Interactions in Yeast Pre-mRNA Splicing

University Of New Mexico Health Sciences Center, Albuquerque NM

Investigators

Abstract

Gene expression in nearly all eukaryotes requires the excision of introns from nuclear precursor messenger RNAs (pre-mRNAs) to produce functional mRNAs. The introns are excised by the spliceosome, which is a very large, macromolecular complex composed of five small nuclear ribonucleoproteins (snRNPs) and many proteins. Each snRNP is itself composed of a small nuclear RNA (snRNA) and several proteins. A fundamental question regarding pre-mRNA splicing is how the active catalytic site of the spliceosome is formed. The focus of this project is on interactions between two spliceosomal components (Snp1p and Prp8p) that may contribute to the formation and activation of the pre-catalytic spliceosome. The yeast Saccharomyces cerevisiae is an ideal organism for these studies because genetics and biochemistry can be used in tandem to study dynamic and transient interactions. An interaction between Snp1p (a protein of the U1 snRNP) and Prp8p (a protein of the U5 snRNP), which was discovered in the previous grant period, is hypothesized to be important for association of the U1 and U5 snRNPs in the spliceosome. It may help dock the U5 snRNP onto the developing spliceosome, and promote rearrangements leading to formation of the spliceosome's catalytic site. One such rearrangement is the 5' splice site (SS) switch when the pre-mRNA's 5' SS switches from pairing with U1 snRNA to U6 snRNA. Preliminary results suggest that the Prp8/Snp1 interaction is important in destabilizing the U1/5'SS duplex as part of this switch. Mutations or inhibitors will be assayed to correlate functional changes with physical changes in the association of Prp8p with Snp1p. To map the region(s) of Prp8p required for interaction with Snp1p, a library of random prp8 mutations will be screened for those altering the interaction via a novel, high-throughput assay. Candidate mutations will be further assessed by a combination of in vivo and in vitro assays. A notable percentage of human genetic diseases including some types of retinitis and thalassemias are due to defects in pre-mRNA splicing. Furthermore, alternative splicing contributes to the development and life cycles of many organisms as well as to biodiversity. Thus understanding the basic mechanism of splicing will contribute to our understanding of several biological systems and, in the long-term, contribute to improving human health. The proposed research will also make several contributions to other areas of science and society. The development of a high-throughput assay will be a collaborative effort using instrumentation invented at the host institution. Results generated by this assay will be disseminated both by traditional scientific publication and by a website specifically designed as a database for yeast spliceosomal components and their interactions. The research will be conducted at an EPSCoR and designated minority institution. Thus it is likely that some of the students participating in the proposed research will be from minority groups.