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. 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 spliceosomal dynamics impact splicing fidelity. Experiments will first investigate binding and positioning of the 3'SS-UAG onto the spliceosome. Binding of the spliceosome to the 3'SS is critical for intron definition, for spliceosome assembly, and for splicing catalysis. Yet, nothing is known of spliceosome?3'SS-UAG interaction, other than the early interaction with U2AF. Here we use an `orthogonal spliceosome' (second-copy, reverse-engineered, designer spliceosome) that we have developed in yeast, to identify both the 3'SS binding site for second-step catalysis and a `loading site' for 3'SS on the assembling spliceosome. Second, two large gaps in our understanding of RNA biology are the identification of RNAs between 50 and 200 nts, which are missing in almost all modern-day sequencing datasets, and the bioinformatic analysis of repetitive sequences ? the snRNAs represent both. We have identified novel U2 snRNA variants that are expressed differentially in cells, and we will investigate the components, function, and substrates of novel U2-variant spliceosomes.

Public Health Relevance

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 U2 snRNP are implicated in myelodysplastic syndrome (MDS), and mutations that alter snRNP levels underlie spinal muscular atrophy (SMA). Our studies will provide a better understanding of U2 snRNPs and their interactions with 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 #
5R01GM057829-20
Application #
9487339
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1999-08-01
Project End
2021-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
20
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine, Inc
Department
Type
DUNS #
079783367
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
Query, Charles C; Konarska, Maria M (2013) Structural biology: Spliceosome's core exposed. Nature 493:615-6
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
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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