Eukaryote-specific insertion elements control human ARGONAUTE slicer activity.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Human ARGONAUTE1 (hAGO1) is a key protein in RNA interference pathways.
• Understanding hAGO1's structure and function is crucial for deciphering gene regulation.
• Evolutionary comparisons reveal insights into protein inactivation mechanisms.

Purpose of the Study:

• To determine the crystal structure of human ARGONAUTE1 (hAGO1) complexed with guide RNA.
• To identify structural differences between inactive hAGO1 and active ARGONAUTE2 (hAGO2).
• To understand the molecular basis for hAGO1's evolutionary inactivation.

Main Methods:

• X-ray crystallography was used to solve the structure of hAGO1 bound to guide RNA.
• Comparative structural analysis between hAGO1 and hAGO2 was performed.
• Site-directed mutagenesis was employed to test hypotheses about hAGO1 activity.

Main Results:

• The crystal structure of hAGO1 with guide RNA was elucidated.
• Inactive hAGO1 features proline residues (P670, P675) in the cS7 loop, creating a kink and steric hindrance.
• Restoring the catalytic tetrad and altering cS7 loop residues (P670S, P675Q) significantly increased hAGO1 cleavage activity.
• Evolutionary changes in hAGO1 were found to be reversible.

Conclusions:

• The kinked cS7 loop in hAGO1 sterically impedes guide-target RNA duplex binding in the active site.
• Specific proline substitutions in hAGO1 are primarily responsible for its loss of catalytic activity.
• The reversibility of these changes suggests that guide RNA loading and miRNA-target RNA pairing influence sequence drift.