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.

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.

Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.

The amoeba Dictyostelium discoideum is a well-established model organism for studying numerous aspects of cellular and developmental functions. Its rather small (~34Mb) chromosomal genome and the high efficiency of gene disruption by homologous recombination have enabled researchers to dissect various specific gene functions. We describe here the use of one-step cloning for the fast and efficient generation of deletion vectors that are produced in a one-step reaction by inserting two PCR products into an organism-specific, generic acceptor system. This worked efficiently for all 16 tested constructs directed against genes in the amoeba Dictyostelium discoideum. Saving cost and time, the used protocol represents a significant advancement in the generation of such plasmids compared to the conventionally applied restriction enzyme/ligation approach. Using appropriate selection markers, similar systems could also be useful in other organisms, where genes can be knocked out by homologous recombination.

The 5' spreading of small RNAs in Dictyostelium discoideum depends on the RNA-dependent RNA polymerase RrpC and on the dicer-related nuclease DrnB.

RNA interference (RNAi) is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs) are required for RNAi. To study the function of the three RdRPs in the amoeba Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced ß-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of ß-gal small interfering RNAs (siRNAs) could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured ß-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5' of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.