Pre-mRNA splicing is an essential component of eukaryotic gene expression. Many metazoans, including humans, regulate alternative splicing patterns to generate expansions of their proteome from a limited number of genes. Importantly, a considerable fraction of human disease causing mutations manifest themselves by altering the sequences that shape the splicing patterns of genes. Nevertheless, the mechanisms by which this complex pathway is regulated remain poorly understood. Understanding how disease-causing mutations impair this ability will require improved knowledge of the mechanisms by which the spliceosome correctly identifies and activates ?cognate? splice site sequences in the background of scores of ?near-cognate? aberrant splice sites, and in the context of changing developmental and environmental cues. At the simplest level, this will require understanding both: (1) the cis-regulatory elements within a transcript (or gene structure) that destine it for regulation; and (2) the mechanistic bases by which trans-regulatory factors can impart this specific regulation. To better understand the mechanisms of alternative pre-mRNA splicing, we have chosen to examine the genetically tractable fission yeast, Schizosaccharomyces pombe. In many ways, splicing in S. pombe looks similar to splicing in higher eukaryotes. Introns have been identified in nearly half of all S. pombe genes, and single genes are interrupted by multiple introns. The splice site sequences found in S. pombe introns do not conform to tight consensus sequences but rather appear much more like human introns in the nucleotide degeneracy found at these positions, a known hallmark of splicing regulation. Building upon our recent demonstrations that S. pombe can catalyze mammalian-like environmentally-regulated alternative splicing, the goals of our current work are to understand the cis-regulatory elements and trans-acting factors that are necessary for this regulation. Toward this end, we will employ high-throughput genetic tools that we have developed to identify the elements within these transcripts that are required for their regulation. Similarly, we will use a forward genetic approach to identify and characterize the splicing factors that are necessary for these regulated events. The combination of these approaches should provide important insights into the mechanisms by which this organism can regulate its gene expression via this pathway. Given the high level of conservation between splicing in S. pombe and humans, this work is likely to provide critical insights into splicing regulation in higher eukaryotes, including its mis-regulation in many human diseases.

Public Health Relevance

Pre-mRNA splicing is an essential component of gene expression for eukaryotic organisms. Recent evidence demonstrates that regulation of pre-mRNA splicing is an important mechanism by which organisms can regulate their gene expression, and that many human diseases involved mutations in this pathway. We are using highly genetically tractable unicellular yeasts to elucidate the mechanisms by which this pathway functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM098634-06
Application #
9384342
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2011-08-01
Project End
2021-07-31
Budget Start
2017-08-15
Budget End
2018-07-31
Support Year
6
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Cornell University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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Yeh, Chung-Shu; Chang, Shang-Lin; Chen, Jui-Hui et al. (2017) The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex. Nucleic Acids Res 45:9679-9693
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Inada, Maki; Nichols, Robert J; Parsa, Jahan-Yar et al. (2016) Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast. Nucleic Acids Res 44:9180-9189
Fair, Benjamin Jung; Pleiss, Jeffrey A (2016) The power of fission: yeast as a tool for understanding complex splicing. Curr Genet :
Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A (2016) Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast. G3 (Bethesda) 6:1513-23
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Awan, Ali R; Manfredo, Amanda; Pleiss, Jeffrey A (2013) Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans. Proc Natl Acad Sci U S A 110:12762-7

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