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.

The RNA exosome is a conserved multiprotein complex that achieves a large number of processive and degradative functions in eukaryotic cells. Recently, mutations have been mapped to the gene encoding one of the subunits of the exosome, EXOSC3 (yeast Rrp40p), which results in pontocerebellar hypoplasia with motor neuron degeneration in human patients. However, the molecular impact of these mutations in the pathology of these diseases is not well understood. To investigate the molecular consequences of mutations in EXOSC3 that lead to neurological diseases, we analyzed the effect of three of the mutations that affect conserved residues of EXOSC3/Rrp40p (G31A, G191C, and W238R; G8A, G148C, and W195R, respectively, in human and yeast) in S. cerevisiae. We show that the severity of the phenotypes of these mutations in yeast correlate with that of the disease in human patients, with the W195R mutant showing the strongest growth and RNA processing phenotypes. Furthermore, we show that these mutations affect more severely pre-ribosomal RNA processing functions of the exosome rather than other nuclear processing or surveillance functions. These results suggest that delayed or defective pre-rRNA processing might be the primary defect responsible for the pathologies detected in patients with mutations affecting EXOSC3 function in residues conserved throughout eukaryotes.

RNA polymerase II (Pol II) transcription termination by the Nrd1p-Nab3p-Sen1p (NNS) pathway is critical for the production of stable noncoding RNAs and the control of pervasive transcription in Saccharomyces cerevisiae. To uncover determinants of NNS termination, we mapped the 3 ′ -ends of NNS-terminated transcripts genome-wide. We found that nucleosomes and specific DNA-binding proteins, including the general regulatory factors (GRFs) Reb1p, Rap1p, and Abf1p, and Pol III transcription factors enhance the efficiency of NNS termination by physically blocking Pol II progression. The same DNAbound factors that promote NNS termination were shown previously to define the 3 ′ -ends of Okazaki fragments synthesized by Pol δ during DNA replication. Reduced binding of these factors results in defective NNS termination and Pol II readthrough. Furthermore, inactivating NNS enables Pol II elongation through these roadblocks, demonstrating that effective Pol II termination depends on a synergy between the NNS machinery and obstacles in chromatin. Consistent with this finding, loci exhibiting Pol II readthrough at GRF binding sites are depleted for upstream NNS signals. Overall, these results underscore how RNA termination signals influence the behavior of Pol II at chromatin obstacles, and establish that common genomic elements define boundaries for both DNA and RNA synthesis machineries.

In Saccharomyces cerevisiae, splicing is critical for expression of ribosomal protein genes (RPGs), which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the RPL22B pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p in vivo and splicing inhibition in vivo and in vitro and can rescue splicing inhibition in vitro when added in trans to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the RPL22B locus. We further demonstrate that the inhibition of RPL22B pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis.

Proteins of the translational apparatus, including ribosomal proteins, release factors, and elongation factors, are known to be modified by a large number of methyltransferases in eukaryotic cells. The majority of these methylation reactions occurs at lysine residues. To date, 12 protein lysine methyltransferase involved in these reactions (Efm1–7; Rkm 1–5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, 5 appear to be specific to elongation factor 1 alpha (EF1A)(Efm1, Efm4–7). Each methyltransferase modifies a specific lysine residue to give mono‐, di‐, and tri‐methyllysine products. Two of these enzymes (Efm4 and Efm5) are highly conserved in nature while the others appear to be fungal specific. In S. cerevisiae the functional implications of lysine methylation are mostly unknown. EF1A's primary function, with its GTP cofactor, is to deliver amino‐acyl tRNA to the A site of the ribosome during translation. Once the correct tRNA is positioned, a conformational change results in the hydrolysis of GTP and its release from the ribosome for another cycle. Studies have also shown the involvement of EF1A in the assembly of ribosomes. Using yeast genetics we have assessed the biological relevance of EF1A lysine methylation on its translational roles using two approaches. The first approach uses site directed mutagenesis to mutate four of the most conserved methylated lysine sites to arginine residues. The second approach involves combinatorial knockouts of the genes that code for each of the five methyltransferases. In each approach, we have then used functional translation assays in yeast to evaluate the physiological impact of the loss of EF1A methylations. We have found that inactivation of multiple methyltransferases can cause a significant reduction of translational fidelity. Additionally, we have shown that the ribosomal assembly function of EF1A does not appear to be affected by the loss of methyltransferase activity. These studies should illuminate why one protein appears to need five different methyltransferases for its functions. Support or Funding Information This work is funded by the National Science Foundation Grant MCB‐1714569 and NSF‐LSAMP Bridge to Doctorate Fellowship. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.