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Insights into RNA Splicing and the Regulation of Gene Expression in Saccharomyces cerevisiae

  • Author(s): Gabunilas, Jason
  • Advisor(s): Chanfreau, Guillaume
  • et al.
Abstract

RNA splicing is a critical component in the regulation of gene expression in all eukaryotes. The work described herein chronicles our investigative efforts into three facets of RNA splicing and their associated mechanisms in the model organism Saccharomyces cerevisiae. Previous work from our group highlighted the ability for nonsense-mediated mRNA decay (NMD) to mask the splicing defects of splicing factor mutants, suggesting that the full repertoire of splicing substrates and products is at least partly occluded by RNA surveillance mechanisms. Continuing this work, we sought to uncover previously unidentified splicing events by performing RNA-sequencing in wild-type yeast as well as strains deficient in NMD. This analysis revealed that alternative splicing at unannotated non-canonical 5’- and 3’-splice sites occurs within a large number pre-mRNAs in yeast, but that these events are not usually observed because they introduce premature termination codons (PTCs) into the translational reading frames of the spliced transcripts, thereby rendering them targets for degradation by NMD. This work demonstrated that the degree of alternative splicing in yeast RNA transcripts is greater than previously appreciated, and that alternative splicing linked to NMD (AS-NMD) serves to regulate overall transcript levels. Notably, this study uncovered a non-productive alternative 5’-splice site for the ribosomal protein gene RPL22B that is activated in a stress-dependent manner. We further investigated the splicing of RPL22B and found that its protein product Rpl22p functions in an extra-ribosomal capacity by inhibiting the splicing of its own pre-mRNA, defining an autoregulatory splicing-mediated negative feedback mechanism that fine-tunes the expression of Rpl22p with potential implications in stress response. Finally, we identified a global role for the second-step splicing factor Prp18p in the suppression of non-canonical alternative 3’-splice sites throughout the yeast transcriptome. Specifically, we found that branchpoint-proximal alternative 3’-splice sites are activated in the absence of Prp18p in a substantial fraction of intron-containing genes. These results suggest that Prp18p is responsible for maintaining the fidelity of RNA splicing. Together, these studies reveal new insights into gene regulation by highlighting the interplay between RNA splicing and quality control.

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