Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.
Drosophila dicer-2 cleavage is mediated by helicase- and dsRNA termini-dependent states that are modulated by Loquacious-PD.
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DOI：
10.1016/j.molcel.2015.03.012
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发表时间：
2015-05-07
期刊：
影响因子：
16
通讯作者：
Bass, Brenda L.
中科院分区：
文献类型：
--
作者：
Sinha, Niladri K.;Trettin, Kyle D.;Aruscavage, P. Joseph;Bass, Brenda L.
在之前的研究中，我们观察到果蝇 Dicer-2 (dmDcr-2) 的解旋酶结构域控制底物识别和切割效率，而 dsRNA 末端是这种区分的关键。我们现在为这些观察结果提供了机制基础。我们表明，末端的辨别发生在初始结合期间。如果没有 ATP，dmDcr-2 会结合 3' 突出末端，但不结合平末端。相比之下，dmDcr-2 与 ATP 结合，结合两种类型的末端，用平端 dsRNA 观察到的结合亲和力最高。在 ATP 存在的情况下，与 3'ovr 末端相比，BLT 末端的结合、裂解和 ATP 水解效果最佳。有限的蛋白水解实验表明，BLT dsRNA 的最佳反应性是由依赖于 ATP 和解旋酶结构域的构象变化介导的。我们发现 dmDcr-2 的伴侣蛋白 Loquacious-PD 改变了末端依赖性，使 dmDcr-2 能够切割通常难以切割的底物，例如具有封闭、结构化或磨损末端的 dsRNA。
In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3′ overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3′ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2’s partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured or frayed ends.
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