Skip to main content
eScholarship
Open Access Publications from the University of California

UC San Diego

UC San Diego Electronic Theses and Dissertations bannerUC San Diego

Splice Site Recognition During Early Spliceosome Assembly

Abstract

Protein coding sequences in most eukaryotic pre-messenger RNAs (pre-mRNA) are interrupted by intervening sequences called introns, while the protein coding sequences are termed exons. The process by which introns are removed, and exons are joined, generating a processed mRNA is called splicing, catalyzed by the spliceosome enzyme. Within pre-mRNA the spliceosome distinguishes exon and intron boundaries, also referred to as splice sites (SS). In addition to SS, there are two essential, but highly degenerate sequence elements within an intron, a branch site (BS) and a poly-pyrimidine tract (PPT) sequence, both near the 3’SS. Lacking such highly conserved features in pre-mRNAs, how the splicing machinery recognizes authentic SS amongst several similar sequences remains unclear. In the first step of spliceosome assembly U1 snRNP recognizes the 5’SS, while the BS, PPT and 3’SS are recognized by their respective partners, SF1, U2AF65 and U2AF35. However, each factor interacts with its partner RNA sequence in vitro with low affinity, suggesting that early pre-mRNA substrate recognition must be more complex. The serine-arginine (SR) protein family is thought to facilitate early spliceosome assembly, but no systematic analysis on SR protein mediated early spliceosome assembly has been done. Using in vitro binding experiments, I found that the interaction between U1 snRNP and pre-mRNA through the 5’SS is assisted by the presence of the PPT, suggesting both the 5’SS and 3’SS are jointly recognized very early in assembly. I further show that an SR protein can support U1 snRNP binding in certain splicing substrates but not all.

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View