Structural basis for gene silencing by siRNAs in humans

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Small interfering RNAs (siRNAs) are crucial for targeted gene silencing via mRNA cleavage mediated by human Argonaute2 (hAgo2).
• Despite their significance in research and therapeutics, the precise structural mechanisms underlying hAgo2's catalytic activity remain poorly understood.

Purpose of the Study:

• To elucidate the structural basis of siRNA-mediated mRNA cleavage by human Argonaute2 (hAgo2).
• To provide high-resolution structural insights into the catalytic mechanism of hAgo2 for therapeutic design.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to determine the structure of hAgo2 with a modified siRNA-target duplex.
• Biochemical assays were used to assess the impact of structural modifications on catalytic activity.

Main Results:

• Disrupting siRNA 3'-end binding to hAgo2 stabilized the enzyme's catalytic conformation, facilitating structural analysis.
• A 3.16 Å cryo-EM reconstruction revealed key interactions, including target RNA entry into the catalytic cleft and the roles of previously unrecognized catalytic residues Lysine-709 and Arginine-710.
• Specific nucleotide positioning and duplex major groove dynamics were shown to be critical for docking the scissile phosphate and facilitating target RNA hydrolysis and release.

Conclusions:

• The study reveals the detailed mechanism of siRNA target hydrolysis by hAgo2, highlighting specific residue interactions and conformational changes.
• A high-resolution structural model of hAgo2's catalytic activity is presented, offering valuable insights for the rational design of RNA interference-based therapeutics.