Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease.
Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease.
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DOI：
10.1016/j.molcel.2011.03.002
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发表时间：
2011-04-22
期刊：
影响因子：
16
通讯作者：
Zamore PD
中科院分区：
文献类型：
--
作者：
Cenik ES;Fukunaga R;Lu G;Dutcher R;Wang Y;Tanaka Hall TM;Zamore PD
果蝇Dicer-2从长双链RNA （dsRNA）产生小干扰RNA (sirna)，而Dicer-1从前microrna产生microrna。是什么使这两种dicer对它们的生物底物具有特异性？我们发现纯化的Dicer-2可以有效地切割pre-miRNA，但无机磷酸盐和Dicer-2伴侣蛋白R2D2抑制pre-miRNA的切割。Dicer-2包含介导RNA切割的c端RNase III结构域和功能尚不清楚的n端解旋酶基序。我们发现Dicer-2是一种dsrna刺激的ATP酶，它将ATP水解成ADP；Dicer-2需要ATP水解来处理长dsRNA，但不需要预处理mirna。野生型Dicer-2，但不是ATP水解缺陷的突变体，产生sirna的速度比从长dsRNA底物解离的速度快。我们提出Dicer-2解旋酶结构域在与底物分离之前使用ATP从单个dsRNA分子中产生许多sirna。
Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from pre-microRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage, and an N-terminal helicase motif whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.
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