Cofactor-assisted dicing: insights from structural snapshots.
Cofactor-assisted dicing: insights from structural snapshots.
复制标题
辅因子辅助切割：结构快照的见解。
DOI：
10.1038/s41422-022-00716-9
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发表时间：
2022
期刊：
影响因子：
44.1
通讯作者：
Patel,DinshawJ
中科院分区：
文献类型：
--
作者：
Du,Jiamu;Patel,DinshawJ
在真核细胞中，大小为 20 至 30 个核苷酸 (nt) 的小 RNA (sRNA) 是 RNA 介导的基因沉默途径的关键参与者，在基因调控、发育、DNA 甲基化和病毒防御中发挥关键功能作用。值得注意的是，dsRNA 衍生的小干扰 RNA (siRNA) 和内源发夹 RNA 衍生的 microRNA (miRNA) 都需要 RNase III 家族核酸内切酶 Dicer 来实现其长度依赖性和末端特异性生物发生。 1 Dicer 核酸酶在 sRNA 生物发生途径的所有步骤中发挥着核心作用，包括底物 RNA 选择和加载、RNA 在活性位点内的定位、长度依赖性切割以及通过参与其蛋白质和 RNA 成分中的一组构象转变将 sRNA 产物释放到下游 Argonaute (Ago) 效应蛋白。自发现以来，DicerLs 功能的分子机制已对 sRNA 领域提出了核心挑战。 2 apo 状态下的极简（缺乏解旋酶结构域）肠贾第鞭毛虫 Dicer 的初始晶体结构定义了蛋白质亚基的排列，这反过来又允许在 Dicer 上定位 dsRNA 的生化验证模型，其中 PAZ 结构域识别的 RNA 3' 突出端精确定位 dsRNA，通过一对 RNase III 结构域形成的复合袋实现长度依赖性 2 碱基对 (bp) 交错切割。 3 接下来，该提议的 3' 计数规则被附加的 5' 计数规则更新，其中切割位点主要根据果蝇系统中的 5'-磷酸进行测量。 4 为了从结构上了解 Dicer 和 dsRNA 之间的排列和分子间接触，我们对人类 5、6 和果蝇 7 Dicer-dsRNA 复合物进行了冷冻电镜研究，从而深入了解该复合物的切割前构象。这些研究随后对植物 Dicer DCL3 与 pre-siRNA 8 结合以及植物 Dicer DCL1 在活性切割能力构象中与 pri-和 pre-miRNA 9 结合进行了冷冻电镜研究，揭示了对底物选择性和 RNA 长度测量基础原理的更多见解。
In eukaryotic cells, small RNAs (sRNAs) ranging in size from~ 20 to 30 nucleotides (nt) are critical players in the RNA-mediated gene silencing pathway, playing key functional roles in gene regulation, development, DNA methylation and viral defense. Notably, dsRNA-derived small interfering RNAs (siRNAs) and endogenous hairpin RNA-derived microRNAs (miRNAs) both require the RNase III family endonuclease Dicer for their lengthdependent and termini-specific biogenesis. 1 The Dicer nuclease plays a central role throughout all steps of the sRNA biogenesis pathway, including substrate RNA selection and loading, positioning of RNA within the active site, length-dependent dicing and release of the sRNA product to downstream Argonaute (Ago) effector proteins, through involvement of a set of conformational transitions in both its protein and RNA components. Since its discovery, the molecular mechanism underlying DicerLs function has posed a central challenge in the sRNA field. 2 The initial crystal structure of minimalist (lacks a helicase domain) Giardia intestinalis Dicer in the apo state defined the alignment of protein subunits, which in turn allowed a biochemically validated model for positioning of dsRNA on Dicer, whereby RNA 3′-overhang recognition by the PAZ domain precisely positioned the dsRNA for length-dependent 2-base pair (bp) staggered cleavage by the composite pockets formed by the pair of RNase III domains. 3 This proposed 3′-counting rule was next updated by an additional 5′-counting rule where the cleavage site was measured primarily from the 5′-phosphate in the Drosophila system. 4 To gain a structure-based understanding of the alignment and intermolecular contacts between Dicer and dsRNA, cryo-EM studies have been undertaken on both the human 5, 6 and Drosophila 7 Dicer-dsRNA complexes, thereby providing insights into the pre-dicing conformation of the complex. These studies were followed by cryo-EM studies of plant Dicer DCL3 bound to pre-siRNA 8 and plant Dicer DCL1 bound to pri-and pre-miRNAs 9 in the active dicing-competent conformation that revealed additional insights into substrate selectivity and principles underlying RNA length measurement.