A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• MicroRNA (miRNA) biogenesis involves the Microprocessor complex (MC), comprising Drosha and DGCR8.
• The exact stoichiometry of Drosha and DGCR8 within the MC was previously unknown.
• Existing models suggested sequential binding of proteins to pri-miRNA, but recent findings challenged this.

Purpose of the Study:

• To determine the precise stoichiometry of Drosha and DGCR8 within the human Microprocessor complex.
• To investigate whether the MC exists as a preformed complex in the absence of RNA.

Main Methods:

• Single-molecule photobleaching assays were employed to analyze protein stoichiometry.
• Experiments utilized full-length proteins expressed and purified from human cells.
• Tagged proteins (fluorescent and hAGT-derived) were used to confirm findings.

Main Results:

• The human MC forms a heterotrimeric complex consisting of two DGCR8 molecules and one Drosha molecule.
• This heterotrimeric complex is preformed and exists independently of RNA binding.
• The stoichiometry was confirmed using different protein tags and expression systems.

Conclusions:

• The Microprocessor complex adopts a stable heterotrimeric structure (2x DGCR8:1x Drosha).
• This complex is likely preassembled before binding to pri-miRNA substrates.
• The findings refine our understanding of miRNA biogenesis initiation.