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Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

ABSTRACT: Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

SUBMITTER: Deng T

PROVIDER: S-EPMC10460399 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

Deng Ting T Su Shichen S Yuan Xun X He Jinqiu J Huang Ying Y Ma Jinbiao J Wang Jia J

Nature communications 20230826 1

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into e ...[more]

PMID: 37633971

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Project description:Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing mechanisms that repress TEs expression: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2 that primarily helps to direct siRNAs for loading into Ago2. We provide deep sequencing evidence consistent with the idea that R2D2 and Loqs-PD can function in part redundantly. Certain transposons display a preference for either dsRBD-protein for production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in the germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway. small RNA sampling experiment; small RNAs were prepared from head & thorax as well as dissected ovaries of Adult female Drosophila melanogaster. We used homozygous mutants of the dsRBD proteins Loqs and r2d2 to determine their contribution to the biogenesis of transposon-derived small RNAs. Heterozygous mutant animals served as control. For each RNA sample, we performed one deep-sequencing run without any treatment, and in parallel one sequencing run after periodate oxidation and beta-elimination. After this treatment, only Ago2, Piwi, Aub and Ago3-loaded small RNAs remain as they carry a 2'-O-methyl modification at their 3'-end. This helps to determine the loading status of the small RNAs detected. In total 8 different RNA samples were prepared and 16 libraries were sequenced.

Dataset Information

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

Publications

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

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