HuR Prevents Amyloid Beta Induced Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodies

Abstract

Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies or P-bodies, the subcellular structures that are formed due to the assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming a complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors that control the compartmentalization of Ago2 and miRNA-repressed mRNAs to P-bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to P-bodies in vitro. miRNA binding of Ago2 is essential for its targeting to P-bodies and the process is ATP-dependent. The P-body compartmentalization of Ago2 is also influenced by the osmolarity and salt concentration of the reaction buffer. Amyloid beta oligomers enhance Ago2-miRNP targeting to RNA processing bodies by retarding the dynamics of cytosolic Ago2 and  inhibiting mTORC1 activity. However, P-body targeting gets retarded by RNA binding protein HuR that reverses P-body localization by “sponging” out Ago2- associated miRNAs.

Keywords: miRNA, RNA processing body, Liquid-liquid phase separation, AGO2, HuR, Amyloid beta.