Project description:We report on the function of the D. dictyostelium argonaute proteins AgnA and AgnB in the silencing mechanism of the retrotransposon DIRS-1. By deep sequencing analysis we found that AgnA is involved in the accumulation of DIRS-1 siRNAs since in respective knockout strains siRNAs are strongly reduced. This was verified by northern blots. Reduction of siRNAs was accompanied by increased transcript accumulation and the appearance of an extrachromosomal single stranded DIRS-1 cDNA. In contrast, disruption of the closely related agnB gene had no effect on DIRS-1 siRNAs. agnA-/agnB- double knockouts suggested positively regulator effects by the AgnB protein on DIRS-1.
Project description:We report on the function of the D. dictyostelium argonaute proteins AgnA and AgnB in the silencing mechanism of the retrotransposon DIRS-1. By deep sequencing analysis we found that AgnA is involved in the accumulation of DIRS-1 siRNAs since in respective knockout strains siRNAs are strongly reduced. This was verified by northern blots. Reduction of siRNAs was accompanied by increased transcript accumulation and the appearance of an extrachromosomal single stranded DIRS-1 cDNA. In contrast, disruption of the closely related agnB gene had no effect on DIRS-1 siRNAs. agnA-/agnB- double knockouts suggested positively regulator effects by the AgnB protein on DIRS-1. Small RNA profiles of the Ax2 wildtype and different mutant strains were generated by deep sequencing on an Illumina platform.
Project description:Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase. Keywords: cDNA library; small RNA sequencing
