Monitoring miRNA-mediated silencing in Drosophila melanogaster S2-cells.
MicroRNAs (miRNAs) are small cellular RNAs that participate in post-transcriptional gene regulation. Even though they were only recently discovered, research on the biogenesis, mechanism of repression and biological significance of miRNAs has already received much attention. In this study, we have compared expression strategies for miRNA-activity reporter constructs and have examined the dependence of silencing by a particular Drosophila miRNA, bantam, on specific argonaute proteins. Consistent with previous biochemical experiments, we found that bantam silencing is strongly dependent on Ago1, but in addition we could detect the activity of Ago2-loaded bantam. Our experiments suggest that a perfectly complementary design and a transient expression strategy for reporter constructs may--in the case of catalytically active Ago-proteins--lead to a disproportionately strong response mediated by a minor fraction of silencing complexes. We present evidence that Drosophila S2-cells of independent sources differ in their RNAi efficiency in response to dsRNA added to the growth medium, and that the selection antibiotic G418 acts as an inhibitor of RNAi induced by soaking.
Shah
Claudio
C
Gene Center, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, D-81377 Munich, Germany.
Förstemann
Klaus
K
D000818:Animals; D002460:Cell Line; D003470:Culture Media; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D005839:Gentamicins / Q000494:pharmacology; D035683:MicroRNAs / Q000378:metabolism*; D034622:RNA Interference / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; D029721:Drosophila Proteins; D005839:Gentamicins; D035683:MicroRNAs; D012330:RNA, Double-Stranded; C010680:antibiotic G 418