Inhibition of micro-RNA-induced RNA silencing by 2'-o-methyl oligonucleotides in Drosophila S2 cells.
More than 90 different micro-ribonucleic acid (miRNA) encoding genes have been identified in Drosophila, yet the function of only two of these, bantam and DmiR-14, has been elucidated. In an effort to develop a general strategy for the analysis of miRNA function in Drosophila, two procedures were developed, in a Schneider line 2 cell culture system, which may be adapted to that end. First, we show that endogenous miRNAs can partially inhibit the expression of a transiently transfected reporter gene that has been modified to contain sequences complementary to that miRNA in the 3' UTR of a target messenger RNA (mRNA). Inhibition occurs by RNA interference (RNAi), which involves mRNA degradation. Second, we demonstrate that this miRNA-induced RNAi can be partially rescued with 2'-O-methyl oligonucleotides that contain sequences complementary to the cognate miRNA. We discuss how these techniques may be used, in vivo, both for localizing the tissue distribution of endogenous miRNAs during Drosophila development and identifying phenotypes associated with a loss of miRNA function.
Berger
Edward M
EM
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA. edward.berger@Dartmouth.edu
Dubrovsky
Edward B
EB
Appleby
Lara
L
Dubrovskaya
Veronica
V
D000818:Animals; D018929:Cell Culture Techniques; D004330:Drosophila / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005821:Genetic Techniques; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D035683:MicroRNAs; D009841:Oligonucleotides; D012333:RNA, Messenger