RNA-seq of temperature sensitive alleles of splicing factors in fission yeast

To identify conditional alleles which disrupt the splicing pathway, we screened a collection of ~2000 chemically mutagenized temperature-sensitive fission yeast isolates for intron accumulation in three introns, including a naturally occurring U2-dependent AT-AC intron. We identified 54 strains which accumulate unspliced message at the non-permissive temperature. A combination of mutation effect prediction and genetic mapping techniques revealed missense mutations in core splicing genes which function at various steps of spliceosome assembly and activation including: prp10, prp28, sap61, ntr1, spp42, sap114, cwf22, and prp22. Interrogation of structural data and genome-wide analysis of the splicing defects in these mutations revealed intron-specific defects which suggest the biochemical basis for these mutations' effects on splicing. Overall design: pre-mRNA splicing profiles of various temperature sensitve strains