The Drosha-DGCR8 complex in primary microRNA processing

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• MicroRNA (miRNA) biogenesis is crucial for gene regulation, involving sequential processing by RNase III enzymes.
• The nuclear RNase III enzyme Drosha (class II) initiates miRNA processing by cleaving pri-miRNAs, but its mechanism is less understood than cytoplasmic Dicer (class III).
• Understanding Drosha's function is vital for comprehending the complete miRNA biogenesis pathway.

Purpose of the Study:

• To dissect the action mechanism of human Drosha, a class II RNase III enzyme.
• To identify and characterize interacting partners of Drosha involved in pri-miRNA processing.
• To propose a model for the function of class II RNase III proteins in miRNA biogenesis.

Main Methods:

• Site-directed mutagenesis to generate Drosha mutants.
• Biochemical assays to characterize Drosha's enzymatic activity and interactions.
• RNA interference (RNAi) to assess the role of interacting partners.
• Biochemical reconstitution experiments.

Main Results:

• Human Drosha functions as a large complex (approximately 650 kDa).
• Drosha interacts with DGCR8, a protein containing double-stranded RNA (dsRNA)-binding domains.
• The catalytic mechanism of Drosha involves its RNase III domains forming an intramolecular dimer, similar to human Dicer.
• DGCR8 is identified as a potentially essential component of the pri-miRNA processing complex with Drosha.

Conclusions:

• Human Drosha utilizes an intramolecularly dimerized RNase III domain structure for pri-miRNA cleavage.
• DGCR8 is a critical cofactor for Drosha, likely facilitating pri-miRNA binding and processing.
• A model for the action mechanism of class II RNase III proteins, including Drosha and DGCR8, is proposed.