UCLA

Using the budding yeast, Saccharomyces cerevisiae as our model system we set out to investigate the role of nonsense-mediated mRNA decay (NMD) pathway in the quality control of premature translation termination codon (PTC) containing transcripts to include those resulting from splicing factor mutations as well as unproductive alternative splicing events. The function of many splicing factors in pre-mRNA splicing and their involvement in the processing of a specific subset of transcripts has often been defined through loss of function analysis. We show that NMD can mask some of the effects of splicing factor mutations, and the full role of the splicing factor cannot be understood unless the RNA degradation system that degrades unspliced precursors are also inactivated. Tiling microarrays showed that inactivation of the NMD factor Upf1p in combination with splicing factor mutants prp17Δ and prp18Δ resulted in a larger spectrum of splicing defects than in the single mutants. Analysis of these double mutants also hinted to the possibility of non-productive alternative splicing. This lead us to seek out and identify splicing defects arising from alternative splice site selection through RNA-Sequencing analysis of wild-type and NMD mutant (upf1Δ, upf2Δ, and upf3Δ) strains. We found that a large fraction of intron containing genes exhibit alternative splicing, but are masked by NMD because they generate PTCs. Analysis of splicing factor mutations combined with upf1Δ revealed the role of specific splicing factors in governing the use of these alternative splice sites. Furthermore, we show the use of a non-productive alternative 5' splice site in RPL22B to be regulated during conditions of stress. These studies bring to light an unexpected flexibility of the spliceosome in splice site selection and the role of NMD in limiting the accumulation of these erroneous transcripts. Finally, we identified a small subset of two intron containing genes whose erroneous transcripts are not targeted to NMD as expected, but rather degraded by the nuclear turnover system. While most S. cerevisiae intron containing genes have only one intron, there are a few numbers of genes that contain two small introns; amongst these are the genes MATa1, DYN2, SUS1, and YOS1. The degradation of the exon2 skipped forms of these transcripts was found to be dependent on the nuclear RNA turnover pathway consisting of the 5' to 3' exonuclease Rat1p and the nuclear exosome. MATa1 was additionally found to be cleaved by the nuclear RNase III endonuclease Rnt1p. These findings show that nuclear degradation mechanisms have also evolved to complement the role of NMD to limit the accumulation of mRNAs that are erroneously spliced by the splicing machinery.