Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites

Abstract

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5′ flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.

A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes

“…Therefore, our results suggest that the cDNA might not be necessary for IN1 recruitment at Pol III‐transcribed genes. A recent study reached a similar conclusion for the recruitment of Ty3 integrase at tRNA genes (Patterson et al , ).…”

The Ty1 integrase nuclear localization signal is necessary and sufficient for retrotransposon targeting to tRNA genes

“…Therefore, our results indicate that the cDNA is not necessary for IN1 recruitment at Pol III. A recent study reached a similar conclusion for the recruitment of Ty3 integrase at tDNA genes (Patterson et al, 2019).…”

“…Very weak or no HA-IN1 binding was observed for RNA170 and ZOD1, previously shown to have low level of Pol III occupancy (Moqtaderi and Struhl, 2004) ( Figure 3C and Table S1). Three tDNAs, tK(CUU)C, tM(CAU)C, and tD(GUC)N, were not recovered: These genes are either absent or transcriptionally inactive in the laboratory strain we used (Kumar and Bhargava, 2013;Patterson et al, 2019). HA-IN1 was absent from most Pol II-transcribed genes (Table S1) or present at a much lower level than at Pol III-transcribed genes ( Figure 3C).…”

Diverse transposable element landscapes in pathogenic and nonpathogenic yeast models: the value of a comparative perspective

“…2) [71,72]. Nearly all newly integrated copies of Ty3 also insert upstream of RNA Polymerase III-transcribed genes, with the great majority occurring 0-20 bp upstream of the 5′ ends of tRNA genes in the vicinity of the transcription start site [73][74][75][76]. There are no functional copies of Ty5 in the reference S. cerevisiae genome, but a functional Ty5 element from S. paradoxus has been shown to preferentially target heterochromatin in subtelomeric regions and near the HML and HMR silent mating loci (Fig.…”

“…This interaction provides the initial localization of IN and cDNA to heterochromatin, but the specific site of insertion then appears to depend on the presence of nucleosome-free regions that are accessible for integration [79]. Ty3 integrates 0-20 bp upstream of tRNA genes, near their transcription start sites [73][74][75][76], which is dependent on the transcriptional competence of the RNA Pol III promoter [74,75]. Recapitulation of targeted insertions in vitro using VLPs, an RNA Pol III target gene, and purified RNA Pol III transcription factors shows that integration depends on the Pol III transcription factors TFIIIB and TFIIIC [176].…”