Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast

Abstract

The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when cotranscriptional. Moreover, we demonstrate that altering the RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimize the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the nonribosomal protein transcripts.[Supplemental material is available for this article.]Splicing is the process of removing introns from precursor messenger RNAs (pre-mRNAs) and joining adjacent exons to produce spliced mRNA. The excised intron, in the form of a branched lariat, is rapidly debranched and discarded. If genes contain multiple introns, alternative splicing pathways can give rise to distinct mRNA and protein isoforms by using alternative splice sites or by including or excluding particular exons. In human cells, ∼95% of transcripts are alternatively spliced, thereby greatly expanding the coding capacity of the genome (Kornblihtt et al. 2013). Moreover, alternative splicing events that introduce translational stop codons in mRNAs are generally coupled with nonsensemediated decay (NMD) to down-regulate that spliced isoform, offering an additional layer of gene expression regulation (Lewis et al. 2003;Sayani et al. 2008;Kawashima et al. 2014). In Saccharomyces cerevisiae (budding yeast) only ∼5% of genes contain an intron, although they produce ∼27% of total mRNA, because many intron-containing genes are highly expressed (Ares et al. 1999). There is extensive evidence that in both metazoans and budding yeast, the process of splicing occurs as soon as the intron is transcribed and before transcription termination, that is, cotranscriptionally (Alexander et al. 2010b;Ameur et al. 2011;Carrillo Oesterreich et al. 2016). As a result of splicing being cotranscriptional, RNA polymerase II (RNAPII) elongation rate can influence splicing. According to one model, referred to as the "kinetic coupling" model, variations in RNAPII elongation rate can alter the time available, or the "window of opportunity" for upstream splice sites to be recognized before competing downstream splice sites are produced (Roberts et al. 1998;de la Mata et al. 2003;Kornblihtt 2007;Naftelberg et al. 2015;Saldi et al. 2016). Consistent with this model, ...

Regulation of yeast RPL22B splicing depends on intact pre-mRNA context of the intron

“…We found that deletion of the ALT5'ss of RPL22Bi impairs splicing efficiency at the major site (Fig2A). In previously published studies, we and others have shown that splicing in vivo uses alternative 5' as well as 3' splice sites, albeit at low frequencies (Abrhámová et al, 2018;Kawashima et al, 2014;Schreiber et al, 2015;Gould et al, 2016;Aslanzadeh et al, 2018). While the alternative site does not lead to a functional product, it may be part of the spliceosome assembly process, e.g., as an adjunct assembly landing platform (Libri et al, 2000;Spingola and Ares, 2000).…”

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes

“…Notably, there is a functional relationship between the transcriptional and the splicing machineries, as evidenced by the role of splicing factors, such as TCERG1, also known as CA150 (Suñ é & Garcia-Blanco, 1999) and SRSF2 (Lin et al, 2008), in stimulating transcriptional elongation. Interestingly, a role for transcription elongation rate influencing splicing fidelity and cotranscriptionality was also observed in yeast (Herzel et al, 2017;Aslanzadeh et al, 2018).…”

“…The co-transcriptional nature of pre-mRNA splicing led to the suggestion that the rate of transcription elongation acts to control AS in mammalian cells (Beyer & Osheim, 1988;Roberts et al, 1998;Pandya-Jones & Black, 2009). Interestingly, a role for transcription elongation rate influencing splicing fidelity and cotranscriptionality was also observed in yeast (Herzel et al, 2017;Aslanzadeh et al, 2018). Interestingly, a role for transcription elongation rate influencing splicing fidelity and cotranscriptionality was also observed in yeast (Herzel et al, 2017;Aslanzadeh et al, 2018).…”

Rapidly evolving protointrons inSaccharomycesgenomes revealed by a hungry spliceosome

“…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.…”

“…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.…”