RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA
RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA
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作者：
Parker, GS;Eckert, DM;Bass, BL
在从拟南芥到人类的生物体中，Dicer需要dsRNA结合蛋白（dsRBP）来执行其在RNA干扰（RNAi）和微小RNA（miRNA）加工中的作用。在秀丽隐杆线虫中，当长dsRNA被切割成小干扰RNA（siRNA）时，dsRBP RDE-4在RNAi起始期间与Dicer起作用。RDE-4在后续步骤中不需要，并且RDE-4如何区分长dsRNA和短siRNA尚不清楚。我们报告的第一个详细的分析RDE-4结合，使用纯化的重组RDE-4和各种截短的蛋白质。我们发现，类似于其他dsRBP，RDE-4不是序列特异性的。然而，与其体内作用一致，RDE-4以更高的亲和力与长双链RNA结合。我们还观察到RDE-4在溶液中是同源二聚体，并且蛋白质的C-末端结构域是二聚化所需的。利用野生型和rde-4突变体C. elegans中，我们表明C-末端二聚化结构域是生产siRNA所必需的。我们的研究结果表明，RDE-4的功能在RNAi的启动模型。
In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.