HuR prevents amyloid beta-induced phase separation of miRNA-bound Ago2 to RNA-processing bodies.

Phase separation into membrane-less organelles regulates protein activity in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins localize to RNA-processing bodies (P-bodies), which are subcellular structures formed by several RNA-binding and regulatory proteins. Ago2, the essential miRNA-binding protein, forms a complex with miRNAs to repress protein synthesis by binding to mRNAs and targeting them to P-bodies. However, factors controlling Ago2 and miRNA-repressed mRNA compartmentalization into P-bodies are not fully understood. We developed a detergent-permeabilized cell-based assay system to observe the phase separation of exogenously added Ago2 into P-bodies in vitro. We observed that miRNA binding to Ago2 is essential for its localization to P-bodies, which is also ATP dependent. Osmolarity and salt concentration also affect Ago2 compartmentalization to P-bodies. Amyloid beta oligomers enhance Ago2 targeting to P-bodies by slowing down cellular Ago2 dynamics and inhibiting mTORC1 activity. However, the RNA-binder HuR disrupts P-body targeting by "sponging" out Ago2-associated miRNAs.