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

Hinas A, Reimegård J, Wagner EG, Nellen W, Ambros VR, Söderbom F - Nucleic Acids Res. (2007)

Bottom Line: 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.Three of these mRNAs are developmentally regulated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.

ABSTRACT
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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Small RNAs derived from the DIRS-1 retrotransposon (A) Distribution of cloned DIRS-1 sequences in the two cDNA libraries in sense (top) and antisense (bottom) orientation relative to the DIRS-1 retrotransposon (middle), which is drawn to scale. The asterisk indicates the location of the small RNA for which expression was analyzed in Figure 3B. (B) Northern blot analysis of one DIRS-1 small RNA during growth (0 h) and development (16 and 24 h) of D. discoideum. Approximately equal loading was visualized by use of a probe against 5.8S rRNA. Radioactively labeled size markers (M) are shown for reference.
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Figure 3: Small RNAs derived from the DIRS-1 retrotransposon (A) Distribution of cloned DIRS-1 sequences in the two cDNA libraries in sense (top) and antisense (bottom) orientation relative to the DIRS-1 retrotransposon (middle), which is drawn to scale. The asterisk indicates the location of the small RNA for which expression was analyzed in Figure 3B. (B) Northern blot analysis of one DIRS-1 small RNA during growth (0 h) and development (16 and 24 h) of D. discoideum. Approximately equal loading was visualized by use of a probe against 5.8S rRNA. Radioactively labeled size markers (M) are shown for reference.

Mentions: DIRS-1 is the most abundant retrotransposon in the D. discoideum genome and DIRS-1-rich regions are found at one end of each chromosome (26). These regions cluster during mitosis and have therefore been suggested to function as centromeres. The complete DIRS-1 retrotransposon encodes a 4.5 kb mRNA and a 900 nt antisense RNA and is flanked by inverted long terminal repeats (LTRs) (43–45). We identified small RNAs along almost the entire retrotransposon, not only from the segments that would be predicted to form dsRNAs, e.g. mRNA-antisense RNA and LTRs (Figure 3A).Figure 3.


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

Hinas A, Reimegård J, Wagner EG, Nellen W, Ambros VR, Söderbom F - Nucleic Acids Res. (2007)

Small RNAs derived from the DIRS-1 retrotransposon (A) Distribution of cloned DIRS-1 sequences in the two cDNA libraries in sense (top) and antisense (bottom) orientation relative to the DIRS-1 retrotransposon (middle), which is drawn to scale. The asterisk indicates the location of the small RNA for which expression was analyzed in Figure 3B. (B) Northern blot analysis of one DIRS-1 small RNA during growth (0 h) and development (16 and 24 h) of D. discoideum. Approximately equal loading was visualized by use of a probe against 5.8S rRNA. Radioactively labeled size markers (M) are shown for reference.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175303&req=5

Figure 3: Small RNAs derived from the DIRS-1 retrotransposon (A) Distribution of cloned DIRS-1 sequences in the two cDNA libraries in sense (top) and antisense (bottom) orientation relative to the DIRS-1 retrotransposon (middle), which is drawn to scale. The asterisk indicates the location of the small RNA for which expression was analyzed in Figure 3B. (B) Northern blot analysis of one DIRS-1 small RNA during growth (0 h) and development (16 and 24 h) of D. discoideum. Approximately equal loading was visualized by use of a probe against 5.8S rRNA. Radioactively labeled size markers (M) are shown for reference.
Mentions: DIRS-1 is the most abundant retrotransposon in the D. discoideum genome and DIRS-1-rich regions are found at one end of each chromosome (26). These regions cluster during mitosis and have therefore been suggested to function as centromeres. The complete DIRS-1 retrotransposon encodes a 4.5 kb mRNA and a 900 nt antisense RNA and is flanked by inverted long terminal repeats (LTRs) (43–45). We identified small RNAs along almost the entire retrotransposon, not only from the segments that would be predicted to form dsRNAs, e.g. mRNA-antisense RNA and LTRs (Figure 3A).Figure 3.

Bottom Line: 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.Three of these mRNAs are developmentally regulated.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden.

ABSTRACT
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.

Show MeSH