The study investigates the action mechanism of the human Drosha enzyme, which is crucial for primary microRNA (pri-miRNA) processing. Drosha, a class II RNase III protein, cleaves pri-miRNAs to generate hairpin-shaped pre-miRNAs, which are then processed by Dicer into mature miRNAs. The researchers generated mutants of Drosha and characterized its interaction with DGCR8, a protein containing two double-stranded RNA (dsRNA)-binding domains. Key findings include: 1. **Action Mechanism of Drosha**: Drosha forms an intramolecular dimer with its two RNase III domains (RIIIDa and RIIDb), where RIIDa cuts the 3′ strand and RIIDb cuts the 5′ strand of the stem. This mechanism is similar to that of human Dicer. 2. **Complex Formation**: Drosha functions as part of a large complex (~650 kDa), which includes DGCR8. DGCR8 is essential for the processing of pri-miRNAs and may interact with Drosha through its middle region and RIIDa. 3. **Domain Mapping**: Deletion mutants of Drosha were used to map the essential domains for processing and complex formation. The middle region, RIIDa, and RIIDb are crucial for these functions. 4. **Homodimerization**: Drosha molecules interact with each other, suggesting that multiple copies of Drosha may be present in the complex. 5. **Model for Pri-miRNA Cleavage**: The study proposes a model where the two RIIDa and RIIDb form a single processing center, with E110 residues playing a key role in catalysis. DGCR8 stabilizes the substrate and helps in the correct orientation of Drosha on the pri-miRNA. These findings provide insights into the fundamental similarities and differences between class II and class I RNase III proteins, and highlight the importance of DGCR8 in the Drosha complex.The study investigates the action mechanism of the human Drosha enzyme, which is crucial for primary microRNA (pri-miRNA) processing. Drosha, a class II RNase III protein, cleaves pri-miRNAs to generate hairpin-shaped pre-miRNAs, which are then processed by Dicer into mature miRNAs. The researchers generated mutants of Drosha and characterized its interaction with DGCR8, a protein containing two double-stranded RNA (dsRNA)-binding domains. Key findings include: 1. **Action Mechanism of Drosha**: Drosha forms an intramolecular dimer with its two RNase III domains (RIIIDa and RIIDb), where RIIDa cuts the 3′ strand and RIIDb cuts the 5′ strand of the stem. This mechanism is similar to that of human Dicer. 2. **Complex Formation**: Drosha functions as part of a large complex (~650 kDa), which includes DGCR8. DGCR8 is essential for the processing of pri-miRNAs and may interact with Drosha through its middle region and RIIDa. 3. **Domain Mapping**: Deletion mutants of Drosha were used to map the essential domains for processing and complex formation. The middle region, RIIDa, and RIIDb are crucial for these functions. 4. **Homodimerization**: Drosha molecules interact with each other, suggesting that multiple copies of Drosha may be present in the complex. 5. **Model for Pri-miRNA Cleavage**: The study proposes a model where the two RIIDa and RIIDb form a single processing center, with E110 residues playing a key role in catalysis. DGCR8 stabilizes the substrate and helps in the correct orientation of Drosha on the pri-miRNA. These findings provide insights into the fundamental similarities and differences between class II and class I RNase III proteins, and highlight the importance of DGCR8 in the Drosha complex.