Abstract
Retrotransposons are mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. Saccharomyces cerevisiae has been invaluable to retrotransposon research due to the presence of an active retroelement known as Ty1. The retromobility of Ty1 is regulated both positively and negatively by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. Paradoxically, previous studies have implicated the nuclear Mediator complex in the regulation of post-translational steps in Ty1 retromobility. To attempt to resolve this apparent paradox, we have examined the effects of deleting non-essential Mediator subunits on the various stages of Ty1 retromobility. The Mediator core complex is organized into genetically and structurally defined head, middle, and tail modules, along with a transiently associated kinase module. We show that with the exception of the kinase module, deleting Mediator subunits has a major impact on Ty1 mobility. Disrupting the Mediator tail module decreases Ty1 activity to undetectable levels, while disrupting the head or middle module increases Ty1 retromobility substantially. These major changes in retromobility are accompanied by insignificant differences in Ty1 RNA or Gag protein levels in Mediator mutants relative to the wild-type strain. Decreased retromobility in tail module gene deletion mutants requires the Ty1 promoter in the 5′ LTR and is correlated with increased expression of an internal transcript known as Ty1i, which encodes a dominant negative inhibitor of Ty1 retromobility. We present evidence that Mediator preferentially associates with the Ty1 or Ty1i promoters in strains lacking specific Mediator head or tail subunits, respectively, indicating that Mediator controls Ty1 retromobility by governing transcription start site selection within Ty1 elements. This work elucidates a mechanism of host control of retrotransposon activity via promoter competition and provides mechanistic insight into transcriptional regulation of Ty1i RNA.
Author Summary Retrotransposons are mobile genetic elements that copy their RNA genomes into DNA and insert the DNA copies into the host genome. These elements contribute to genome instability, control of host gene expression and adaptation to changing environments. Retrotransposons depend on numerous host factors for their own propagation and control. The retrovirus-like retrotransposon, Ty1, in the yeast Saccharomyces cerevisiae has been an invaluable model for retrotransposon research, and hundreds of host factors that regulate Ty1 retrotransposition have been identified. Non-essential subunits of the Mediator transcriptional co-activator complex have been identified as one set of host factors implicated in Ty1 regulation. Here, we report a systematic investigation of the effects of loss of these non-essential subunits of Mediator on Ty1 retrotransposition. Our findings reveal a heretofore unknown mechanism by which Mediator influences the choice of transcription start site (TSS) in Ty1 to modulate expression of an autoinhibitory transcript known as Ty1i RNA. Our results provide new insights into host control of retrotransposon activity via TSS selection and elucidate a novel mechanism by which different promoters compete for the Mediator co-activator.