Department of Biochemistry, University of Utah, Salt Lake City, UT 84112.
Department of Biochemistry, University of Utah, Salt Lake City, UT 84112
Proc Natl Acad Sci U S A. 2017 Sep 19;114(38):E7939-E7948. doi: 10.1073/pnas.1707063114. Epub 2017 Sep 5.
Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs in In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2's helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.
Loquacious-PD（Loqs-PD）是许多内源性 siRNA 生物发生所必需的。在体外，Loqs-PD 增强了 Dicer-2 切割双链 RNA 的速度，并且还能够处理通常对切割有抗性的底物。使用纯化的组分和 Loqs-PD 截断物，我们为 Loqs-PD 的功能提供了一个机制基础。我们的研究表明，Loqs-PD 的 C 末端的 22 个氨基酸，包括一个 FDF 样基序，直接与 Dicer-2 的解旋酶结构域的 Hel2 亚结构域相互作用。这种相互作用是 RNA 非依赖性的，但我们发现 Dicer-2 切割的调节还需要 Loqs-PD 与双链 RNA 的结合。此外，虽然 Loqs-PD 的第一个双链 RNA 结合基序对于增强最佳底物的切割不是必需的，但对于增强次优底物的切割是必需的。最后，我们的研究定义了一个以前未被识别的 Dicer 相互作用界面，并表明 Loqs-PD 很好地定位以将底物募集到 Dicer-2 的解旋酶结构域中。
