Functional Genomics Reveals Relationships Between the Retrovirus-Like Ty1 Element and Its Host Saccharomyces cerevisiae

Abstract

Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription

“…Indeed, when we inactivated NUP84 in the original strain transformed with an integrative plasmid carrying the Ty1-his3AI reporter [49,87], we quantified increased retrotransposition levels, in agreement with our data obtained with the chromosomal reporter (S5A and S5B Fig). However, our comparative assays with alternative reporters [48][49][50][51][52] generally indicated that the increased mobility scored in nup mutants (S5A and S5D Fig) was all the less pronounced that the reporter expression and transposition frequency were elevated in WT cells (S5E Fig). This is exemplified by Ty1 reporters under the control of the strong GAL1 promoter [48,51], with which Nup84 complex mutants confirmedly displayed lower transposition frequencies (S5D Fig).…”

“…However, our comparative assays with alternative reporters [48][49][50][51][52] generally indicated that the increased mobility scored in nup mutants (S5A and S5D Fig) was all the less pronounced that the reporter expression and transposition frequency were elevated in WT cells (S5E Fig). This is exemplified by Ty1 reporters under the control of the strong GAL1 promoter [48,51], with which Nup84 complex mutants confirmedly displayed lower transposition frequencies (S5D Fig). This could similarly account for the unchanged Ty1 mobility previously scored in nup mutants using a centromeric plasmid-borne Ty1 reporter [52], which led to higher retrotransposition rates in WT cells as compared to our assay performed at 25˚C (S5F Fig) . In this view, the slight increase in retrotransposition that we scored in nup mutants carrying this reporter (S5C Fig) is not inconsistent with the previously described transposition frequencies [52].…”

“…When we further investigated the mechanism involved in this regulation, we highlighted that the Nup84 complex restricts Ty1 transcription through SUMOylation-dependent processes and not by modulating nuclear transport (Fig 3). We further scored an increase in Ty1 mobility in Nup84 complex mutants (Fig 4), in contrast with previous reports using alternative retrotransposition reporters [48][49][50][51][52] (S5 Fig) . In particular, one of these earlier studies identified the nup84Δ mutant in a large-scale screen based on the observation of a decreased number of His + papillae arising from retrotransposition events [49]. Yet, this phenotype was not validated in quantitative assays, a critical issue for such poorly-growing mutants [31,86].…”

“…Multiple systematic studies for regulators of the replication of LTR-retroelements in budding yeast had previously uncovered several NPC components [48][49][50]53], identifying in particular a post-transcriptional role for the nuclear basket [52]. In this report, we have focused on the contribution of a unique core sub-complex of the NPC, the Nup84 complex, to the expression and the retrotransposition of endogenous Ty elements.…”

“…In addition, dozens of cellular factors identified as regulators of Ty1 replication still await mechanistic characterization (reviewed in [47]). Among them, subunits of the Nup84 complex were previously scored as necessary for Ty1 retrotransposition in genomewide screens [48][49][50], as further validated for the Nup84 subunit [51]. In contrast, a systematic analysis of nucleoporin mutants did not confirm this requirement of the Nup84 complex for Ty1 replication using a distinct reporter system [52].…”