Widespread use of non-productive alternative splice sites in Saccharomyces cerevisiae.

Tadashi Kawashima,Guillaume F Chanfreau,Stephen Douglass,Jason Gabunilas,Matteo Pellegrini,Hiten D Madhani

doi:10.1371/journal.pgen.1004249

Tadashi Kawashima, Guillaume F Chanfreau + Show 4 more

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https://doi.org/10.1371/journal.pgen.1004249

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Journal: PLoS Genetics	Publication Date: Apr 10, 2014
Citations: 127	License type: CC BY 4.0

Affiliation: University of California, Los Angeles

Abstract

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

Highlights

Nonsense-mediated mRNA decay (NMD) is an RNA degradation system that degrades RNAs containing premature termination codons [1,2]
The yeast Saccharomyces cerevisiae has long been used as a model system to investigate the mechanisms of pre-mRNA splicing, as many components of the splicing machinery were identified through genetic screens in S. cerevisiae [14], and most splicing factors are highly conserved from yeast to mammalian cells [15]
Alternative splicing events were detected more frequently in RNA samples obtained from the NMD mutants (Fig. 1A and 1B; Table S2), consistent with the fact that most of these alternative splicing events result in the introduction of a premature termination codons (PTC), either by inducing a translational frameshift or by inserting an intronic PTC-containing sequence (Table S2)

Summary

Introduction

Nonsense-mediated mRNA decay (NMD) is an RNA degradation system that degrades RNAs containing premature termination codons [1,2]. In mammalian cells and higher eukaryotes, NMD can be used to regulate gene expression, for instance by reducing the level of alternatively spliced isoforms containing premature termination codons [3,4,5,6,7,8]. This interplay between alternative splicing and NMD is involved in the autoregulation of SR proteins [3,4,5]. Recent work analyzing alternative splicing across fungal species has shown that S. cerevisiae has lost some of the alternative splicing events through gene duplication and sub-functionalization of the duplicated genes, which are otherwise produced by alternative splicing in other species [20]

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