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 #
5R01GM070503-09
Application #
8103948
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2004-07-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
9
Fiscal Year
2011
Total Cost
$378,914
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Kielkopf, Clara L (2017) Insights from structures of cancer-relevant pre-mRNA splicing factors. Curr Opin Genet Dev 48:57-66
Jenkins, Jermaine L; Kielkopf, Clara L (2017) Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures. Trends Genet 33:336-348
Glasser, Eliezra; Agrawal, Anant A; Jenkins, Jermaine L et al. (2017) Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs. Biochemistry 56:4757-4761
Chatrikhi, Rakesh; Wang, Wenhua; Gupta, Ankit et al. (2016) SF1 Phosphorylation Enhances Specific Binding to U2AF65 and Reduces Binding to 3'-Splice-Site RNA. Biophys J 111:2570-2586
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
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
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
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
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
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

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