Retrotransposon profiling of RNA polymerase III initiation sites

Abstract

Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.

In Vitro Targeting of Strand Transfer by the Ty3 Retroelement Integrase

“…Although this activity is interesting in terms of intasome structure-function, it may have minimal in vivo significance. In vivo, integration into Pol III initiation sites clearly dominates (40) and the concentration of MnCl 2 required for IR targeting was significantly greater than the reported physiologic concentration (41).…”

Everyman's Guide to Bacterial Insertion Sequences

“…Another potential interaction, in this case with RNA polymerase, is suggested by the recent observation that the transposon TnGBS (an ICE from Streptococcus agalactiae) and members of the closely related ISLre2 family insert preferentially 15-17 bp upstream of σA promoters (50,51). Targeting of upstream regions of transcription units has also been extensively documented for certain eukaryotic transposons (121).…”

Ty3, a Position-specific Retrotransposon in Budding Yeast

“…One of the striking observations enabled by next generation sequencing of Ty3 insertion sites was that tDNA families, within which sequences are similar or identical, generated widely different numbers of sequencing reads (38). Ty3 could use identical genes at widely differing frequencies because of different chromatin contexts.…”

“…Next generation sequencing enables genomewide assessment of retroelement targeting and use of Ty3 as a probe for RNAP III-transcribed genes, as well as an entrée to additional features of RNAP III activation (38). Next generation sequencing analysis of genomic DNA from cells representing ∼10,000 independent Ty3-HIS3 retrotransposition events identified ∼300 significant insertion sites.…”

“…This impediment was circumvented by introducing a short repeat of the downstream end of POL3, which supplies a second, noncoding, copy of the cis-acting polypurine tract (PPT) and LTR sequences required for replication and integration. A genetic marker, for example, HIS3, can be inserted between these repeats (38). This Ty3 can be expressed from a URA3-marked plasmid so that in a his3 ura3 background, retrotransposed cells can be selected on medium lacking histidine and containing 5-fluoro-uracil, which selects against the URA3-marked plasmid (39) but for the transposed element.…”