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Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

机译：fulgidus Piwi蛋白对指导RNA进行5'-末端特异性识别的结构基础

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摘要

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago 2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.
机译：RNA干扰（RNAi）是由RNA诱导的沉默复合物（RISC）介导的保守序列特异性基因调控机制，该复合物由单链引导RNA和Argonaute蛋白质组成。 PIWI结构域是Argonaute中一个高度保守的基序，已显示出对RISC的核酸内切酶裂解活性至关重要的RNase H折叠。在这里，我们报告与双链RNA结合的古细菌粉红色Piwi蛋白的晶体结构，从而确定用于引导链5'端识别的结合袋，并提供对引导链介导的信使RNA目标识别的见解。指导RNA的磷酸化5'端锚定在高度保守的碱性口袋中，并辅以羧基末端的羧酸盐和结合的二价阳离子。来自指导RNA 5'端的第一个核苷酸不成对，并堆积在一个保守的酪氨酸残基上，而连续的核苷酸形成一个四碱基对的RNA双链体。与人Ago 2中的5'磷酸接触的相应氨​​基酸的突变导致mRNA切割活性减弱。我们的Piwi-RNA复合物的结构以及在其他地方确定的结构为作为靶标mRNA配对的成核位点的指导RNA的5'区域提供了直接支持，并且将RISC介导的mRNA切割位点与指导RNA的锚定的5'端。

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