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MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo.

TitleMicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo.
Publication TypeJournal Article
Year of Publication2008
AuthorsTang F, Hajkova P, O'Carroll D, Lee C, Tarakhovsky A, Lao K, M Surani A
JournalBiochem Biophys Res Commun
Volume372
Issue1
Pagination24-9
Date Published2008 Jul 18
ISSN1090-2104
KeywordsAnimals, Cell Line, Mice, MicroRNAs, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleotides, RNA-Induced Silencing Complex
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.

DOI10.1016/j.bbrc.2008.04.137
Alternate JournalBiochem. Biophys. Res. Commun.
PubMed ID18474225
Grant List / / Biotechnology and Biological Sciences Research Council / United Kingdom
/ / Wellcome Trust / United Kingdom
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