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 snRNAs and over 100 protein factors. The five snRNAs (U1, U2, U4, U5, U6) and associated proteins form snRNPs, which play important roles in intron recognition, splice site definition, and the splicing reaction. U5 snRNP is a core spliceosomal component that contains U5 snRNA and three protein factors (Prp8, Brr2, and Snu114) that form a stable core even after chaotropic salt treatment. U5 snRNA interacts with both exons and is critical for aligning the exons for ligation in the second catalytic reaction. Prp8 lies in the heart of the spliceosome and is hypothesized to help form/stabilize the active site or even contribute functional groups to the catalytic reaction. Brr2 is a DExD/H-box protein responsible for U4/U6 unwinding, an essential step for spliceosomal activation. Snu114 is the only GTPase in the spliceosome and is thought to regulate the activity of Brr2. Crystal structures of several domains of Prp8 and Brr2 are known, but there is no structure of any full-length protein or U5 snRNA. It is not clear how proteins in U5 snRNP interact with each other, with U5 snRNA, or with the spliceosome catalytic core. The goal of this proposal is to determine the structure and function of components of the U5 snRNP, as well as their complexes with other proteins and RNAs (including those at the spliceosome catalytic core), using a combination of cutting edge cryo-electron microscopy, crystallography, biochemistry, and yeast genetics approaches. A thorough understanding of the structure and function of U5 snRNP will significantly advance our understanding of the molecular mechanisms of pre-mRNA splicing in general.

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. U5 snRNP contains the U5 snRNA and several spliceosomal proteins that play critical roles in spliceosomal assembly, activation, and the splicing reaction. Studies of the structure and function of the U5 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 #
5R01GM114178-03
Application #
9247839
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
2015-04-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
3
Fiscal Year
2017
Total Cost
$309,103
Indirect Cost
$85,231
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Li, Xueni; Liu, Shiheng; Jiang, Jiansen et al. (2017) CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing. Nat Commun 8:1035
Liu, Shiheng; Li, Xueni; Zhang, Lingdi et al. (2017) Structure of the yeast spliceosomal postcatalytic P complex. Science 358:1278-1283
Espinosa, Sara; Zhang, Lingdi; Li, Xueni et al. (2017) Understanding pre-mRNA splicing through crystallography. Methods 125:55-62