Non-canonical argonaute loading of extracellular vesicle-derived exogenous single-stranded miRNA in recipient cells

Abstract

MicroRNAs, the tiny regulators of gene expression, can be transferred between neighbouring cells via Extracellular Vesicles (EV) to control the expression of genes in both donor and recipient cells. How the EV-derived miRNAs get internalized and become functional in target cells is an unresolved question. We have expressed liver specific microRNA, miR-122, in non-hepatic cells for packaging in the released EVs. With these EVs, we have followed the trafficking of miR-122 to recipient HeLa cells that otherwise don't express this miRNA. We found that EV-associated miR-122 are primarily single stranded and, to become functional, get loaded onto the recipient cell Ago proteins without requiring host Dicer1. Following endocytosis, EV-associated miR-122 get loaded onto the host cell Ago on the endosomal membrane where the release of internalized miRNAs occurs in a pH-dependent manner facilitating the formation of the exogenous miRNP pool in the recipient cells. Endosome maturation defect affects EV-mediated entry of exogeneous miRNAs in mammalian cells.

Inhibition of extracellular vesicle-associated MMP2 abrogates intercellular hepatic miR-122 transfer to liver macrophages and curtails inflammation

“…We have confirmed the luminal presence of EV-associated miR-122 in an RNase protection assay where the EV-associated miR-122 remains largely protected from RNases unless the EVs were treated with the detergent Triton X-100 to disrupt the membranous structure ( Figure S2 A). The functionality of the EV-derived miR-122 in recipient cells has been confirmed before ( Ghoshal et al., 2021 ), and we have reconfirmed the repression of miR-122 targets in recipient cells treated with miR-122-containing EVs in subsequent assay using RAW264.7 cells as the recipient cells ( Figure S2 B). To test whether EVs isolated from lipid-treated hepatic cells could increase the inflammatory response in recipient macrophage cells, we added isolated EVs to various macrophages corresponding in origin to the donor hepatocyte ( Figure 2 A).…”

Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation

“…Interestingly, treatment of cells with EVs containing miR-122 has no significant effect on endosome number or Rab5a expression in infected RAW264.7 cells. The infection also had no effect on Dynamin 2 expression and thus cannot account for the reduced miR-122 entry that is known to be a dynamin 2 dependent process in mammalian cells ( Fig S2B and C ; [ Ghoshal et al, 2021 ]).…”

“…After centrifugation the non-polysome fractions were collected and the rest of the solution was mixed followed by centrifugation at 31,200 rpm for 30 min at 4°C in a SW61Ti rotor. The pelleted polysome was suspended in polysome buffer (10 mM Hepes, 25 mM KCL, 5 mM MgCl 2 , 1 mM DTT, 5 mM VRC, and 1× PMSF) and kept for RNA isolation and Western blot ( Ghoshal et al, 2021 ).…”

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

“…Upon reaching the target cells, exosomes are internalized by fusion of exosomal membrane with the plasma membrane or by endocytosis. The release of both fRNAa and miRNAs [83] from endosomes into cytoplasm can occur in a pH-dependent manner through the internal fusion of the exosomal and endosomal membranes, allowing the therapeutic domain activity. The proposed procedure does not require exosome isolation.…”