Understanding the specificity and mechanism of gene silencing by Argonaute2 phosphorylation in Neurons.

Abstract

The RNA induced silencing complex (RISC) has an essential role in the regulation of protein translation in neurons. Specific miRNAs are involved in NMDA receptor
(NMDAR)-dependent synaptic plasticity by regulating the protein synthesis involved in dendritic spine morphogenesis. Rapid NMDAR-dependent silencing of Limk1 via miR134 is essential for spine shrinkage, these are modulated by S387 phosphorylation of Ago2, and the mechanisms that govern the selection of these specific mRNAs for silencing downstream of S387 phosphorylation are still unknown.
In this study, using RNA immunoprecipitation technique, it was demonstrated
that NMDAR-dependent S387 phosphorylation causes a rapid and transient increase in the association of Ago2 with Limk1, but not Apt1 mRNA, and the RNA helicase DDX6 is required for this phosphorylation-dependent increase. Furthermore, studying the effect of a DDX6 mutant (R386E) that disrupts the DDX6-CNOT1 interaction and hence DDX6 association with RISC, and a DDX6 helicase mutant (E247Q) showed that the specific increase in Limk1 mRNA binding to Ago2 S387 phosphomimic mutant (S387D) requires RISC-associated DDX6 helicase activity. In addition, dual luciferase assays and dendritic spine analysis in the presence of these DDX6 mutants demonstrated that RISC-associated DDX6 helicase activity is essential for NMDAR dependent Limk1 silencing via miR-134, but not Apt1 silencing via miR-138, and is
also essential for NMDAR-dependent dendritic spine shrinkage. This study elucidates a novel mechanism of NMDAR-dependent miRNA-mediated translational repression of specific genes to control dendritic spine morphology

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jonathan G Hanley (Supervisor)