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The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human–yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach.

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease., Author Summary Cancer results from mutations that alter the function of normal genes. The results of these mutations directly lead to the known properties of cancer cells, for example, over-proliferation or resistance to cellular death signals. An additional property of most tumors is chromosome instability (CIN)—the unequal distribution of DNA to the daughter cells following cell division. While CIN appears to be important for the formation of many tumors, the mutations that can lead to CIN are not well understood. Here we used a simple model cell, budding yeast, to systematically identify genes whose mutation can lead to CIN. Our data identify previously uncharacterized CIN genes that we show play a role in the stability of important cellular signaling proteins. Moreover, our results directly predict human CIN candidate genes, hundreds of which are mutated in tumors. Together our data create a resource for understanding new chromosome biology and the genetic basis of CIN in human cancers.