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Plant and Cell Physiology Advance Access originally published online on March 14, 2008
Plant and Cell Physiology 2008 49(4):493-500; doi:10.1093/pcp/pcn043
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Rapid paper

The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins

Atsushi Takeda1, Shintaro Iwasaki1, Toshiaki Watanabe2,3, Maki Utsumi1 and Yuichiro Watanabe1,*

1Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902 Japan
2Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima, 411-8540 Japan
3Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Mishima, 411-8540 Japan

*Corresponding author: E-mail, solan{at}bio.c.u-tokyo.ac.jp; Fax, +81-3-5454-6776.


  Abstract

Double-stranded RNA induces RNA silencing and is cleaved into 21–24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO–small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390*) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390*. Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

Keywords: Arabidopsis - Argonaute (AGO) - Cucumber mosaic virus (CMV) - MicroRNA (miRNA) - 5' Nucleotide of small RNA - RNA silencing

Abbreviations: AGO, Argonaute; CMV, cucumber mosaic virus; HA, hemagglutinin; IP, immunoprecipitation; miRNA, microRNA; nat-siRNA, siRNA derived from a pair of natural cis-antisense transcripts; RISC, RNA-induced silencing complex; siRNA, small interfering RNA; tasiRNA, trans-acting siRNA; vi-siRNA, virus-derived siRNA.

(Received February 22, 2008; Accepted March 12, 2008)
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