Role of RNA Polymerase III Transcription Factors in the Selection of Integration Sites by the Dictyostelium Non-Long Terminal Repeat Retrotransposon TRE5-A

Abstract

In the compact Dictyostelium discoideum genome, non-long terminal repeat (non-LTR) retrotransposons known as TREs avoid accidental integration-mediated gene disruption by targeting the vicinity of tRNA genes. In this study we provide the first evidence that proteins of a non-LTR retrotransposon interact with a target-specific transcription factor to direct its integration. We applied an in vivo selection system that allows for the isolation of natural TRE5-A integrations into a known genomic location upstream of tRNA genes. TRE5-A frequently modified the integration site in a way characteristic of other non-LTR retrotransposons by adding nontemplated extra nucleotides and generating small and extended target site deletions. Mutations within the B-box promoter of the targeted tRNA genes interfered with both the in vitro binding of RNA polymerase III transcription factor TFIIIC and the ability of TRE5-A to target these genes. An isolated B box was sufficient to enhance TRE5-A integration in the absence of a surrounding tRNA gene. The RNA polymerase III-transcribed ribosomal 5S gene recruits TFIIIC in a B-box-independent manner, yet it was readily targeted by TRE5-A in our assay. These results suggest a direct role of an RNA polymerase III transcription factor in the targeting process.

Protein Interactions Involved in tRNA Gene-Specific Integration of Dictyostelium discoideum Non-Long Terminal Repeat Retrotransposon TRE5-A

“…TRE5-A is the first non-LTR retrotransposon that has been shown to use direct protein contacts with chromatin constituents for integration site selection. Recent experiments have revealed a role for DdTFIIIC in the targeting of genes transcribed by RNA Pol III (i.e., tRNA genes and the ribosomal 5S gene) by TRE5-A (40). Here, we provide direct evidence that DdTFIIIB is the target of TRE5-A ORF1p, which suggests that the role of DdTFIIIC in tRNA gene targeting by TRE5-A is to recruit DdTFIIIB.…”

“…We have shown recently that integration of TRE5-A upstream of D. discoideum tRNA genes requires a functional tRNA gene promoter, suggesting active involvement of DdTFIIIC in the targeting process (40). Since DdTFIIIB is predicted to cover the 5Ј end of a tRNA gene close to the natural integration site of TRE5-A, we proposed that TFIIIC may be required only to recruit DdTFIIIB.…”

“…discoideum tRNA genes are scattered on all chromosomes and do not show significant DNA sequence conservation in their flanking regions (11). Thus, we hypothesized that protein interactions between a TRE5-A-encoded protein and an RNA polymerase III (Pol III)-specific transcription factor may be responsible for the recognition of tRNA genes as integration sites (40). We have recently shown that targeting of tRNA genes by TRE5-A requires an active binding site-the B box-for the Pol III transcription factor IIIC (TFIIIC) (40).…”

“…Thus, we hypothesized that protein interactions between a TRE5-A-encoded protein and an RNA polymerase III (Pol III)-specific transcription factor may be responsible for the recognition of tRNA genes as integration sites (40). We have recently shown that targeting of tRNA genes by TRE5-A requires an active binding site-the B box-for the Pol III transcription factor IIIC (TFIIIC) (40). The ribosomal 5S gene, which is a typical Pol III gene, is also targeted by TRE5-A under experimental conditions (40).…”

“…We have recently shown that targeting of tRNA genes by TRE5-A requires an active binding site-the B box-for the Pol III transcription factor IIIC (TFIIIC) (40). The ribosomal 5S gene, which is a typical Pol III gene, is also targeted by TRE5-A under experimental conditions (40). Although the 5S gene lacks a B box promoter, its transcription does also depend on TFIIIC.…”