Limits...
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.

Show MeSH

Related in: MedlinePlus

miRNA candidates in D. discoideum (A) northern blot analysis of mica1190 and mica1198 using RNA isolated from growing cells (0 h) and developed cells (16 and 24 h). The 5.8S rRNA is used as a loading control and radioactively labeled size markers (M) are shown. (B) Predicted secondary structure (Mfold) of sequences surrounding miRNA candidates mica1190 and mica1198. (C) Northern blot analysis showing the expression of mica1198 in strains where genes encoding putative Dicer homologs (drnA− and drnB−), RdRp (rrpA−, rrpB− and rrpC−) and RNA helicase (helF−) have been disrupted. Wild-type (wt) represents D. discoideum AX2 strain, which is the background strain of all knockout strains except rrpC−. rrpC− was constructed in the AX4 wild-type strain, which displayed similar expression levels as AX2 for all RNAs analyzed in this study (data not shown). The same membrane was stripped and reprobed with the DIRS-1 small RNA which is expressed approximately equally in all strains analyzed and was therefore used as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175303&req=5

Figure 2: miRNA candidates in D. discoideum (A) northern blot analysis of mica1190 and mica1198 using RNA isolated from growing cells (0 h) and developed cells (16 and 24 h). The 5.8S rRNA is used as a loading control and radioactively labeled size markers (M) are shown. (B) Predicted secondary structure (Mfold) of sequences surrounding miRNA candidates mica1190 and mica1198. (C) Northern blot analysis showing the expression of mica1198 in strains where genes encoding putative Dicer homologs (drnA− and drnB−), RdRp (rrpA−, rrpB− and rrpC−) and RNA helicase (helF−) have been disrupted. Wild-type (wt) represents D. discoideum AX2 strain, which is the background strain of all knockout strains except rrpC−. rrpC− was constructed in the AX4 wild-type strain, which displayed similar expression levels as AX2 for all RNAs analyzed in this study (data not shown). The same membrane was stripped and reprobed with the DIRS-1 small RNA which is expressed approximately equally in all strains analyzed and was therefore used as a loading control.

Mentions: Until very recently, miRNAs had only been identified in multicellular organisms and their viruses (11,13). To explore the possibility that miRNAs were represented in our cDNA libraries, we computationally predicted secondary structures of RNA sequences flanking intergenic, intronic and antisense small RNA loci (see Materials and Methods section). Five out of 167 folded sequences fulfilled standard criteria for miRNA precursors; four were derived from intergenic regions, and one was in antisense orientation to an intron of a predicted gene. By northern blot analysis, expression of ∼22 nt RNAs was detected for two of the intergenic small RNAs, mica1190 and mica1198. Furthermore, both are up-regulated during development (16 h slugs and 24 h fruiting bodies) (Figure 2A). The predicted precursors of the miRNA candidates are shown in Figure 2B. No signals corresponding to pre-miRNAs could be detected in the northern blot experiment; however, this is also a common situation in plants where miRNA precursors are rarely seen (40).Figure 2.


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)

miRNA candidates in D. discoideum (A) northern blot analysis of mica1190 and mica1198 using RNA isolated from growing cells (0 h) and developed cells (16 and 24 h). The 5.8S rRNA is used as a loading control and radioactively labeled size markers (M) are shown. (B) Predicted secondary structure (Mfold) of sequences surrounding miRNA candidates mica1190 and mica1198. (C) Northern blot analysis showing the expression of mica1198 in strains where genes encoding putative Dicer homologs (drnA− and drnB−), RdRp (rrpA−, rrpB− and rrpC−) and RNA helicase (helF−) have been disrupted. Wild-type (wt) represents D. discoideum AX2 strain, which is the background strain of all knockout strains except rrpC−. rrpC− was constructed in the AX4 wild-type strain, which displayed similar expression levels as AX2 for all RNAs analyzed in this study (data not shown). The same membrane was stripped and reprobed with the DIRS-1 small RNA which is expressed approximately equally in all strains analyzed and was therefore used as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: miRNA candidates in D. discoideum (A) northern blot analysis of mica1190 and mica1198 using RNA isolated from growing cells (0 h) and developed cells (16 and 24 h). The 5.8S rRNA is used as a loading control and radioactively labeled size markers (M) are shown. (B) Predicted secondary structure (Mfold) of sequences surrounding miRNA candidates mica1190 and mica1198. (C) Northern blot analysis showing the expression of mica1198 in strains where genes encoding putative Dicer homologs (drnA− and drnB−), RdRp (rrpA−, rrpB− and rrpC−) and RNA helicase (helF−) have been disrupted. Wild-type (wt) represents D. discoideum AX2 strain, which is the background strain of all knockout strains except rrpC−. rrpC− was constructed in the AX4 wild-type strain, which displayed similar expression levels as AX2 for all RNAs analyzed in this study (data not shown). The same membrane was stripped and reprobed with the DIRS-1 small RNA which is expressed approximately equally in all strains analyzed and was therefore used as a loading control.
Mentions: Until very recently, miRNAs had only been identified in multicellular organisms and their viruses (11,13). To explore the possibility that miRNAs were represented in our cDNA libraries, we computationally predicted secondary structures of RNA sequences flanking intergenic, intronic and antisense small RNA loci (see Materials and Methods section). Five out of 167 folded sequences fulfilled standard criteria for miRNA precursors; four were derived from intergenic regions, and one was in antisense orientation to an intron of a predicted gene. By northern blot analysis, expression of ∼22 nt RNAs was detected for two of the intergenic small RNAs, mica1190 and mica1198. Furthermore, both are up-regulated during development (16 h slugs and 24 h fruiting bodies) (Figure 2A). The predicted precursors of the miRNA candidates are shown in Figure 2B. No signals corresponding to pre-miRNAs could be detected in the northern blot experiment; however, this is also a common situation in plants where miRNA precursors are rarely seen (40).Figure 2.

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
Related in: MedlinePlus