The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway

Nucleic Acids Research, Nov 2007

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.

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The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway

Andrea Hinas 2 Johan Reimega rd 1 E. Gerhart H. Wagner 1 Wolfgang Nellen 0 Victor R. Ambros 3 Fredrik So derbom 2 0 Department of Genetics, Kassel University , Heinrich Plett Strasse 40, 34132 Kassel, Germany 1 Department of Cell and Molecular Biology, Biomedical Center, Uppsala University , Box 596, SE-75124 Uppsala, Sweden 2 Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences , Box 590, SE-75124 Uppsala, Sweden 3 Department of Genetics, Dartmouth Medical School , Hanover, NH 03755, USA 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. - Since their initial discovery in worms, 1826 nt small RNAs have now been identified in many eukaryotes (1,2). By antisense complementarity, they confer specificity to associated protein complexes and can thereby regulate expression of their target genes at the level of transcription, mRNA stability or translation (35). These small RNAs can be divided into two main classes; microRNAs (miRNAs), which are processed from imperfectly basepaired hairpin transcripts, and small interfering RNAs (siRNAs), which are derived from long double-stranded RNAs (dsRNAs) (57). The dsRNA precursors of siRNAs originate e.g. from viruses, repetitive elements or are synthesized by an RNA-dependent RNA polymerase (RdRP) using a single-stranded RNA as a template (2,810). Despite their different origin, miRNA and siRNA pathways share many similarities. In both cases, these small RNAs are processed from precursors into mature small RNAs by the RNase III-type Dicer proteins, and are subsequently incorporated in an effector complex that contains an ArgonautePiwi family protein (5,6). The miRNA/siRNA guides the protein complex to its complementary target RNA and induces cleavage of the target if the small RNA and target RNA form a perfectly or close to perfectly base-paired duplex. In plants, most miRNAs exert their effect in this way. If base pairing is only partial, as is the common feature of animal miRNA target interactions, the main effect seems to be inhibition of translation, although some mRNA degradation is also frequently observed (4). miRNAs are common in multicellular organisms and animal viruses and were only recently discovered in a unicellular organism, the green alga Chlamydomonas reinhardtii (6,1113). In animals and plants, miRNAs play important cellular roles by modulating the expression of endogenous genes and it has been estimated that up to onethird of all human genes may be regulated by miRNAs (5,14). The physiological role of miRNAs in C. reinhardtii is still not known, however, verified targets include genes encoding flagellum-associated proteins (11,13). siRNAs are present in eukaryotes from all major phylogenetic branches including plants, animals and fungi where they act e.g. in a defense mechanism against viral RNAs and mobilization of transposons (2,15). Repetitive elements, such as transposons and retrotransposons, constitute substantial parts of the centromeres in many eukaryotes, and the RNAi machinery is required for silencing of centromeric repeats in e.g. fission yeast (16,17). Although most studies on natural siRNAs have focused on their roles in defense against viruses and repetitive elements, an increasing number of reports point to an additional role in regulation of non-transposon genes. For example, large-scale cloning of small RNAs from Caenorhabditis elegans and Arabidopsis thaliana has identified many small RNAs with antisense complementarity to genes other than repetitive elements (1820). Recently, such small RNAs were demonstrated to be involved in regulation of overlapping A. thaliana genes during salt stress and bacterial infection (20,21). Furthermore, whole-genome microarray analyses and (...truncated)


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Andrea Hinas, Johan Reimegård, E. Gerhart H. Wagner, Wolfgang Nellen, Victor R. Ambros, Fredrik Söderbom. The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway, Nucleic Acids Research, 2007, pp. 6714-6726, 35/20, DOI: 10.1093/nar/gkm707