MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo.
Previous work has shown that synthesized siRNA/miRNA is tightly associated with RNA-induced Gene Silencing Complexes (RISCs) in vitro. However, it is unknown if the endogenous miRNAs are also stably bound to RISC complexes in vivo in cells under physiological conditions. Here we describe the use of the looped real-time PCR-based method to trace the location of endogenous miRNAs in intact cells. We found that most of the endogenous miRNAs are tightly bound to RISC complexes, and only a very small proportion of them are free in cells. Furthermore, synthesized single-stranded mature miRNA or hairpin miRNA precursor cannot replace endogenous miRNAs already present in RISC complexes. However, we found that modified 2-O-Methyl-ribonucleotides were able to dissociate the target miRNA specifically from the RISC complex. These findings have important implications for understanding the basis for the stability and metabolism of miRNAs in living cells.
Tang
Fuchou
F
Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
Hajkova
Petra
P
O'Carroll
Dónal
D
Lee
Caroline
C
Tarakhovsky
Alexander
A
Lao
Kaiqin
K
Surani
M Azim
MA
D000818:Animals; D002460:Cell Line; D051379:Mice; D035683:MicroRNAs / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012265:Ribonucleotides / Q000737:chemistry
D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; D012265:Ribonucleotides