The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

Abstract

Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC– strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC– strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5′ and 3′ directions.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…The great majority of the 21 nt RNAs from both wt and drnB - strains (68,4% and 67,2%, respectively) derive from the retrotransposon element DIRS-1, which also may function as centromeres in D. discoideum [37,38]. This result corroborates our previous finding of large numbers of DIRS-1 small RNAs in D. discoideum wt cells [22,28,39,40]. Furthermore, about equal numbers of 21 nt RNAs from both strains (~ 30%) have their origin in intergenic regions, where genes for most of the previously identified miRNAs resides (see below) [22,28].…”

Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1

“…Overexpressing constructs to monitor 3Ј-spreading activity in the DIRS-1 element were published previously (21). To analyze 5Ј-spreading activity, the DIRS-1 ORFs were cut out from the pJET.1.2/blunt cloning vectors (21) with BamHI and SpeI and ligated in the extrachromosomal vector pDM317 (37) that was previously linearized by BglII and SpeI.…”

“…To analyze 5Ј-spreading activity, the DIRS-1 ORFs were cut out from the pJET.1.2/blunt cloning vectors (21) with BamHI and SpeI and ligated in the extrachromosomal vector pDM317 (37) that was previously linearized by BglII and SpeI. The ORFs were thus fused to an N-terminal GFP tag.…”

“…discoideum is suitable in studying RNA-mediated gene silencing pathways (19) as all RNAi key components, namely the Dicer homologues DrnA and DrnB, the RdRP homologues RrpA to RrpC, and five Argonaute proteins AgnA to AgnE, homologous to the Piwi-clade, are encoded in the genome (20). RrpC is known to be involved in the accumulation of DIRS-1 siRNAs and transposon silencing (21).…”

“…DIRS-1 generates a population of several polyadenylated RNAs that accumulate during development and upon heat shock (26). Recently, a long antisense transcript driven by the right ITR has been identified in the D. discoideum wild type strain and in several mutants (21). Bidirectional transcription and Dicer cleavage of the resulting double-stranded RNAs could, therefore, be the source of primary siRNAs.…”