Publication Date:
2015-03-31
Description:
Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Iwasaki, Shintaro -- Sasaki, Hiroshi M -- Sakaguchi, Yuriko -- Suzuki, Tsutomu -- Tadakuma, Hisashi -- Tomari, Yukihide -- England -- Nature. 2015 May 28;521(7553):533-6. doi: 10.1038/nature14254. Epub 2015 Mar 30.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. ; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. ; Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. ; 1] Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. [2] Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25822791" target="_blank"〉PubMed〈/a〉
Keywords:
Animals
;
Argonaute Proteins/metabolism
;
Drosophila Proteins/metabolism
;
Drosophila melanogaster/*enzymology/*genetics
;
Heat-Shock Proteins/metabolism
;
In Vitro Techniques
;
Janus Kinases/metabolism
;
Protein Binding
;
RNA Helicases/metabolism
;
*RNA Interference
;
RNA, Guide/genetics/metabolism
;
RNA, Small Interfering/genetics/metabolism
;
RNA-Binding Proteins/metabolism
;
RNA-Induced Silencing Complex/*chemistry/*metabolism
;
Ribonuclease III/metabolism
;
Transcription Factors/metabolism
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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