MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATION

Summary

Principal Investigator: Klemens J Hertel
Abstract: DESCRIPTION (provided by applicant): With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 90% of human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. This renewal application focuses on understanding the mechanisms of splice site selection with the long-term goal to generate a splicing code that permits alternative splicing predictions based on sequence analysis. The experiments outlined in this application build on the most exciting discoveries made during the previous funding period;charting highly significant correlations between alternative splicing and cis-acting RNA splicing elements, the discovery of a mechanism that describes position-dependent activities of splicing regulators, and the discovery that the retention of spliceosomal components along ligated exons ensures efficient processing. The proposal has three major goals: 1) To investigate experimentally and computationally the complexity of exon selection. A systems approach is proposed to examine methodically how the combination of cis-acting RNA splicing elements influences splice site selection with the objective to generate a splicing code that permits alternative splicing predictions based on sequence analysis. 2) To test the hypothesis that position dependent splicing activation or repression is due to unique interactions between splicing regulatory complexes and spliceosomal components. 3) To determine the mechanisms that ensure highly efficiency processing of multi-intron containing pre-mRNAs. Because defects in splicing lead to many human genetic diseases, including a number of genes that have been implicated in cancer, the proposed research will greatly enhance our understanding of regulated gene expression and human disease. In particular, the results from these studies will provide a quantitative framework for exon recognition and they will permit splicing predictions to classify exonic mutations, thus paving the way for the design of alternative therapeutic approaches to combat disease.
Funding Period: 2001-02-01 - 2014-06-30
more information: NIH RePORT

Top Publications

  1. pmc Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns
    Martin J Hicks
    Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, USA
    PLoS Biol 4:e147. 2006
  2. pmc G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site
    Erming Wang
    Department of Neurology, University of Kentucky, Lexington, Kentucky 40536, USA
    J Biol Chem 286:4059-71. 2011
  3. pmc Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, University of California at Irvine, Irvine, California 92697, USA
    RNA 17:761-72. 2011
  4. pmc Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Nucleic Acids Res 39:8928-37. 2011
  5. pmc Evolution of SR protein and hnRNP splicing regulatory factors
    Anke Busch
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Wiley Interdiscip Rev RNA 3:1-12. 2012
  6. pmc Extensive regulation of NAGNAG alternative splicing: new tricks for the spliceosome?
    Anke Busch
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697 4025, USA
    Genome Biol 13:143. 2012
  7. pmc HEXEvent: a database of Human EXon splicing Events
    Anke Busch
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
    Nucleic Acids Res 41:D118-24. 2013
  8. pmc Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms
    Steffen Erkelenz
    Institute of Virology, Heinrich Heine University, D 40225 Dusseldorf, Germany
    RNA 19:96-102. 2013
  9. ncbi Preparation of splicing competent nuclear extracts
    Chiu Ho T Webb
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    Methods Mol Biol 1126:117-21. 2014
  10. ncbi In vitro assay of pre-mRNA splicing in mammalian nuclear extract
    Maliheh Movassat
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    Methods Mol Biol 1126:151-60. 2014

