An RNA polymerase III subunit determines sites of retrotransposon integration

Abstract

Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host.

Ty1 Integrase Interacts with RNA Polymerase III-specific Subcomplexes to Promote Insertion of Ty1 Elements Upstream of Polymerase (Pol) III-transcribed Genes

“…Because the cell lysis procedure disrupts the nuclear and cytoplasmic compartments, we do not think the lack of interaction is due to IN-NLS1ϩ2 being excluded from the nucleus, although we cannot entirely rule out this possibility. However, when combined with the Ty1-IN truncation analysis, our data suggest that the C-terminal 75 amino acids of Ty1-IN interact with RNA Pol III, which concurs with a recent studying showing a two-hybrid interaction between Rpc40 and Ty1-IN that was abrogated by removal of the C-terminal amino acids 578 -635 (24). Although BridierNahmias et al (24) found that Ty1-IN amino acids 578 -635 were also sufficient to interact with Rpc40 using a two-hybrid assay, we were unable to detect an interaction of a C-terminal Ty1-IN fragment (amino acids 422-636; IN-B) with Rpc82 or Rpc53 by co-IP (Fig.…”

“…A standard 2-h comparative PCR analysis was performed using a 7500 Real Time PCR System (Applied Biosystems). The primers for the tLEU, tLEU de novo, and tGLY genes have been described (24). qPCR analysis was performed using the comparative C T method (⌬⌬C T method, Applied Biosystems) with TAF10 used as an internal control.…”

“…This exciting new discovery provides significant insight into the mechanism of Ty1 element insertion. However, despite the lack of interaction between S. pombe AC40 and Ty1-IN, overall Ty1 mobility was not affected, suggesting that other factors could still mediate Ty1 element insertion (24).…”

“…Recently, a yeast two-hybrid screen using the RNA Pol III AC40 subunit as bait identified an interaction with Ty1-IN (24). Replacing the S. cerevisiae AC40 subunit with Schizosaccharomyces pombe AC40 disrupted the interaction with Ty1-IN and redirected Ty1 element insertion to telomeric and subtelomeric regions (24). This exciting new discovery provides significant insight into the mechanism of Ty1 element insertion.…”

“…However, despite the fact that separase mutants have reduced and cohesin mutants have increased Ty1 insertion frequency, no change in targeting specificity was demonstrated (23). Recently, a yeast two-hybrid screen using the RNA Pol III AC40 subunit as bait identified an interaction with Ty1-IN (24). Replacing the S. cerevisiae AC40 subunit with Schizosaccharomyces pombe AC40 disrupted the interaction with Ty1-IN and redirected Ty1 element insertion to telomeric and subtelomeric regions (24).…”