title: MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo.
abstract: 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.
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
authors.forename: Fuchou
authors.initials: F
authors.lastname: Tang
authors.forename: Petra
authors.initials: P
authors.lastname: Hajkova
authors.forename: Dónal
authors.initials: D
authors.lastname: O'Carroll
authors.forename: Caroline
authors.initials: C
authors.lastname: Lee
authors.forename: Alexander
authors.initials: A
authors.lastname: Tarakhovsky
authors.forename: Kaiqin
authors.initials: K
authors.lastname: Lao
authors.forename: M Azim
authors.initials: MA
authors.lastname: Surani
mesh_terms: 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
chemical_list: D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; D012265:Ribonucleotides