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| Subject Categories: RNA |
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| The EMBO Journal
(2002) 21, 5864–5874, doi: 10.1093/emboj/cdf578 |
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| Ribonuclease activity and RNA binding of recombinant human Dicer |
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| Patrick Provost1, 2, David Dishart1, Johanne Doucet1, 2, David Frendewey3, Bengt Samuelsson1 and Olof Rådmark1 |
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1 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 2, Stockholm, S-171 77, Sweden 2 Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd Laurier, Ste-Foy, Quebec, G1V 4G2, Canada 3 Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6707, USA To whom correspondence should be addressed Patrick Provost, patrick.provost@crchul.ulaval.ca Olof Rådmark, olof.radmark@mbb.ki.se Received 9 July 2002; Revised 9 September 2002; Accepted 10 September 2002. |
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| Abstract |
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RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated  21−23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes. |
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| Keywords: Dicer, dsRNA, ribonuclease III, RNA binding, RNA processing |
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