The 5′ Spreading of Small RNAs in Dictyostelium discoideum Depends on the RNA-Dependent RNA Polymerase RrpC and on the Dicer-Related Nuclease DrnB

Abstract

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 β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-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 β-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.

A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms

“…Based on these strong opposing promoters of a genetic element that is naturally silenced by RNAi, we have established a convenient gene silencing system in extrachromosomal vectors which is easier to use and apparently more reliable than the previously described antisense and hairpin constructs [ 21 , 23 ]. We have tested seven genes and find RNAi effects on all of them: the generation of siRNAs, reduction on the mRNA and the protein level and expected phenotypes and combinations of all of the above wherever it was examined.…”

“…Knock-down strategies that may circumvent these problems have previously been reported using antisense [ 20 ] or hairpin constructs [ 21 – 23 ]. Driving sense and antisense RNA from opposing promoters has also been employed with variable and mostly unsatisfactory results.…”

G17-modified hammerhead ribozymes are active in vitro and in vivo

“…To analyze the in vivo performance of the different motifs, we made use of a recently established β-galactosidase (β-Gal) reporter system in the amoeba Dictyostelium discoideum (Wiegand and Hammann 2013), in which we cloned a HHRz cassette between the stop codon and the poly(A) signal sequences of the lacZ gene (Fig. 6A,B), similar to a reporter described earlier (Khvorova et al 2003).…”

“…The primers of these PCR reactions are listed in Supplemental Table S1 and add XhoI sites at the 5 ′ and 3 ′ ends of the fragment for subsequent cloning in expression vector pDneo2a lacZ (Wiegand and Hammann 2013).…”

“…For the generation of the β-galactosidase-expressing strains with the HHRz cassette, we used the added XhoI restriction site to excise the HHRz cassette from the cloning vector pGEM-T easy and inserted this construct in the expression vector pDneo2a lacZ (Wiegand and Hammann 2013), which also had been cleaved by XhoI. The localization of the HHRz cassette between the stop codon and the poly (A) signal sequences of the lacZ gene was confirmed by sequencing.…”