The Drosha-DGCR8 complex in primary microRNA processing
The Drosha-DGCR8 complex in primary microRNA processing
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DOI：
10.1101/gad.1262504
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发表时间：
2004-12-15
影响因子：
10.5
通讯作者：
Kim, VN
中科院分区：
文献类型：
--
作者：
Han, JJ;Lee, Y;Kim, VN
RNase III蛋白在microRNA（miRNA）生物合成中起关键作用。核RNase III Drosha切割初级miRNA（pri-miRNA）以释放发夹形的pre-miRNA，其随后被细胞质RNase III Dicer切割以产生成熟的miRNA。虽然Dicer（III类）和其他简单的RNase III蛋白（I类）已被深入研究，但II类酶Drosha仍有待表征。在这里，我们通过产生突变体和表征其新的相互作用伴侣DGCR 8来剖析人类Drosha的作用机制。发现Drosha的基本作用机制与人Dicer相似; RNase III结构域A和B形成分子内二聚体并分别切割茎的3'和5'链。人Drosha在650 kDa处分离，表明Drosha作为一个大的复合物起作用。在这个复合物中，Drosha与DGCR 8相互作用，DGCR 8包含两个双链RNA（dsRNA）结合结构域。通过RNAi和生化重建，我们发现DGCR 8可能是pri-miRNA加工复合物的重要组成部分，沿着Drosha。基于这些结果，我们提出了一个模型的II类RNase III蛋白的作用机制。
RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs. While Dicer (class III) and other simple RNase III proteins (class I) have been studied intensively, the class II enzyme Drosha remains to be characterized. Here we dissected the action mechanism of human Drosha by generating mutants and by characterizing its new interacting partner, DGCR8. The basic action mechanism of Drosha was found to be similar to that of human Dicer; the RNase III domains A and B form an intramolecular dimer and cleave the 3' and 5' strands of the stem, respectively. Human Drosha fractionates at similar to650 kDa, indicating that Drosha functions as a large complex. In this complex, Drosha interacts with DGCR8, which contains two double-stranded RNA (dsRNA)-binding domains. By RNAi and biochemical reconstitution, we show that DGCR8 may be an essential component of the pri-miRNA processing complex, along with Drosha. Based on these results, we propose a model for the action mechanism of class II RNase III proteins.