Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Argonaute proteins are central to microRNA-mediated gene silencing.
• Post-translational modifications, like phosphorylation, regulate Argonaute protein function.
• Understanding these modifications is key to deciphering gene regulation.

Purpose of the Study:

• To investigate the role of phosphorylation in Argonaute protein function.
• To identify specific phosphorylation sites and their impact on microRNA and mRNA interactions.
• To elucidate the contribution of Argonaute phosphorylation to gene silencing.

Main Methods:

• Quantitative mass spectrometry was employed to identify phosphorylation sites.
• Functional assays were performed on wild-type and mutant Argonaute proteins (Ago2 and ALG-1).
• MicroRNA binding, mRNA binding, localization, and gene silencing activity were assessed.

Main Results:

• A C-terminal serine/threonine cluster is phosphorylated at five residues in human and C. elegans Argonaute proteins.
• In human Ago2, hyper-phosphorylation affects mRNA binding but not microRNA binding or cleavage activity.
• Phosphorylation is essential for microRNA function in C. elegans ALG-1, with single mutations phenocopying full phosphorylation loss.

Conclusions:

• The phosphorylation state of the Argonaute C-terminal cluster is critical for gene silencing.
• Phosphorylation primarily influences Argonaute-mRNA interactions, particularly at later stages of gene silencing.
• These findings highlight a novel regulatory mechanism in microRNA-mediated gene silencing.