Azuma-Mukai Asuka, Oguri Hideo, Mituyama Toutai, Qian Zhi Rong, Asai Kiyoshi, Siomi Haruhiko, Siomi Mikiko C
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan.
Proc Natl Acad Sci U S A. 2008 Jun 10;105(23):7964-9. doi: 10.1073/pnas.0800334105. Epub 2008 Jun 4.
Small RNAs triggering RNA silencing are loaded onto Argonautes and then sequence-specifically guide them to target transcripts. Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, and hAgo4) are associated with siRNAs and miRNAs, but only epitope-tagged hAgo2 has been shown to have Slicer activity. Contrarily, how endogenous hAgos behave with respect to small RNA association and target RNA destruction has remained unclear. Here, we produced monoclonal antibodies for individual hAgos. High-throughput pyrosequencing revealed that immunopurified endogenous hAgo2 and hAgo3 associated mostly with miRNAs. Endogenous hAgo3 did not show Slicer function but localized in P-bodies, suggesting that hAgo3 endogenously expressed is, like hAgo2, involved in the miRNA pathway but antagonizes the RNAi activity of hAgo2. Sequence variations of miRNAs were found at both 5' and 3' ends, suggesting that multiple mature miRNAs containing different "seed" sequences can arise from one miRNA precursor. The hAgo antibodies we raised are valuable tools for ascertaining the functional behavior of endogenous Argonautes and miRNAs in RNA silencing.
触发RNA沉默的小RNA被装载到AGO蛋白上，然后通过序列特异性引导它们靶向转录本。带有表位标签的人类AGO蛋白（hAgo1、hAgo2、hAgo3和hAgo4）与小干扰RNA（siRNA）和微小RNA（miRNA）相关联，但只有带有表位标签的hAgo2被证明具有核酸酶活性。相反，内源性AGO蛋白在小RNA结合和靶RNA破坏方面的行为仍不清楚。在这里，我们制备了针对单个AGO蛋白的单克隆抗体。高通量焦磷酸测序显示，免疫纯化的内源性hAgo2和hAgo3主要与miRNA相关联。内源性hAgo3不显示核酸酶功能，但定位于P小体，这表明内源性表达的hAgo3与hAgo2一样，参与miRNA途径，但拮抗hAgo2的RNA干扰活性。在miRNA的5'和3'末端均发现了序列变异，这表明一个miRNA前体可能产生多个包含不同“种子”序列的成熟miRNA。我们制备的hAgo抗体是确定内源性AGO蛋白和miRNA在RNA沉默中功能行为的有价值工具。
