Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations

Abstract

Genetic approaches in Saccharomyces cereviasine have identified 38 genes required for efficient RNA splicing. The majority have been found by screening (high) temperature-sensitive (ts) mutants for those defective in splicing, an approach limited by the presence of ts hotspots and by the fact that many essential genes rarely mutate to the ts phenotype. To identify novel genes, we screened a collection of 340 cold-sensitive (cs) mutants for those that exhibited diminished splicing of several pre-mRNAs. We isolated 12 mutants in nine complementation groups. Four of these affected known genes (PRP8, PRP16, PRP22, PRP28), three of which encode RNA helicase homologues. Five genes are novel (BRR1, BRR2, BRR3, BRR4, BRR5; Bad Response to Refrigeration); mutations in these genes inhibited splicing before the first chemical step of the reaction. Analysis of BBR2 revealed it to encode an essential member of a new class of RNA helicase-like proteins that includes the yeast antiviral protein SKi2. These data validate the use of cs mutants in genetic screens and raise the possibility that RNA helicase family members are particularly prone to mutation to cold sensitivity.