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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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Two cDNA libraries representing 18–26 nt RNAs from D. discoideum. (A) Classification of small RNAs represented in the cDNA libraries constructed by 5′-ligation-dependent cloning (left) and 5′-ligation-independent cloning (right). Numbers of sequences (total as well as unique) are indicated in brackets. (B) Size distribution of small RNAs represented in the cDNA libraries. Sequences (percentage of each library) from the 5′-ligation-dependent and 5′-ligation-independent libraries are represented by black and gray bars, respectively.
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Figure 1: Two cDNA libraries representing 18–26 nt RNAs from D. discoideum. (A) Classification of small RNAs represented in the cDNA libraries constructed by 5′-ligation-dependent cloning (left) and 5′-ligation-independent cloning (right). Numbers of sequences (total as well as unique) are indicated in brackets. (B) Size distribution of small RNAs represented in the cDNA libraries. Sequences (percentage of each library) from the 5′-ligation-dependent and 5′-ligation-independent libraries are represented by black and gray bars, respectively.

Mentions: Of the 5009 cloned sequences from the 5′-ligation-dependent cDNA library, 48% (2387 sequences) matched the published D. discoideum genome sequence (26) whereas the corresponding number for the 5′-ligation-independent library was 36% (1432 sequences) of a total of 4002 sequenced clones. The relative representation of different RNA classes in the two libraries is shown in Figure 1A, along with the numbers of unique sequences identified. Most clones represented small RNAs derived from the DIRS-1 retrotransposon (68.7% and 37.5% in the 5′-ligation-dependent and 5′-ligation-independent libraries, respectively). Another large fraction was derived from putative degradation products of rRNAs, tRNAs, small nucleolar RNAs, small nuclear RNAs and other non-coding RNAs (22.6% and 46.7%). A substantial portion (2.7% and 10.2%) matched the intergenic region between the 26S rRNA and 5S rRNA genes, which in D. discoideum is present on the 88 kb extrachromosomal rDNA palindrome (39). Out of these clones, 90% were derived from a 27 nt region situated ∼450 bp downstream and ∼1500 nt upstream of the 26S rRNA and 5S rRNA genes, respectively. This region has previously been referred to as non-transcribed (39). Northern blot analysis of this region yielded several different hybridization signals of which none was shorter than ∼40 nt in length (data not shown).Figure 1.


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)

Two cDNA libraries representing 18–26 nt RNAs from D. discoideum. (A) Classification of small RNAs represented in the cDNA libraries constructed by 5′-ligation-dependent cloning (left) and 5′-ligation-independent cloning (right). Numbers of sequences (total as well as unique) are indicated in brackets. (B) Size distribution of small RNAs represented in the cDNA libraries. Sequences (percentage of each library) from the 5′-ligation-dependent and 5′-ligation-independent libraries are represented by black and gray bars, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2175303&req=5

Figure 1: Two cDNA libraries representing 18–26 nt RNAs from D. discoideum. (A) Classification of small RNAs represented in the cDNA libraries constructed by 5′-ligation-dependent cloning (left) and 5′-ligation-independent cloning (right). Numbers of sequences (total as well as unique) are indicated in brackets. (B) Size distribution of small RNAs represented in the cDNA libraries. Sequences (percentage of each library) from the 5′-ligation-dependent and 5′-ligation-independent libraries are represented by black and gray bars, respectively.
Mentions: Of the 5009 cloned sequences from the 5′-ligation-dependent cDNA library, 48% (2387 sequences) matched the published D. discoideum genome sequence (26) whereas the corresponding number for the 5′-ligation-independent library was 36% (1432 sequences) of a total of 4002 sequenced clones. The relative representation of different RNA classes in the two libraries is shown in Figure 1A, along with the numbers of unique sequences identified. Most clones represented small RNAs derived from the DIRS-1 retrotransposon (68.7% and 37.5% in the 5′-ligation-dependent and 5′-ligation-independent libraries, respectively). Another large fraction was derived from putative degradation products of rRNAs, tRNAs, small nucleolar RNAs, small nuclear RNAs and other non-coding RNAs (22.6% and 46.7%). A substantial portion (2.7% and 10.2%) matched the intergenic region between the 26S rRNA and 5S rRNA genes, which in D. discoideum is present on the 88 kb extrachromosomal rDNA palindrome (39). Out of these clones, 90% were derived from a 27 nt region situated ∼450 bp downstream and ∼1500 nt upstream of the 26S rRNA and 5S rRNA genes, respectively. This region has previously been referred to as non-transcribed (39). Northern blot analysis of this region yielded several different hybridization signals of which none was shorter than ∼40 nt in length (data not shown).Figure 1.

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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