Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNPs (U1, U2, U4, U5, U6) and many non-snRNP related protein factors. Despite the recent determination of multiple high resolution cryo EM structures of spliceosome complexes at later stages of the splicing pathway, there is a lack of structural and mechanistic understanding of the initial intron recognition and early spliceosome assembly events. U1 snRNP is critical for the initial recognition of 5' splice site (ss). Due to its important role in 5' ss recognition, U1 snRNP is a frequent target of the action of alternative splicing factors that either facilitate or prevent U1 snRNP from binding to 5' ss. Much of what we know today about the molecular mechanism and regulation of 5' ss recognition comes from genetic, biochemical, and structural studies of two commonly used model systems, S. cerevisiae (yeast) and human U1 snRNP. Intriguingly, the yeast U1 snRNP is much more complex than the human U1 snRNP. Yeast U1 snRNA contains a 3.5-fold larger RNA and seven additional proteins, most of which have human homologs that are weakly associated with U1 snRNP and are involved in alternative splicing. We have recently determined the cryoEM structure of yeast U1 snRNP to 3.6 resolution, generating many interesting hypotheses on how human alternative splicing factors recruit U1 snRNP and the unexpected role of human PrpF39 in alternative splicing. We will test these hypotheses using a combination of biochemistry and molecular biology approaches. We will also decipher the molecular mechanism of concerted recognition of all intron elements through a combination of structural biology and biochemical approaches. Results from this project will significantly advance our understanding of the molecular mechanism of intron definition and alternative splicing.

Public Health Relevance

Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. U1 snRNP is critical for the 5' splice site (ss) recognition and is the frequent target of the action of alternative splicing factors that either facilitate or prevent U1 snRNP from binding to the 5' ss. Studies of the structure and function of the U1 snRNP will lead to a better understanding of the molecular mechanisms of pre-mRNA splicing, which has the potential to provide useful approaches for human disease therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM126157-02
Application #
9751902
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Bender, Michael T
Project Start
2018-08-01
Project End
2022-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045