Department of Pathology and Laboratory Medicine, Division of Neuropathology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Cell. 2012 May 25;46(4):507-17. doi: 10.1016/j.molcel.2012.03.010. Epub 2012 Apr 12.
Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.
miRNA 核糖核蛋白（miRNPs）或 RNA 诱导沉默复合物（RISCs）的组装对于 miRNA 的功能至关重要，并且从 miRNA 前体（pre-miRNAs）的 Dicer 或 Ago2 加工开始。在这里，我们报告了一种由哺乳动物细胞裂解物中的 pre-miRNA 编程的体外 miRNP/RISC 组装测定。结合在 Dicer 敲除细胞中的体内研究，这些细胞用野生型或无催化活性的 Dicer 重建，我们发现 miRNA 加载复合物（miRLC）是将 pre-miRNA 加工与 miRNA 加载联系起来的主要机制。我们表明，miRNA 前体沉积复合物（miPDC）在 Dicer 非依赖性 miRNA 生物发生中起着关键作用，并促进某些 Dicer 依赖性 miRNA 的 miRNP 组装。此外，我们发现 pre-miRNA 内的 5'-尿嘧啶、3'-中碱基配对和 5'-中碱基错配促进它们组装成 miPDC。我们的研究提供了哺乳动物中 miRNP/RISC 组装途径的全面视图，并且我们的测定为进一步在哺乳动物中对这些途径的机制进行剖析提供了一个通用平台。
