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

Small antisense RNAs may originate from longer transcripts. (A) Schematic drawing (left) of structures of genes analyzed by RT-PCR (right). The positions of sequences represented in the cDNA libraries are indicated for hatA, rsmF and DDB0230011. Primers used in the study are represented by small arrows. Letters in brackets following the oligo numbers indicate whether the oligo was used for reverse transcription of mRNA (mR) or asRNA (asR), PCR (P), or to construct templates for in vitro transcription (I). +/− RT indicates the presence or absence of reverse transcriptase in the reaction, respectively. The faint band at ∼300 bp for the PCR reaction performed with genomic DNA (gDNA) and hatA primers is probably due to cross-hybridization of primers to hatC. (B) Northern blot analysis during growth (0 h) and development (16 h) using in vitro transcribed, strand-specific probes. The probes constructed against hatA will most likely also hybridize to hatB and hatC and are therefore designated hatA*. The arrowhead next to the hatA* asRNA panel indicates a signal corresponding to the size of the hatA* mRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Small antisense RNAs may originate from longer transcripts. (A) Schematic drawing (left) of structures of genes analyzed by RT-PCR (right). The positions of sequences represented in the cDNA libraries are indicated for hatA, rsmF and DDB0230011. Primers used in the study are represented by small arrows. Letters in brackets following the oligo numbers indicate whether the oligo was used for reverse transcription of mRNA (mR) or asRNA (asR), PCR (P), or to construct templates for in vitro transcription (I). +/− RT indicates the presence or absence of reverse transcriptase in the reaction, respectively. The faint band at ∼300 bp for the PCR reaction performed with genomic DNA (gDNA) and hatA primers is probably due to cross-hybridization of primers to hatC. (B) Northern blot analysis during growth (0 h) and development (16 h) using in vitro transcribed, strand-specific probes. The probes constructed against hatA will most likely also hybridize to hatB and hatC and are therefore designated hatA*. The arrowhead next to the hatA* asRNA panel indicates a signal corresponding to the size of the hatA* mRNA.

Mentions: hatA encodes the actin-binding protein hisactophilin I. A closely related gene, encoding hisactophilin II, (hatB), is located ∼600 bp downstream of hatA. Gene disruption of both hatA and hatB renders D. discoideum more sensitive to hyperosmotic stress than wild-type cells (47). A third, highly similar gene, hatC, is located on another chromosome although there is no previously reported evidence for its expression. hatA and hatB each contain one intron, whereas hatC lacks introns (Figure 6A). Although these three genes are very similar, the small RNA identified in our cDNA library is fully complementary only to hatA.Figure 6.


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 antisense RNAs may originate from longer transcripts. (A) Schematic drawing (left) of structures of genes analyzed by RT-PCR (right). The positions of sequences represented in the cDNA libraries are indicated for hatA, rsmF and DDB0230011. Primers used in the study are represented by small arrows. Letters in brackets following the oligo numbers indicate whether the oligo was used for reverse transcription of mRNA (mR) or asRNA (asR), PCR (P), or to construct templates for in vitro transcription (I). +/− RT indicates the presence or absence of reverse transcriptase in the reaction, respectively. The faint band at ∼300 bp for the PCR reaction performed with genomic DNA (gDNA) and hatA primers is probably due to cross-hybridization of primers to hatC. (B) Northern blot analysis during growth (0 h) and development (16 h) using in vitro transcribed, strand-specific probes. The probes constructed against hatA will most likely also hybridize to hatB and hatC and are therefore designated hatA*. The arrowhead next to the hatA* asRNA panel indicates a signal corresponding to the size of the hatA* mRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Small antisense RNAs may originate from longer transcripts. (A) Schematic drawing (left) of structures of genes analyzed by RT-PCR (right). The positions of sequences represented in the cDNA libraries are indicated for hatA, rsmF and DDB0230011. Primers used in the study are represented by small arrows. Letters in brackets following the oligo numbers indicate whether the oligo was used for reverse transcription of mRNA (mR) or asRNA (asR), PCR (P), or to construct templates for in vitro transcription (I). +/− RT indicates the presence or absence of reverse transcriptase in the reaction, respectively. The faint band at ∼300 bp for the PCR reaction performed with genomic DNA (gDNA) and hatA primers is probably due to cross-hybridization of primers to hatC. (B) Northern blot analysis during growth (0 h) and development (16 h) using in vitro transcribed, strand-specific probes. The probes constructed against hatA will most likely also hybridize to hatB and hatC and are therefore designated hatA*. The arrowhead next to the hatA* asRNA panel indicates a signal corresponding to the size of the hatA* mRNA.
Mentions: hatA encodes the actin-binding protein hisactophilin I. A closely related gene, encoding hisactophilin II, (hatB), is located ∼600 bp downstream of hatA. Gene disruption of both hatA and hatB renders D. discoideum more sensitive to hyperosmotic stress than wild-type cells (47). A third, highly similar gene, hatC, is located on another chromosome although there is no previously reported evidence for its expression. hatA and hatB each contain one intron, whereas hatC lacks introns (Figure 6A). Although these three genes are very similar, the small RNA identified in our cDNA library is fully complementary only to hatA.Figure 6.

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