An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

Authors
Golden RJ1,2, Chen B3,4, Li T1, Braun J1, Manjunath H1, Chen X1, Wu J5, Schmid V6, Chang TC1, Kopp F1, Ramirez-Martinez A1, Tagliabracci VS1, Chen ZJ1,7, Xie Y3,4,8, Mendell JT1,7,8,9.
02-08-2017
12:00pm
PST
Categories
Chemical Biology of RNA
Speaker
John Gagnon
Abstract
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824–S834), followed by rapid dephosphorylation by the ANKRD52–PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNAmediated silencing.