Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage
Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage
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DOI：
10.1074/jbc.ra117.001051
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发表时间：
2018-04-27
影响因子：
4.8
通讯作者：
Nakanishi, Kotaro
中科院分区：
文献类型：
--
作者：
Dayeh, Daniel M.;Kruithoff, Bradley C.;Nakanishi, Kotaro
一些基因转录本具有作为调节性非编码 RNA 的细胞功能。例如，类似于 23 个核苷酸 (nt) 长的 siRNA 被加载到 Argonaute 蛋白中。由此产生的核糖核蛋白组装体，即 RNA 诱导的沉默复合物 (RISC)，会切割与加载的 siRNA 广泛碱基配对的 RNA。迄今为止，碱基互补性被认为是特定目标切割（或切片）的主要决定因素，但人们对 Argonaute 如何在切割前检查碱基配对知之甚少。 Argonaute 蛋白的一个标志是其双叶结构，但尽管这种结构对于减少针对不匹配靶标的切片活性具有重要意义，但其分子机制仍然难以捉摸。在这里，我们对双叶酵母 Argonaute 蛋白及其分离的催化 C 末端叶 (C-lobe) 的结构和功能研究表明，单独的 C 叶保留了双叶 Argonaute 的几乎所有特性：siRNA 双链体加载、过客裂解/喷射和 siRNA 依赖性 RNA 裂解。 2.1 埃分辨率的晶体结构表明，催化 C 叶在引导 RNA 识别方面反映了双叶 Argonaute，并且转变为催化活性构象的所有要求都位于 C 叶中。然而，我们发现，在缺乏 N 末端叶 (N-lobe) 的情况下，在核酸内切切割之前，仅在 23-nt 指导 RNA 上的 5-14 位处扫描靶 RNA 的互补性，从而允许一些脱靶切割。值得注意的是，N 叶的获取将用于检查靶标互补性的指导 RNA 链的范围扩大到了位置 2-23。这些发现为催化活性 Argonaute 蛋白双叶结构的进化提供了线索。
Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, similar to 23-nucleotide (nt)-long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. A 2.1 angstrom-resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5-14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2-23. These findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.