Structural basis for chaperone-guided assembly of RNA-induced silencing complex.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The RNA-induced silencing complex (RISC) is crucial for RNA silencing, mediated by Argonaute (AGO) proteins and small RNAs.
• The precise mechanism of small RNA loading onto AGO and subsequent RISC assembly is not fully understood.
• Heat shock proteins (HSP70 and HSP90) are known to be involved in this process.

Purpose of the Study:

• To elucidate the molecular mechanism of RISC assembly.
• To identify and characterize the complex involved in AGO maturation and RNA loading.
• To provide insights into the rational design of small interfering RNA (siRNA) therapeutics.

Main Methods:

• Identification and purification of the human AGO-HSP90-p23 complex, termed the AGO maturation complex (AMC).
• Cryogenic electron microscopy (cryo-EM) to determine the structure of AMC bound to a microRNA duplex.
• Biochemical assays to investigate the role of RNA duplexes in AGO folding.

Main Results:

• The AMC captures AGO in an RNA-free, open conformation, distinct from its RISC-bound state.
• Cryo-EM revealed AGO domains detached and anchored to HSP90, connected by an unfolded linker, exposing a cleft for RNA binding.
• Small RNA duplexes, particularly those with a 5'-terminal phosphate, act as chaperone-like cofactors to facilitate AGO domain assembly and folding.

Conclusions:

• The study elucidates the de novo assembly mechanism of RISC, highlighting the AMC's role.
• The findings establish the AMC as a tool for optimizing RNA features for siRNA therapeutics.
• The research sheds light on chaperone-ligand interactions in guiding protein folding.