MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo |
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Authors: | Tang Fuchou Hajkova Petra O'Carroll Dónal Lee Caroline Tarakhovsky Alexander Lao Kaiqin Surani M Azim |
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Institution: | a Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK b The Laboratory for Lymphocyte Signaling and the Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA c Molecular Cell Biology, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA |
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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. |
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Keywords: | MicroRNAs RNA-induced gene silencing complexes Embryonic stem cells Dicer Argonaute |
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