GENOMIC ANALYSIS OF PRE-MRNA SPLICING MUTANTS IN YEAST

Brandeis University, Waltham MA

Investigators

Abstract

DESCRIPTION The goal of this research is to understand the precise roles of individual splicing factors with respect to all the endogenous introns in yeast. An essential step in eukaryotic gene expression is the precise removal of introns from pre-mRNA. Pre-mRNA splicing requires action of the spliceosome, a complex of 5 snRNAs and over 50 proteins that must assemble anew on each pre-mRNA substrate. Both the mechanism and the machinery of splicing are conserved among eukaryotes, yet the precise function of many spliceosome components has been difficult to elucidate. Most experiments typically follow splicing of one or at most several genes making it difficult to determine whether a given splicing factor is equally required for all introns. Without this knowledge, our understanding of splicing, and hence gene expression, is limited, at best. To address this gap directly, DNA microarrays will be constructed specifically to detect the approximately 220 known and predicted introns and their related exons, as well as all genes presumably lacking introns (approximately 6000) in the yeast S. cerevisiae. RNA from conditional lethal splicing mutants will be examined at timepoints after shift from permissive to restrictive temperature. Various RNA labeling techniques and hybridization procedures will be examined at timepoints after shift from permissive to restrictive temperature. Various RNA labeling techniques and hybridization procedures will be compared to optimize the procedure for quantitative detection of pre-mRNA species. These techniques will be applied to a large collection of splicing mutants, yielding profiles of "expression" for every intron, exon, and complete gene. Such analysis will make it possible to determine if certain splicing factors have gene-specific effects, the features of the RNAs affected, and the consequences on overall gene expression wrought by each splicing mutation.