Small RNAs in four Argonaute complexes in Arabidopsis

The core components of RNA silencing effector complexes include small RNAs and members of the Argonaute (AGO) protein family. Arabidopsis encodes ten AGOs and complex population of small RNAs. It remains largely unknown how these small RNAs are recognized and recruited by each AGO complex. Here we purified four AGO complexes and identified small RNAs in each complex by deep sequencing. Keywords: Small RNA sorting Small RNAs were prepared from Arabidopsis AGO1, AGO2, AGO4 and AGO5 complexes, ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of solexa cloning primers and then provided for sequencing. For technical details, see Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. 2006. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114): 1008-1012.

RNA沉默效应复合物的核心组成成分包括小RNA（small RNAs）与Argonaute（AGO）蛋白家族成员。拟南芥（Arabidopsis）编码十种AGO蛋白，并拥有复杂多样的小RNA种群。目前学界仍未明确这些小RNA如何被各AGO复合物识别并招募。本研究纯化了四种AGO复合物，并通过深度测序（deep sequencing）鉴定了各复合物中的小RNA。
关键词：小RNA分选（Small RNA sorting）
小RNA样品制备自拟南芥AGO1、AGO2、AGO4及AGO5复合物，依次连接3'接头与5'受体，随后经逆转录PCR（RT-PCR）扩增。PCR产物使用一对Solexa克隆引物进行二次扩增，随后用于测序。技术细节详见Qi,Y.、He,X.、Wang,X.J.、Kohany,O.、Jurka,J.与Hannon,G.J.于2006年发表于《Nature》的论文：《ARGONAUTE4在RNA指导的DNA甲基化中的独特催化与非催化功能》，Nature 443(7114): 1008-1012.