Mechanisms of Phosphorylation‐Mediated Regulation of the RNAi Effector Protein Argonaute 2

Abstract

Argonaute (Ago) proteins play a central role in gene regulation through a process called RNA interference (RNAi). Ago binds to small RNAs (sRNAs) such as small interfering RNA (siRNA) and microRNA (miRNA) to form the functional core of the RNA Induced Silencing Complex (RISC), which targets mRNAs with complementary sequences, ultimately leading to down‐regulation of the corresponding genes. It was previously shown that phosphorylation of a cluster of residues (824–834) in the “eukaryotic insertion” (EI) of Ago by CK1α can alleviate this repression in a variety of cell types. However, the mechanisms of such regulation were unknown. Using in‐vitro phosphorylation assays, we show that binding of miRNA‐loaded Ago2 to target RNA primes the EI for phosphorylation. Furthermore, we show that this phosphorylation reduces the affinity of the Ago‐miRNA complex to its target mRNA. While the EI of Ago2 contains 5 potential phosphorylation sites, we propose that specific residues in the EI are more susceptible to phosphorylation and dictate the pattern and sequence of the phosphorylation events. We also examined the structural requirements for the target to induce phosphorylation. These findings shed light on the manner in which phosphorylation of the EI acts to regulate the RNAi pathway central to gene regulation. The high conservation of potential phosphosites in the EI (both among Ago homologs and throughout eukaryotes) suggests that such a regulatory strategy may be a shared mechanism with broader impact.Support or Funding InformationThis work was supported by the NSF Graduate Research Fellowship Program and Cold Spring Harbor Laboratory’s Watson School of Biological Sciences. L.J. is an Investigator of the Howard Hughes Medical Institute.