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
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Wang, Wenhua; Maucuer, Alexandre; Gupta, Ankit et al. (2013) Structure of phosphorylated SF1 bound to U2AFýýýýýý in an essential splicing factor complex. Structure 21:197-208
Bauer, William J; Heath, Jason; Jenkins, Jermaine L et al. (2012) Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1. J Mol Biol 415:727-40
Gupta, Ankit; Jenkins, Jermaine L; Kielkopf, Clara L (2011) RNA induces conformational changes in the SF1/U2AF65 splicing factor complex. J Mol Biol 405:1128-38
McLaughlin, Krystle J; Jenkins, Jermaine L; Kielkopf, Clara L (2011) Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins. Biochemistry 50:1429-31
Gupta, Ankit; Kielkopf, Clara L (2011) Purification, crystallization and preliminary X-ray crystallographic analysis of a central domain of human splicing factor 1. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:486-90
Manceau, Valerie; Kielkopf, Clara L; Sobel, Andre et al. (2008) Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors. J Mol Biol 381:748-62
Jenkins, Jermaine L; Shen, Haihong; Green, Michael R et al. (2008) Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65. J Biol Chem 283:33641-9
Lin, Yuan; Kielkopf, Clara L (2008) X-ray structures of U2 snRNA-branchpoint duplexes containing conserved pseudouridines. Biochemistry 47:5503-14
Kumar, Amit O; Swenson, Matthew C; Benning, Matthew M et al. (2008) Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution. Biochem Biophys Res Commun 367:813-9

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