Probing the Splicing and Enzymatic Function of Fission Yeast Prp16 A DEAD Box RNA Helicase

Abstract

Nuclear pre-mRNA splicing occurs at precisely conserved sequence elements at and around the splice sites which result in ligation of exons and release of intron as lariat. The Schizosaccharomyces pombe genome is an apt model to study splicing where genes have many short introns per transcript and splicing occurs by intron-definition model. Prp16 (a DExD box RNA helicase) functions are largely unexplored in Schizosaccharomyces pombe. Here, we present functional studies on the essential spprp16+, through our studies on several missense alleles with mutations in the DEAH box motif containing helicase domain. In this study we show functional conservation of C-terminal region by expressing chimeric Prp16 protein (N-terminal from budding yeast ScPrp16 was translationally fused to the C-terminal of SpPrp16) in budding yeast scprp16-2 temperature sensitive recessive mutant. Prior studies done by collaborator in laboratory created two mis-sense alleles spprp16F528S and spprp16G515A mutants in the essential fission yeast spprp16+ gene, characterized their growth and effects on genome-wide splicing and their in vitro dsRNA helicase activities using purified helicase domain of SpPrp16WT, SpPrpF528S and SpPrp16G515A. In this study their ATP hydrolysis activity was analysed. The SpPrp16F528S helicase showed near normal ATPase activity which was comparable to the wild-type while the ATP hydrolysis activity was compromised for SpPrp16G515A mutant. RNA binding ability of the wild-type and SpPrp16G515A helicase proteins was assessed by electro-mobility shift assays using a 47 nucleotide ssRNA substrate. The SpPrp16G515A protein exhibited poor ssRNA binding over a wide range of protein concentrations compared to the wild-type helicase protein. These data suggest that the poor dsRNA unwinding activity by SpPrp16G515A mutant protein could be mainly due to its distinguishably weaker in vitro RNA binding or it may also be augmented by other interactions in the spliceosome. Taking leads from the transcriptome deep sequencing data ba...