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A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

ABSTRACT: Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition - one to remove cohesin and the second to target Ty1-IN to chromatin.

SUBMITTER: Ho KL

PROVIDER: S-EPMC4378997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well-understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report a novel intrinsically disordered N-terminal pr ion-like d omain (PrLD) within Gag that is required for transposition. This domain contains amino-acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wildtype to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays, and host modulators developed to study Ty1 retromobility. Our work invites study into the prevalence of PrLDs in additional mobile elements.SignificanceRetrovirus-like retrotransposons help shape the genome evolution of their hosts and replicate within cytoplasmic particles. How their building blocks associate and assemble within the cell is poorly understood. Here, we report a novel pr ion-like d omain (PrLD) in the budding yeast retrotransposon Ty1 Gag protein that builds virus-like particles. The PrLD has similar sequence properties to prions and disordered protein domains that can drive the formation of assemblies that range from liquid to solid. We demonstrate that the Ty1 PrLD can function as a prion and that certain prion sequences can replace the PrLD and support Ty1 transposition. This interchangeable system is an effective platform to study additional disordered sequences in living cells.

Project description:UnlabelledSaccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation.ImportanceRetrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both "host and parasite." To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate retroelement replication.

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A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

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