Research Grants

Detail Information

Publications23

  1. pmc Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns
    Martin J Hicks
    Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, USA
    PLoS Biol 4:e147. 2006
    ..We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs...
  2. pmc G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site
    Erming Wang
    Department of Neurology, University of Kentucky, Lexington, Kentucky 40536, USA
    J Biol Chem 286:4059-71. 2011
    ..The data suggest that different mechanisms underlie G run-mediated recognition of 5' splice sites and that context and position play a critical role...
  3. pmc Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, University of California at Irvine, Irvine, California 92697, USA
    RNA 17:761-72. 2011
    ..Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation...
  4. pmc Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Nucleic Acids Res 39:8928-37. 2011
    ....
  5. pmc Evolution of SR protein and hnRNP splicing regulatory factors
    Anke Busch
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Wiley Interdiscip Rev RNA 3:1-12. 2012
    ....
  6. pmc Extensive regulation of NAGNAG alternative splicing: new tricks for the spliceosome?
    Anke Busch
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697 4025, USA
    Genome Biol 13:143. 2012
    ..A recent study using massive parallel sequencing demonstrates unequivocally that alternative tandem acceptor splicing is tissue-specifically regulated...
  7. pmc HEXEvent: a database of Human EXon splicing Events
    Anke Busch
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
    Nucleic Acids Res 41:D118-24. 2013
    ..Importantly, the generated data sets are downloadable for further analysis...
  8. pmc Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms
    Steffen Erkelenz
    Institute of Virology, Heinrich Heine University, D 40225 Dusseldorf, Germany
    RNA 19:96-102. 2013
    ..Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection...
  9. ncbi Preparation of splicing competent nuclear extracts
    Chiu Ho T Webb
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    Methods Mol Biol 1126:117-21. 2014
    ..Nuclear extracts derived from HeLa cells provide all of the obligatory components to carry out intron removal in vitro. This chapter describes the large-scale preparation of nuclear extract from HeLa cells. ..
  10. ncbi In vitro assay of pre-mRNA splicing in mammalian nuclear extract
    Maliheh Movassat
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    Methods Mol Biol 1126:151-60. 2014
    ..Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis. ..
  11. pmc Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns
    Tara L Crabb
    Department of Microbiology and Molecular Genetics, University of California at Irvine, Irvine, California 92697 4025, USA
    RNA 16:1786-96. 2010
    ..Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites...
  12. pmc Competing upstream 5' splice sites enhance the rate of proximal splicing
    Martin J Hicks
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Mol Cell Biol 30:1878-86. 2010
    ..These results reveal the discovery of a new splicing regulatory element, an upstream 5' splice site functioning as a splicing enhancer...
  13. pmc Restoration of full-length SMN promoted by adenoviral vectors expressing RNA antisense oligonucleotides embedded in U7 snRNAs
    Till Geib
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, Irvine, California, United States of America
    PLoS ONE 4:e8204. 2009
    ..It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping...
  14. pmc Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site
    Yimeng Dou
    Department of Information and Computer Sciences, University of California, Irvine, Irvine, California 92697 4025, USA
    RNA 12:2047-56. 2006
    ..This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity...
  15. pmc The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicing
    Kathryn S Sciabica
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
    Nucleic Acids Res 34:6612-20. 2006
    ..Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the 'all or none' splicing switch that is required during Drosophila sexual differentiation...
  16. ncbi Combinatorial control of exon recognition
    Klemens J Hertel
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
    J Biol Chem 283:1211-5. 2008
    ..The relative contributions of each of these parameters control how efficiently splice sites are recognized and flanking introns are removed...
  17. pmc Conserved RNA secondary structures promote alternative splicing
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, Institute for Genomics and Bioinformatics, University of California at Irvine, Irvine, California 92697 4025, USA
    RNA 14:1463-9. 2008
    ..Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease...
  18. pmc Spliceosome assembly pathways for different types of alternative splicing converge during commitment to splice site pairing in the A complex
    Matthew V Kotlajich
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697 4025, USA
    Mol Cell Biol 29:1072-82. 2009
    ..These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point...
  19. pmc Splice-site pairing is an intrinsically high fidelity process
    Kristi L Fox-Walsh
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697 4025, USA
    Proc Natl Acad Sci U S A 106:1766-71. 2009
    ..We conclude that all multi-intron-containing genes are alternatively spliced and that the reduction of SMN results in a general splicing defect that is mediated through alterations in the fidelity of splice-site pairing...
  20. pmc Spliceosomes walk the line: splicing errors and their impact on cellular function
    Shu Ning Hsu
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    RNA Biol 6:526-30. 2009
    ..Here we review recent progress made in understanding the extent of alternative splicing within the human genome with particular emphasis on splicing fidelity...
  21. pmc The SR protein family
    Peter J Shepard
    Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697 4025, USA
    Genome Biol 10:242. 2009
    ..These activities point up the importance of SR proteins during the regulation of mRNA metabolism...
  22. pmc Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly
    Sissada Tannukit
    Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033 1004, USA
    Biochem Biophys Res Commun 390:1044-50. 2009
    ..These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function...
  23. ncbi Kinetic analysis of in vitro pre-mRNA splicing in HeLa nuclear extract
    William F Mueller
    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
    Methods Mol Biol 1126:161-8. 2014
    ..This chapter will describe the rationale and approach employed to use kinetic analysis to evaluate an in vitro splicing reaction using radiolabeled pre-mRNA incubated in splicing-competent HeLa nuclear extract (NE). ..

Research Grants30

  1. Mechanisms of Signal-Induced Alternative Splicing: CD45
    Kristen W Lynch; Fiscal Year: 2013
    ....
  2. Development and Evaluation of Dual Compartment Combination Microbicides
    ROBERT WALTER BUCKHEIT; Fiscal Year: 2013
    ..Through our industrial partnership with ImQuest Pharmaceuticals our methodology and products will have an immediate outlet to further clinical development. ..
  3. Spliceosome Mechanism Dissected at the Single Molecule Level
    Nils G Walter; Fiscal Year: 2013
    ..Taken together, these advances will pave the way for, over the funding period, extensive mechanistic studies of yeast splicing and for studying alternative splicing in humans in the longer term. ..
  4. Regulation of Ovarian Cancer Multidrug Resistance by MAP Kinases
    Scott T Eblen; Fiscal Year: 2013
    ..Elucidation of this synergistic mechanism of drug resistance would provide important information for treating drug-resistant ovarian cancer. ..
  5. SMN Control of snRNP Biogenesis: Role in RNA Splicing and Motor Neuron Survival
    Livio Pellizzoni; Fiscal Year: 2013
    ..These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development. ..
  6. The Role of Caspase 9b Expression in NSCLC Survival/Chemotherapeutic Sensitivity
    Charles E Chalfant; Fiscal Year: 2013
    ..g. cisplatinum and paclitaxel). ..
  7. SPLICING OF MRNA PRECURSORS
    Michael R Green; Fiscal Year: 2013
    ....
  8. BIOCHEMISTRY OF PRE-MRNA SPLICING
    Adrian R Krainer; Fiscal Year: 2013
    ..abstract_text> ..
  9. HORMONAL REGULATION OF BLOOD PRESSURE
    Michal Laniado Schwartzman; Fiscal Year: 2013
    ..ular tone, in the pathophysiology of hypertension and cardiovascular disease. ..
  10. Alternative pre-mRNA Splicing in Drosophila
    Donald C Rio; Fiscal Year: 2013
    ....
  11. STEM CELL GENE THERAPY FOR HEMOGLOBINOPATHIES
    George Stamatoyannopoulos; Fiscal Year: 2013
    ..The focus of this Program Project, Gene Therapy, can provide a new paradigm for the treatment of these hemoglobinopathies as well as for other blood diseases. (End of Abstract) ..
  12. Vertebrate Non-Coding RNPs: Integrating function, biogenesis and survival
    Joan A Steitz; Fiscal Year: 2013
    ..Together, this understanding will contribute to the development of therapeutic approaches to combat multiple genetic diseases that derive from faulty RNA processing and surveillance, as well as cancer. ..
  13. Functional RNA elements in the human genome
    EUGENE WEI MING YEO; Fiscal Year: 2013
    ..This research has the potential to fundamentally change our view on splicing control and its contribution to human disease. ..
  14. Electron Microscopy of Biological Macromolecules
    Kenneth H Downing; Fiscal Year: 2013
    ....