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.

The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release ∼70 nt pre‐miRNAs that are subsequently processed by Dicer to generate mature ∼22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, our database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. Our study provides a novel example of eukaryotic gene organization and RNA‐processing control.

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Significance: MicroRNAs (miRNAs) are noncoding RNAs with diverse roles in development and pathogenesis. Biogenesis of canonical miRNA requires nuclear processing by DROSHA, nuclear export by Exportin 5, and cytoplasmic processing by DICER. To gain a deeper understanding of the maturation processes, we here ablated the DROSHA , Exportin 5 , and DICER genes using the same human cell line. Canonical miRNA production was abolished in DROSHA -deleted cells, revealing an irreplaceable role of DROSHA. Interestingly, however, some canonical miRNAs were still produced without DICER albeit at markedly reduced levels, and many were detected in Exportin 5 -deleted cells at only modestly decreased levels. Our study allows us to understand differential contributions of key biogenesis factors, and provides valuable resources for miRNA research.