Exonuclease domain mutants of yeast DIS3 display genome instability.

The exosome functions to regulate the cellular transcriptome through RNA biogenesis, surveillance, and decay. Mutations in Dis3, a catalytic subunit of the RNA exosome with separable endonuclease and exonuclease activities, are linked to multiple myeloma. Here we report that a cancer-associated DIS3 allele, dis3E729K, provides evidence for DIS3 functioning in mitotic fidelity in yeast. This dis3E729K allele does not induce defects in 7S→5.8S rRNA processing, although it elicits a requirement for P-body function. While it does not significantly influence cell cycle progression alone, the allele reduces the efficiency of cell cycle arrest in strains with defects in kinetochore assembly. Finally, point mutations in the exonuclease domains of yeast Dis3 elicit genome instability phenotypes; however, these DIS3 mutations do not increase DNA damage or RNA processing defects that lead to the accumulation of polyadenylated RNA in the nucleus. These data suggest that specific DIS3 activities support mitotic fidelity in yeast.

Selective defects in gene expression control genome instability in yeast splicing mutants.

RNA processing mutants have been broadly implicated in genome stability, but mechanistic links are often unclear. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. In both cases, alterations in gene expression, rather than direct cis effects, are likely to contribute to instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, differing penetrance and selective effects on the transcriptome can lead to a range of phenotypes in conditional mutants of the spliceosome, including multiple routes to genome instability.

Selective aggregation of the splicing factor Hsh155 suppresses splicing upon genotoxic stress.

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery.

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.