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 contributions of the U1-U2 protein-interaction network and the ATPase Prp5 to intron definition and to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a unique commitment to spliceosome assembly and a simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the binding site of the 3'splice site substrate for the second step of splicing, which will use an 'orthogonal spliceosome'system in yeast, and on the mechanism by which the second-step substrate replaces first-step product in the catalytic core. Finally, we are investigating the role of novel RNA-RNA interactions in the transition from the first-to-second step of splicing. Together, the models proposed in these aims will greatly improve our understanding of the dynamic range of RNA-RNA interactions at consecutive stages of splicing.

Public Health Relevance

PUBLIC HEALTH RELEVANCE STATEMENT Excision of introns from pre-mRNA is critical in the pathway of gene expression. This excision occurs within spliceosomes and requires precise recognition - during both spliceosome assembly and splicing catalysis - of three sites (two splice sites and a branch site) to appropriately join exonic coding sequences. Mechanisms by which spliceosomes recognize the exact sites and catalyze the reactions are not well understood. Mutations in components of the U1-U2 snRNP interaction network during spliceosome assembly are implicated in myelodysplastic syndrome (MDS). Our studies will provide a better understanding of interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057829-15
Application #
8518358
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1999-08-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
15
Fiscal Year
2013
Total Cost
$561,613
Indirect Cost
$225,318
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
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
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
Smith, Duncan J; Konarska, Maria M; Query, Charles C (2009) Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Mol Cell 34:333-43
Smith, Duncan J; Query, Charles C; Konarska, Maria M (2008) "Nought may endure but mutability": spliceosome dynamics and the regulation of splicing. Mol Cell 30:657-66
Liu, Li; Query, Charles C; Konarska, Maria M (2007) Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat Struct Mol Biol 14:519-26
Xu, Yong-Zhen; Query, Charles C (2007) Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly. Mol Cell 28:838-49

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