A Quantitative, High-Throughput Reverse Genetic Screen Reveals Novel Connections between Pre–mRNA Splicing and 5′ and 3′ End Transcript Determinants

Abstract

Here we present the development and implementation of a genome-wide reverse genetic screen in the budding yeast, Saccharomyces cerevisiae, that couples high-throughput strain growth, robotic RNA isolation and cDNA synthesis, and quantitative PCR to allow for a robust determination of the level of nearly any cellular RNA in the background of 5,500 different mutants. As an initial test of this approach, we sought to identify the full complement of factors that impact pre–mRNA splicing. Increasing lines of evidence suggest a relationship between pre–mRNA splicing and other cellular pathways including chromatin remodeling, transcription, and 3′ end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. Moreover, it is unclear whether all pathways that are coupled to splicing have been identified. As expected, our approach sensitively detects pre–mRNA accumulation in the vast majority of strains containing mutations in known splicing factors. Remarkably, however, several additional candidates were found to cause increases in pre–mRNA levels similar to that seen for canonical splicing mutants, none of which had previously been implicated in the splicing pathway. Instead, several of these factors have been previously implicated to play roles in chromatin remodeling, 3′ end processing, and other novel categories. Further analysis of these factors using splicing-sensitive microarrays confirms that deletion of Bdf1, a factor that links transcription initiation and chromatin remodeling, leads to a global splicing defect, providing evidence for a novel connection between pre–mRNA splicing and this component of the SWR1 complex. By contrast, mutations in 3′ end processing factors such as Cft2 and Yth1 also result in pre–mRNA splicing defects, although only for a subset of transcripts, suggesting that spliceosome assembly in S. cerevisiae may more closely resemble mammalian models of exon-definition. More broadly, our work demonstrates the capacity of this approach to identify novel regulators of various cellular RNAs.

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

“…The splicing defects we observe are similar in magnitude to those observed when other histone modifications are perturbed. [27][28][29][30]64 In addition, gene-specific splicing defects in budding yeast have been described previously [12][13][14] and could possibly be a result of features of the gene or intron such as the length of the gene, and/or its transcription frequency. Nevertheless, we do observe a trend of decreased splicing efficiency upon removal of Set2, as compared to the wild-type strain ( Fig.…”

“…The intron-containing genes we analyzed included DBP2, TEF5, and RPS21B, which have previously been analyzed in studies investigating co-transcriptional splicing. 14,[27][28][29]64 Yeast were grown at the permissive temperature prior to RNA extraction and the splicing mutant snu66D served as a positive control.…”

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

“…These studies invoked conditional Slu7 functions based on BrPto-3=ss distances, but global substrates are not known in either species (12). While transcriptome analyses of S. pombe grown under varied conditions have provided extensive information on regulated gene expression (47,52), genome-wide transcript isoform analyses have been used to deduce global splicing substrates for only spprp2 ϩ , the U2AF 59 homolog (34). This study found a range of splicing deficiencies on inactivation of this essential factor and, surprisingly, revealed that features other than the 3= Pyn tract confer efficient splicing of specific introns on spprp2-1 inactivation (34).…”

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

“…The efficiency of meiotic intron removal is either regulated or governed by the actions of splicing factors or RNA modifying enzymes that are inessential for vegetative growth (e.g., Mer1, Nam8, Tgs1, Cbc2) (Engebrecht et al 1991;Spingola and Ares 2000;Munding et al 2010;Qiu et al 2011aQiu et al ,b,c, 2012. In addition, comparative genome-wide profiling of vegetative RNA splicing in wild-type and mutant strains deleted for inessential splicing factors, or in conditional mutants of essential factors after shift to restrictive temperature, has fortified the theme that yeast splicing factors are differentially dedicated to splice particular pre-mRNA "clients" (Pleiss et al 2007;Albulescu et al 2012). …”