Title:
Dissection of RISC assembly and function

 

Yukihide Tomari
Institute of Molecular and Cellular Biosciences, The University of Tokyo
1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

 

Abstract:
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) mediate posttranscriptional gene silencing of their target mRNAs via the RNA-induced silencing complex (RISC). These small RNAs are born double-stranded and loaded into Argonaute proteins, the core component of RISC, in an ATP-dependent manner. Subsequently, the two strands of a small RNA duplex are separated within Ago protein in an ATP-independent manner. Why is ATP hydrolysis required for duplex loading—apparently simple binding between the RNA duplex and Argonaute protein—but not for strand separation—a process that disrupts ~20 base pairs between the two strands? We showed that the Hsc70/Hsp90 chaperone machinery is essential for ATP-dependent loading of small RNA duplexes, but not for strand separation, target cleavage or release of the cleavage product [1]. Accordingly, we proposed that the chaperone machinery consumes ATP and mediates a dynamic conformational change of Ago proteins so that they can receive small RNA duplexes. I would like to discuss such action of the chaperone machinery as the driving force for the RISC assembly pathway, in light of our recent progress in single-molecule imaging of RISC assembly and function [2,3].

 

References:
[1] Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Iwasaki S, Kobayashi M, Yoda M, Sakaguchi Y, Katsuma S, Suzuki T, *Tomari Y. Mol Cell. 2010 Jul 30;39(2):292-299.
[2] Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex. Iwasaki S, Sasaki HM, Sakaguchi Y, Suzuki T, *Tadakuma H, *Tomari Y. Nature. 2015 May 28;521(7553):533-6.
[3] Single-molecule analysis of the target cleavage reaction by Drosophila RNAi enzyme complex. Yao C, Sasaki HM, Ueda T, *Tomari Y, *Tadakuma H. Mol Cell. 2015 July 2 (in press)