Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages.

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.

HuR-miRNA complex activates RAS GTPase RalA to facilitate endosome targeting and extracellular export of miRNAs.

Extracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity, thus regulating the 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 poorly studied due to the lack of an in vitro assay system. Here, we have developed an in vitro assay 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 the in vitro reaction 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 the endosome maturation process that is controlled by Rab5 protein and ATP. In summary, we provide an in vitro method to aid in the investigation of the mechanism of miRNA packaging process for its export from mammalian macrophage cells.