Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. It is known that this process requires a splicing machine (spliceosome) composed of ~100 proteins and 5 small nuclear (sn)RNAs. The early stages of spliceosome assembly on pre-mRNA splice sites are key regulated steps that often go awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. Hence, the overall goal of this proposal is to understand the sequential three- dimensional interactions that guide 3'splice site selection in the early stages of spliceosome activation. A complex of the essential splicing factors U2AF and SF1 recognizes pre-mRNA sequences adjacent the 3'splice site, and in turn stabilizes association of the core spliceosome. Pre-mRNA contacts by an arginine-serine (RS) region of U2AF, and the U2AF-associated ATPase UAP56, are required to accomplish this task.
Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) By what means does U2AF recognize diverse splice sites? (2) By what means does SF1 enhance splice site recognition by U2AF? (3) By what means does U2AF recruit UAP56, and in turn, what is the action of UAP56 on U2AF at the splice site? (4) By what means does an RS region promote association of spliceosomal snRNAs with the pre-mRNA? We have already made significant advances towards these aims by (i) determining three- dimensional structures of U2AF bound to splice sites, (ii) evaluating thermodynamic and structural contributions of SF1 domains to U2AF binding, and (iii) characterizing RNA interactions by RS domains. These studies will significantly advance our understanding of this fundamental step of gene expression.

Public Health Relevance

Errors in pre-mRNA splicing contribute to major human diseases, including cancers, leukemias, myotonic dystrophies, neurological and metabolic disorders. The investigation of normal splice site recognition to be gained by the proposed work would serve as a basis for understanding, and in the long term developing treatments against, harmful splice variants of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070503-10
Application #
8296694
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2004-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
10
Fiscal Year
2012
Total Cost
$366,434
Indirect Cost
$129,260
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Loerch, Sarah; Kielkopf, Clara L (2016) Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise. RNA 22:1795-1807
Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh et al. (2016) Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival. PLoS Genet 12:e1006384
Agrawal, Anant A; Salsi, Enea; Chatrikhi, Rakesh et al. (2016) An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal. Nat Commun 7:10950
Loerch, Sarah; Kielkopf, Clara L (2015) Dividing and Conquering the Family of RNA Recognition Motifs: A Representative Case Based on hnRNP L. J Mol Biol 427:2997-3000
Okeyo-Owuor, T; White, B S; Chatrikhi, R et al. (2015) U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing. Leukemia 29:909-17
Agrawal, Anant A; McLaughlin, Krystle J; Jenkins, Jermaine L et al. (2014) Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA. Proc Natl Acad Sci U S A 111:17420-5
Loerch, Sarah; Maucuer, Alexandre; Manceau, Valérie et al. (2014) Cancer-relevant splicing factor CAPERα engages the essential splicing factor SF3b155 in a specific ternary complex. J Biol Chem 289:17325-37
Jenkins, Jermaine L; Agrawal, Anant A; Gupta, Ankit et al. (2013) U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs. Nucleic Acids Res 41:3859-73
Wang, Wenhua; Maucuer, Alexandre; Gupta, Ankit et al. (2013) Structure of phosphorylated SF1 bound to U2AF⁶⁵ in an essential splicing factor complex. Structure 21:197-208
Jenkins, Jermaine L; Laird, Kholiswa M; Kielkopf, Clara L (2012) A Broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF(65). Biochemistry 51:5223-5

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