HuR-miRNA complex Activates RAS GTPase RalA to Facilitate Endosome Targeting and Extracellular Export of miRNAs

Abstract

AbstractExtracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity and thus to regulate expression of miRNA-target genes in both donor and recipient cells. Despite tremendous excitement related to extracellular vesicles-associated miRNAs as biomarkers or having therapeutic potential, the mechanism of selective packaging of miRNAs into endosomes and multivesicular bodies for subsequent extracellular export is a poorly studied area due to lack of assay system to study such processesin vitro. We have developed anin vitroassay with endosomes isolated from mammalian macrophage cells to follow miRNA packaging into endocytic organelles. The synthetic miRNAs, used in the assay, get imported inside the isolated endosomes during thein vitroreaction and become protected from RNase in a time and concentration dependent manner. The selective miRNA accumulation inside endosomes requires both ATP and GTP hydrolysis and the miRNA binding protein HuR. The HuR-miRNA complex binds and stimulates the endosomal RalA GTPase to facilitate the import of miRNAs into endosomes and their subsequent export as part of the extracellular vesicles. The endosomal targeting of miRNAs is also very much dependent on endosome maturation process that is controlled by Rab5 protein and ATP.Graphical Abstract◦miRNAs get imported to endosomesin vitro andare become protected to RNase◦Endosomal miRNA import is sequence specific and requires miRNA exporter protein HuR◦HuR-miRNA complex activates RalA GTPases to complete miRNA import.◦Rab5 protein and ATP hydrolysis is required for endosome maturation and miRNA import