Sarkar Sucharita, Gebert Luca F R, MacRae Ian J
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
These authors contributed equally.
bioRxiv. 2024 Dec 6:2024.12.05.627081. doi: 10.1101/2024.12.05.627081.
Small interfering RNAs (siRNAs) guide mRNA cleavage by human Argonaute2 (hAgo2), leading to targeted gene silencing. Despite their laboratory and clinical impact, structural insights into human siRNA catalytic activity remain elusive. Here, we show that disrupting siRNA 3'-end binding by hAgo2 accelerates target cleavage and stabilizes its catalytic conformation, enabling detailed structural analysis. A 3.16 Å global resolution cryo-EM reconstruction reveals that distortion of the siRNA-target duplex at position 6 allows target RNA entry into the catalytic cleft and shifts Lysine-709, a previously unrecognized catalytic residue, into the active site. A pyrimidine at target nucleotide t10 positions another unrecognized catalytic residue, Arginine-710, for optimal cleavage. Expansion of the guide-target duplex major groove docks the scissile phosphate for hydrolysis and subsequent groove compression after position 16 permits target RNAs to exit the catalytic cleft. These findings reveal how hAgo2 catalyzes siRNA target hydrolysis, providing a high-resolution model for therapeutic design.
小分子干扰RNA（siRNA）引导人类AGO2蛋白（hAgo2）切割信使核糖核酸（mRNA），从而实现靶向基因沉默。尽管siRNA在实验室研究和临床应用方面都有重要影响，但人类siRNA催化活性的结构解析仍然不够深入。在此，我们发现，hAgo2对siRNA 3'端结合的破坏会加速靶标切割并稳定其催化构象，从而能够进行详细的结构分析。一项分辨率为3.16 Å的全分辨率冷冻电镜重建显示，siRNA与靶标双链体在第6位的扭曲使得靶标RNA能够进入催化裂隙，并将赖氨酸-709（一个此前未被识别的催化残基）转移到活性位点。靶标核苷酸t10处的嘧啶将另一个未被识别的催化残基精氨酸-710定位到最佳切割位置。引导-靶标双链体大沟的扩展使可切割磷酸基团对接以进行水解，在第16位之后随后的沟压缩允许靶标RNA离开催化裂隙。这些发现揭示了hAgo2如何催化siRNA靶标水解，为治疗设计提供了一个高分辨率模型。
