Extensive splicing across the Saccharomyces cerevisiae genome

Abstract

Pre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression. Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome components and its catalytic activity. Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes. Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae. Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes. By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage. Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions. Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae, thus expanding our view of the regulatory potential of RNA splicing in yeast.

Rapidly evolving protointrons inSaccharomycesgenomes revealed by a hungry spliceosome

“…A splicing index relating change in the ratio of junction/intron reads over time (SJ index = log2[junctions-t60/intron-t60] -log2[junctions-t0/intron-t0]) increases for most introns in transcripts whose total transcript levels change less than two-fold during the experiment (Fig 1A, blue circles, Table S1, NB: RPGs are repressed >2 fold and are excluded). In addition to the standard introns, we observe more than 600 splicing events that result from use of alternative 3' or 5' splice sites overlapping the standard introns (see also the accompanying study by Douglass et al [43]). Some of these splicing events have been previously characterized [38,41,42], and produce out of frame mRNAs that are more easily detected in the upf1Δ strain due to the loss of NMD (Table S2).…”

“…Here we characterize a class of splicing events in yeast we call protointrons. Many previous studies have noted "novel" introns in yeast under a variety of experimental conditions and genetic backgrounds [37][38][39][40][41][42][43][44]57]. Here we distinguish protointrons by several criteria, most importantly that they reside at locations not overlapping known standard introns.…”

“…We suggest that the small subset of more efficiently spliced protointrons may be advanced in the process of becoming clade-or species-specific standard introns.This intermediate class of introns tend to occur in 5'UTRs where they might buffer the negative effects of RNA secondary structure or micro-ORFs (uORFs) on translation, however the adaptive value of any of these emerging introns remains to be determined. This and the accompanying study [43] reveal the extent to which the spliceosome recognizes and splices intron-like sequences, thus expanding the information contained in the genome. This contribution of the spliceosome to the information content of…”

“…Some of these events appear to be activated in response to stress [37], which down-regulates RPG expression and creates hungry spliceosome conditions. Still others are more readily detected during meiosis [37,44], or when cells are deleted for RNA decay pathway components that degrade unstable transcripts [37,38,43]. Our interest in understanding the evolution of splicing prompted us to focus on these distinct new introns.…”

“…These protointrons are found in mRNAs and noncoding RNAs such as promoter-associated transcripts, CUTs, SUTs, XUTs and other noncoding RNAs [28][29][30][31][32][33][34][35][36]. Other studies have found undocumented splicing events [37][38][39][40][41][42][43][44], including alternative splicing of known introns. Here we discover, collect, and reclassify splicing events into two main categories, standard introns and protointrons, based on efficiency, conservation, location, and relevance to extant gene expression, in order to understand intron creation.Related yeasts S. bayanus and S. mikatae also have protointrons, but most are species-specific, indicating that protointrons appear and disappear during evolution.…”