UCLA

Pre-mRNA splicing is an essential step in expression of eukaryotic genes. In Saccharomyces cerevisiae, pre-mRNA splicing plays an important role in regulating proliferation and the cellular response to changes in nutrient availability. To study how defects in splicing affect cellular viability we performed a genetic screen to identify mutations that enhance growth of the temperature sensitive strain prp2-1, which contains a point mutation in the gene encoding the essential DEAH-box ATPase, Prp2. We identified intragenic mutations within PRP2 that enhance growth of the temperature sensitive allele prp2-1. A majority of these intragenic suppressor mutations were clustered around the ATP binding site of Prp2. The intragenic prp2 mutations identified by the screen also rescued the splicing and rRNA processing defects observed in prp2-1. This work provides insight into the mechanism by which the prp2-1, a commonly used allele to study the function of Prp2 and pre-mRNA splicing, affects RNA processing and cellular viability. Our genetic screen also identified mutations in the stress granule component, Pbp1, which improved the growth of prp2-1, but did not enhance splicing. We find that in addition to decreased production of functional mRNAs, inhibition of splicing in yeast leads to sequestration of Pbp1 in the nucleus during nutrient deprivation and prevention of Pbp1 from localizing to starvation-induced cytoplasmic stress granules. We find that removal of the putative RNA binding domain of Pbp1 prevents sequestration of Pbp1 in the nucleus and restores Pbp1 cytoplasmic condensate formation during nutrient deprivation. Together this work provides insight into the ways defects in pre-mRNA splicing affect cellular function and has implications for the study of human diseases that are impacted by mutations in splicing factors, such as various cancers and neurodegenerative diseases.