. ncbi Splicing fidelity, enhancers, and disease
    Amanda S Solis
    Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
    Front Biosci 13:1926-42. 2008
  5. pmc Growth hormone deficiency and splicing fidelity: two serine/arginine-rich proteins, ASF/SF2 and SC35, act antagonistically
    Amanda S Solis
    Department of Biological Sciences, Vanderbilt University, 2301 Vanderbilt Pl, Nashville, TN 37235, USA
    J Biol Chem 283:23619-26. 2008

Detail Information

Publications5

  1. ncbi siRNA therapeutics: big potential from small RNAs
    R C C Ryther
    Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
    Gene Ther 12:5-11. 2005
    ..While much work remains to optimize delivery and maintain specificity, the therapeutic advantages of siRNAs for treatment of viral infection, dominant disorders, cancer, and neurological disorders show great promise...
  2. pmc Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F
    J Barrett Crawford
    Department of Biological Sciences, Box 1820 Station B, Vanderbilt University, Nashville, TN 37235, USA
    Mol Cell Biol 26:8791-802. 2006
    ..These data suggest that the activation of exon 2 is dependent on the antagonistic activities of 9G8 and hnRNPs H and F...
  3. ncbi The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs
    Rui Peng
    Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA
    RNA Biol 3:69-76. 2006
    ..Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components...
  4. ncbi Splicing fidelity, enhancers, and disease
    Amanda S Solis
    Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
    Front Biosci 13:1926-42. 2008
    ..This review provides an overview of the splicing reaction and mechanisms of alternative splicing and provides examples of enhancer defects that cause disease...
  5. pmc Growth hormone deficiency and splicing fidelity: two serine/arginine-rich proteins, ASF/SF2 and SC35, act antagonistically
    Amanda S Solis
    Department of Biological Sciences, Vanderbilt University, 2301 Vanderbilt Pl, Nashville, TN 37235, USA
    J Biol Chem 283:23619-26. 2008
    ..Although the precedent for SR proteins acting as repressors is established, this is the first example of a patient mutation that creates a site through which an SR protein represses splicing...