Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins

Katherine S Bridge,Kunal M Shah,Yigen Li,Daniel E Foxler,Sybil C.K Wong,Duncan C Miller,Kathryn M Davidson,John G Foster,Ruth Rose,Michael R Hodgkinson,Paulo S Ribeiro,A Aziz Aboobaker,Kenta Yashiro,Xiaozhong Wang,Paul R Graves,Michael J Plevin,Dimitris Lagos,Tyson V Sharp

doi:10.1016/j.celrep.2017.06.027

Abstract

SummaryAs core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

Highlights

MicroRNAs are $22 nucleotide non-coding RNA molecules that silence gene expression post-transcriptionally (Bartel, 2004)
We show that Akt3-mediated phosphorylation of AGO2 (S387) promotes a phospho-dependent interaction with LIMD1 that is vital for enabling engagement of AGO2 with TNRC6A, revealing the mechanism by which AGO-S387 phosphorylation regulates microRNA-induced silencing complex (miRISC) function
AGO Utilization Is Determined by LIMD1 Members of the LIMD1 family (LIMD1, AJUBA, and WTIP) of LIMdomain-containing proteins associate with miRISC and are required for miRNA-mediated silencing (James et al, 2010)

Summary

Introduction

MicroRNAs are $22 nucleotide non-coding RNA molecules that silence gene expression post-transcriptionally (Bartel, 2004) Their loading onto Argonaute (AGO) proteins facilitates base-pairing to mRNA targets with partial complementarity and translational repression and mRNA degradation (Huntzinger and Izaurralde, 2011; Hutvagner and Simard, 2008). While the majority of miRNAs were found in low-molecular-weight miRISC complexes in resting T cells, stimulation of phosphatidylinositol 3-kinase (PI3K) signaling caused an increased association of miRNAs with high-molecular-weight miRISC complexes and an enhancement of miRNA-mediated silencing (La Rocca et al, 2015).

Results

Discussion

Conclusion