The Drosha-DGCR8 complex in primary microRNA processing

TLDR

RNase III enzymes are central to microRNA biogenesis, with Drosha cleaving primary miRNAs into pre‑miRNAs, yet the class II enzyme Drosha remains poorly characterized compared to Dicer and other RNase III proteins. The study aimed to dissect human Drosha’s catalytic mechanism and identify its interacting partner DGCR8. Using mutant analysis and biochemical reconstitution, the authors revealed that Drosha forms a ~650 kDa complex with DGCR8, whose two dsRNA‑binding domains associate with Drosha’s intramolecular dimeric RNase III domains that cleave the 3′ and 5′ strands of the pri‑miRNA stem. These results demonstrate that DGCR8 is an essential component of the pri‑miRNA processing complex, that Drosha’s action resembles Dicer’s intramolecular dimer cleavage, and support a model for class II RNase III enzyme function.

Abstract

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 ∼650 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.