Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain
Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain
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DOI：
10.1002/embj.201387176
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发表时间：
2014-02-18
期刊：
影响因子：
11.4
通讯作者：
Zamore, Phillip D.
中科院分区：
文献类型：
--
作者：
Fukunaga, Ryuya;Colpan, Cansu;Zamore, Phillip D.
在果蝇中，Dice-1从前miRNAs产生microRNAs(MiRNAs)，而Dice-2从长双链RNA(DsRNA)产生小干扰RNA，这是一个需要ATP水解的过程。我们先前的研究表明，无机磷可以抑制前miRNAs的DICER-2切割，但不能抑制长dsRNAs。在这里，我们报告了DICER-2对磷酸依赖的底物的识别反映了dsRNA底物的长度。DICER-2对短dsRNA的有效处理需要一个5‘末端的磷酸和一个两个核苷酸的3’悬垂，但不需要ATP。磷酸盐抑制这种短底物的裂解。相比之下，切割较长的dsRNA需要ATP，但不需要特定的末端结构：磷酸盐不抑制这些底物的切割。DICER-2 PAZ结构域中一对保守的精氨酸残基的突变阻止了短dsRNA的切割，但不能阻止长dsRNA的切割。我们认为无机磷酸占据了与短底物的5‘端磷酸结合所需的PAZ结构域口袋，阻止了它们的使用，并限制了果蝇的前miRNA加工到Dice-1。我们的研究有助于解释一个小分子如何改变核酸处理酶的底物专一性。
In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5 ' terminal phosphate and a two-nucleotide, 3 ' overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5 ' terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme.