The role of non-canonical intronic motifs in splicing

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

Pre-mRNA splicing is a critical component of gene expression in eukaryotic organisms and understanding how splice sites are recognized is vital to deciphering the regulation and mechanism of this fundamental process. It has become apparent that alternative pre-mRNA splicing plays an important role in increasing the diversity of the proteome in higher eukaryotes. The majority of alternative splicing is regulated at the level of intron recognition. Introns are defined by multiple RNA elements, some of which reside in the intron and some of which reside in flanking exons. The known intronic elements are the 5' splice site, the branchpoint sequence, polypyrimidine (PY) tract and 3' splice site; the latter three elements are usually found in the last 30-40 nucleotides of the intron. The goals of this project are two-fold, (1) determine the mechanisms through which introns that lack a canonical PY tract are recognized and spliced and (2) what is the role of U2AF65, the PY tract binding protein, in the recognition and splicing of these introns lacking a canonical PY tract. Bioinformatics work has revealed three classes of intronic motifs that may function as intronic splicing enhancers (ISEs) that could compensate in the splicing of introns lacking the canonical PY tract. In vivo and in vitro splicing assays are being used to test the hypothesis that these putative ISEs function to compensate for the noncanonical PY tract. Preliminary data indicates that two of the three ISEs function as splicing enhancers and trans-acting factors will be identified using cross-linking and mass spectrometry. Mutant U2AF65 molecules have been created that retain the ability to make the necessary protein-protein interactions for intron recognition but have weakened RNA binding to test the hypothesis that RNA binding of U2AF65 to non-canonical PY tracts is not necessary for intron recognition and splicing. Broader Impacts: Most human genes are more intron than exon, yet the majority of intron sequences serve no known purpose. An interesting possibility is that many introns harbor a variety of regulatory elements that dictate when splicing will take place and which potential splice sites will be used. This study focuses on novel intronic sequence motifs, and will provide information about the relatively unexplored sequence diversity of introns. The bioinformatics work has lead to the creation of a webtool termed the Intron Motif Finder (introns.uoregon.edu), which allows researchers without programming skills to manipulate large sequence databases to easily identify introns containing motifs of interest. This tool is also being used in the classroom to teach undergraduate students how to begin performing bioinformatics analysis. For example, a class of 45 biochemistry students read a paper describing how the splicing factor NOVA regulates splicing through intronic UCAY motifs, the students then went on to use the Intron Motif Finder to identify potentially new exons that NOVA might regulate by searching for introns with multiple UCAY motifs downstream and upstream of exons.