Charbonnel Cyril, Niazi Adnan K, Elvira-Matelot Emilie, Nowak Elzbieta, Zytnicki Matthias, de Bures Anne, Jobet Edouard, Opsomer Alisson, Shamandi Nahid, Nowotny Marcin, Carapito Christine, Reichheld Jean-Philippe, Vaucheret Hervé, Sáez-Vásquez Julio
CNRS, Laboratoire Génome et Développement des Plantes, UMR 5096, 66860 Perpignan, France.
University of Perpignan Via Domitia, Laboratoire Génome et Développement des Plantes, UMR 5096, F-66860 Perpignan, France.
Nucleic Acids Res. 2017 Nov 16;45(20):11891-11907. doi: 10.1093/nar/gkx820.
RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.
核糖核酸酶III（RNase III）可切割双链（ds）RNA。这是调控信使核糖核酸（mRNA）、核糖体RNA（rRNA）、核仁小RNA（snoRNA）以及包括小干扰RNA（siRNA）和微小RNA（miRNA）在内的其他小RNA加工过程的关键步骤。拟南芥编码9种RNase III：4种类似Dicer（DCL）和5种类似核糖核酸酶III（RTL）。为了更好地理解植物中RNase III的分子功能，我们以RTL1为模型开发了一种生化检测方法。我们发现RTL1并非随机降解dsRNA，而是识别特定的双链序列以指导精确切割。此外，我们证明了dsRNA切割需要RNase III和双链RNA结合结构域（dsRBD）。有趣的是，携带dsRBD的4种DCL和3种RTL在其dsRBD中共享一个保守的半胱氨酸（拟南芥RTL1中的C230）。C230对RTL1和DCL1的活性至关重要，且会发生转录后修饰。实际上，在氧化条件下，C230的谷胱甘肽化以一种涉及谷氧还蛋白的可逆方式抑制RTL1的切割活性。我们得出结论，dsRBD的氧化还原状态确保了植物RNase III对dsRNA加工的精细调控。
