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Plant and Cell Physiology Advance Access published online on March 14, 2008
Plant and Cell Physiology, 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 e-mail: journals.permissions@oxfordjournals.org

The mechanism selecting the guide strand from small RNA duplexes is different among Argonaute proteins

Atsushi Takeda1,2, Shintaro Iwasaki1, Toshiaki Watanabe3,4, 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.
2JSPS Research Fellow for Young Scientist.
3Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan.
4Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Mishima 411-8540, Japan

Corresponding author: Dr. Yuichiro Watanabe, Address: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan. Tel: 81-3-5454-6776, Fax: 81-3-5454-6776, E-mail: solan{at}bio.c.u-tokyo.ac.jp


  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 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 ten AGO genes, roles and molecular mechanisms of them remains obscure. In this study, we analyzed roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that of AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from 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: 5' nucleotide of small RNA - Arabidopsis - Argonaute (AGO) - Cucumber mosaic virus (CMV) - microRNA (miRNA) - RNA silencing

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