Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

Area of Science:

• Molecular Biology
• RNA Interference
• Enzymology

Background:

• Dicer-1 and Dicer-2 are key enzymes in Drosophila RNA processing, producing microRNAs (miRNAs) and small interfering RNAs (siRNAs), respectively.
• Previous work indicated inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs but not long double-stranded RNA (dsRNA).
• The precise mechanism of Dicer-2 substrate discrimination and phosphate inhibition remained unclear.

Purpose of the Study:

• To investigate the role of inorganic phosphate in Dicer-2 substrate specificity.
• To elucidate the structural and biochemical basis for Dicer-2's differential processing of short versus long dsRNA.
• To understand how small molecules can modulate nucleic acid processing enzyme activity.

Main Methods:

• Biochemical assays measuring Dicer-2 cleavage activity on short and long dsRNA substrates with varying end structures.
• ATP hydrolysis assays to assess energy requirements for processing different dsRNA lengths.
• Site-directed mutagenesis of conserved arginine residues in the Dicer-2 PAZ domain.

Main Results:

• Dicer-2 processing of short dsRNA requires a 5' terminal phosphate and a 3' overhang, is ATP-independent, and is inhibited by inorganic phosphate.
• Dicer-2 processing of long dsRNA is ATP-dependent, does not require specific end structures, and is not inhibited by phosphate.
• Mutating specific PAZ domain arginine residues abolished short dsRNA processing but spared long dsRNA processing.

Conclusions:

• Inorganic phosphate acts as a regulator of Dicer-2 activity, discriminating between short and long dsRNA substrates.
• Phosphate likely binds to a PAZ domain pocket, blocking the interaction with the 5' phosphate of short dsRNA substrates.
• This mechanism ensures Dicer-1 exclusively processes pre-miRNAs and Dicer-2 processes longer dsRNAs, maintaining distinct RNAi pathways in Drosophila.