[
Science,
2007]
In C. elegans, an effective RNA interference (RNAi) response requires the production of secondary siRNAs by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22 nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex) cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch, but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5'' di- or triphosphates and are only in minority associated with RDE-1, the RNAi-specific argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.
[
J Bacteriol,
2013]
Burkholderia cenocepacia J2315 is a highly epidemic and transmissible clinical isolate of the Burkholderia cepacia complex (Bcc), a group of bacteria causing life-threatening respiratory infections among cystic fibrosis patients. This work describes the functional analysis of the 136-nucleotide (nt)-long MtvR small noncoding RNA (sRNA) from the Bcc member B. cenocepacia J2315, with homologues restricted to the genus Burkholderia. Bioinformatic target predictions revealed a total of 309 mRNAs to be putative MtvR targets. The mRNA levels corresponding to 17 of 19 selected genes were found to be affected when MtvR was either overexpressed or silenced. Analysis of the interaction between MtvR and the hfq mRNA, one of its targets, showed that the sRNA binds exclusively to the 5' untranslated region (UTR) of the hfq mRNA. This interaction resulted in decreased protein synthesis, suggesting a negative regulatory effect of MtvR on the RNA chaperone Hfq. Bacterial strains with MtvR silenced or overexpressed exhibited pleiotropic phenotypes related to growth and survival after several stresses, swimming and swarming motilities, biofilm formation, resistance to antibiotics, and ability to colonize and kill the nematode Caenorhabditis elegans. Together, the results indicate that the MtvR sRNA is a major posttranscriptional regulator in B. cenocepacia.