Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD.
Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD.
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Drosophila Dicer-2-Loqs-PD对dsRNA加工的结构见解。
DOI：
10.1038/s41586-022-04911-x
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发表时间：
2022-07
期刊：
影响因子：
64.8
通讯作者：
中科院分区：
文献类型：
--
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小干扰RNA （sirna）是RNA干扰（RNAi）的关键组成部分，RNAi是许多真核生物中保守的RNA沉默机制。在果蝇中，一种RNase III酶Dicer-2 （Dcr-2）在其辅因子Loqs-PD的辅助下，在长双链rna （dsRNAs）生成21 bp的siRNA双链中起着重要作用。Dcr-2解旋酶结构域的ATP水解是成功将长dsRNA加工成连续的siRNA双链的关键。在这里，我们报道了Dcr-2-Loqs-PD在载脂蛋白状态和处理50 bp dsRNA底物的多种状态下的低温电镜结构。这些结构阐明了Dcr-2和Loqs-PD之间的相互作用，以及Dcr-2在dsrna加工周期中的实质性构象变化。初始dsRNA结合后，n端解旋酶和未知功能域283 （DUF283）结构域发生构象变化，形成atp结合袋和5 ' -磷酸结合袋。在ATP存在的情况下，Dcr-2-Loqs-PD沿dsRNA易位过程中的整体构象是相对刚性的，而DUF283和RIIIDb结构域之间的相互作用通过阻断dsRNA进入RNase活性中心来防止易位过程中的非特异性切割。额外的atp依赖的构象变化需要形成一个活跃的切割状态，并精确地将dsRNA切割成一个21 bp的siRNA双工，这被切割后状态的结构所证实。总的来说，本研究揭示了dcr -2 - loq - pd加工atp依赖性dsRNA整个周期的分子机制。Dcr-2 - loq - pd复合物在加工双链RNA （dsRNA）底物时的结构阐明了Dcr-2和loq - pd之间的相互作用，并表明Dcr-2在dsRNA加工周期中经历了实质性的构象变化。
Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD. Structures of the Dcr-2–Loqs-PD complex while it is processing a double-stranded RNA (dsRNA) substrate elucidate the interactions between Dcr-2 and Loqs-PD, and show that Dcr-2 undergoes substantial conformational changes during a dsRNA-processing cycle.
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