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 based global splicing profile in spprp16+ and spprp16F528S strains (Drisya V., IISc; Pushpendra Singh Bawa, IBAB), in this study we carried out experimental analyses to investigate the contribution of splice site and snRNA interactions towards determining the dependence of a fission yeast intron on SpPrp16 for efficient splicing. Test were carried out to assess if the weakening of 5’SS and U6 snRNA interaction can render the Prp16 dependent intron to be spliced in spliceosomes with mutant helicase SpPrp16F528S. We observe the poor splicing of mini transcript of one such candidate seb1intron1 in cells of the spprp16F528S mutant was partially rescued with 5’SS mutation in this intron. Thus, this data supports the bioinformatic prediction. We also analysed if complementarity between BS and U2 snRNA could be an additional intronic determinant contributing to SpPrp16 requirement for splicing. The splicing of mini-gene transcript with wild type BS or two other mini-genes mutated either at the BS −4 or at the −3 position of a candidate intron tif313 intron2 BP were examined where it was found that U2 snRNA interactions of the −4 residue of tif313+ I2 BS is pivotal as mini-transcript with BS mutant -4 residue was spliced efficiently in spprp16F528S cells. These data suggest global splicing dependence on Prp16 is dictated by the strength of splice site-snRNA interactions for individual introns in cellular transcripts. Taken together in vitro enzymatic studies together with the studies of in vivo splicing efficiencies of various intronic transcripts hints at the involvement of some SpPrp16 aminoacid residues in indispensable spliceosomal interactions and in other instances splicing status and efficiency is possibly related to its intrinsic enzymatic activity.