A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding

First author: Benoît Derrien; Affiliations: Swiss Federal Institute of Technology Zurich (苏黎世联邦理工学院): Zurich, Switzerland

Corresponding author: Pascal Genschik(Institut de Biologie Moleculaire des Plantes )

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses (抗病毒响应). The polerovirus (马铃薯卷叶病毒属) F-box P0 protein triggers AGO1 degradation as a viral counter-defence (反防御). Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0-degron (降解决定子) is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening 正向遗传学筛查) compromises recognition by SCFP0. Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding (解链), leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes (双链体), but is mostly dispensable (不必要) for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and thein vivofunction of DUF1785 in RNA silencing.

在拟南芥中，AGO1在microRNA和siRNA介导的基因沉默方面发挥着重要作用，同时也是植物抗病毒响应的关键元件。马铃薯卷叶病毒属的F-box P0蛋白会诱导AGO1的降解，作为病毒的一种反防御措施。本文鉴定了AGO1上的一个基序对于其与S期激酶相关蛋白SKP1-cullin蛋白CUL1-F-box蛋白SCFP0复合物的互作以及随后AGO1的降解至关重要。AGO1 P0降解决定子非常保守，因而赋予了P0能够降解其它AGO蛋白的能力。有趣的是，降解子基序定位在AGO1的DUF1785结构域上，在该结构域上通过正向遗传筛查的一个点突变ago1-57破坏了SCFP0识别AGO1的能力。在一个cell-free的系统中利用RNA诱导沉默复合物揭示了该突变会破坏RNA的解链，导致损伤的AGO1仍然结合到双链的RNA上。在体内，DUF1785对于解链完美匹配siRNA双链体十分必要，但对于解链不完美匹配miRNA双链体可有可无。因此，这个突变近乎废除了phased siRNA的形成和感知转基因的转录后基因沉默。综上，本文的工作促进了对于P0识别AGO1的认知，以及体内DUF1785在RNA沉默中的功能。

通讯：Pascal Genschik (http://www.ibmp.cnrs.fr/annuaire/pascal-genschik/)

doi: https://doi.org/10.1105/tpc.18.00111

Journal: Plant Cell

First Published: 30 May, 2018

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