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.

Public Health Relevance

Alternative pre-mRNA splicing is considered one of the most efficient and widespread avenues to generate multiple protein isoforms from individual genes. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how they influence each other mediate exon inclusion or exclusion. In this application we propose to use a systems approach to define a splicing code that predicts the probability of exons to be recognized by the spliceosome and to determine the mechanisms that ensure efficient processing of multi-intron pre-mRNAs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062287-12
Application #
8294676
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2001-02-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
12
Fiscal Year
2012
Total Cost
$291,941
Indirect Cost
$89,932
Name
University of California Irvine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Movassat, Maliheh; Mueller, William F; Hertel, Klemens J (2014) In vitro assay of pre-mRNA splicing in mammalian nuclear extract. Methods Mol Biol 1126:151-60
Webb, Chiu-Ho T; Hertel, Klemens J (2014) Preparation of splicing competent nuclear extracts. Methods Mol Biol 1126:117-21
Mueller, William F; Hertel, Klemens J (2014) Kinetic analysis of in vitro pre-mRNA splicing in HeLa nuclear extract. Methods Mol Biol 1126:161-8
Erkelenz, Steffen; Mueller, William F; Evans, Melanie S et al. (2013) Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms. RNA 19:96-102
Busch, Anke; Hertel, Klemens J (2013) HEXEvent: a database of Human EXon splicing Events. Nucleic Acids Res 41:D118-24
Busch, Anke; Hertel, Klemens J (2012) Evolution of SR protein and hnRNP splicing regulatory factors. Wiley Interdiscip Rev RNA 3:1-12
Shepard, Peter J; Choi, Eun-A; Busch, Anke et al. (2011) Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites. Nucleic Acids Res 39:8928-37
Shepard, Peter J; Choi, Eun-A; Lu, Jente et al. (2011) Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17:761-72
Wang, Erming; Mueller, William F; Hertel, Klemens J et al. (2011) 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. J Biol Chem 286:4059-71
Hicks, Martin J; Mueller, William F; Shepard, Peter J et al. (2010) Competing upstream 5' splice sites enhance the rate of proximal splicing. Mol Cell Biol 30:1878-86

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