Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events - which are not understood mechanistically - will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which altered spliceosomal dynamics impact splicing fidelity. Experiments will investigate the contribution of the ATPase Prp5 to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a uniquely simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the mechanism of identification of the branch site nucleophile, which will use an 'orthogonal spliceosome'system in yeast. Finally, using a genetic screen, we are investigating components of the spliceosome that strongly alter the transition from the 1st to 2nd step of splicing. This has led to a new view of the mechanism by which non-consensus splice sites are used, through alteration of the equilibrium between 1st and 2nd step conformations. These studies will help elucidate important features of spliceosome function - as these new mutants in Cef1, Prp8, and U6 snRNA have the feature of strongly altering the function of the spliceosome on mutated splice/branch sites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057829-13
Application #
8111992
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1999-08-01
Project End
2012-07-31
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
13
Fiscal Year
2011
Total Cost
$472,690
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Kosmyna, Brian; Query, Charles C (2016) Structural biology: Catalytic spliceosome captured. Nature 537:175-176
Tang, Qing; Rodriguez-Santiago, Susana; Wang, Jing et al. (2016) SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing. Genes Dev 30:2710-2723
Wu, Guowei; Adachi, Hironori; Ge, Junhui et al. (2016) Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly. EMBO J 35:654-67
Chen, Weijun; Shulha, Hennady P; Ashar-Patel, Ami et al. (2014) Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes. RNA 20:308-20
Basak, Anindita; Query, Charles C (2014) A pseudouridine residue in the spliceosome core is part of the filamentous growth program in yeast. Cell Rep 8:966-73
Yang, Fei; Wang, Xiu-Ye; Zhang, Zhi-Min et al. (2013) Splicing proofreading at 5' splice sites by ATPase Prp28p. Nucleic Acids Res 41:4660-70
Query, Charles C; Konarska, Maria M (2013) Structural biology: Spliceosome's core exposed. Nature 493:615-6
Shao, Wei; Kim, Hyun-Soo; Cao, Yang et al. (2012) A U1-U2 snRNP interaction network during intron definition. Mol Cell Biol 32:470-8
Query, Charles C; Konarska, Maria M (2012) CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome. RNA 18:1001-13
Trcek, Tatjana; Larson, Daniel R; Moldon, Alberto et al. (2011) Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell 147:1484-97

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