Extracellular vesicles (EVs) are small membrane-bound particles that are loaded with various proteins, RNA, DNA, and lipids, and secreted by cells. Interest in these vesicles has grown in recent years with mounting evidence that EVs act as intercellular communication systems, transferring their selected cargo to other cells to confer specific effects on target cell biology. However, the processes that direct specific RNAs and proteins into these specialized vesicles remain largely unknown. In the April 25th, 2022 edition of Developmental Cell, Alissa Weaver, M.D., Ph.D. and her research team at the Vanderbilt Center for Extracellular Vesicle Research have uncovered subcellular hubs of EV formation that selectively assemble RNA-containing EVs. These hubs are located at membrane contact sites (MCS) where the endoplasmic reticulum (ER) interacts with EV biogenesis membranes, including late endosomal multivesicular bodies (MVBs) (ER-MVB MCS). Shedding much needed mechanistic light, the group further pinpointed the ER MCS membrane tether protein VAP-A and its binding partner ceramide transfer protein (CERT) as key drivers in this process.
Alissa Weaver will present the research outlined in this blog at the May ERCC webinar. Join us!
RNA-containing EVs are of particular interest to the biomedical research and pharmaceutical communities given their capacity to dictate changes in gene expression in a cell population. Many studies have validated that several types of RNA can be transferred via EVs from donor to recipient cells and then carry out specific effects on gene expression. For instance, microRNAs –– very short, specific RNA strands that disrupt the expression of target gene transcripts –– carried by cancer cell EVs have been shown to shape the tumor microenvironment in cancers of the brain and head and neck. This newly revealed native cellular EV biogenesis pathway, if properly harnessed, has great potential to halt disease pathogenesis and progression.
First author Bahnisikha Barman and her colleagues first contemplated whether ER-MVB MCS might be key sites for formation of RNA-containing EVs based on proteomics data showing enrichment of ER-associated RNA-binding proteins in EVs. Those data, together with Dr. Barman’s previous work showing that complexes of miRNA and RNA-binding proteins assemble on the ER, led Drs. Barman and Weaver to investigate their theory. Soon thereafter, the group confirmed by confocal cell microscopy that the microRNAs miR-100 and let-7a do indeed associate with ER-MVB MCS.
Looking to the molecular players that are essential to these MCS interactions, Dr. Barman depleted the ER tether protein VAP-A in cells and found a sharp loss in the number and RNA content of both small and large EVs. Small EVs impacted by VAP-A depletion were notably distinguished from other small EVs by their differential segregation on density gradients and enrichment in RNA and ceramide, an essential lipid component of EV formation. Using a fluorescent marker of MVBs, the group directly observed the effect of VAP-A depletion on the presence in the MVB lumen of microRNAs, RNA-binding proteins, the ceramide transporter CERT, and other EV cargoes. Knockdown of CERT exerted similar effects on EV formation as VAP-A. In addition, VAP-A colocalized with the ceramide synthesis enzyme neutral sphingomyelinase 2, suggesting that VAP-A and CERT might function together at ER MCS to direct the budding and cargo loading of this unique population of small EVs.
The novel role for ER-MCS as a platform for organizing EV biogenesis has widespread implications for future studies. “Since extracellular vesicles are secreted from all cells, we expect that this basic understanding of how cells package RNA into EVs will impact multiple fields. ER MCS are regulated by diverse signaling and metabolic states, which will influence the formation of this important subtype of EVs. In addition, EVs are considered a highly desirable packing material for drugs, including RNA-based ones, to protect them from degradation in the circulation and target them to specific recipient cells. Thus, our finding that RNA packaging into EVs takes place at ER MCS may help in the development of next-generation RNA therapeutics”, said Dr. Weaver. “Our next goals are to investigate how this pathway is regulated in cancer and other pathological states and how to exploit it for drug delivery”.
This work was funded by NIH Extracellular Research Communication Consortium grant U19CA179514, NIH Program Project grant P01CA229123, and NSF-2036809.
Barman B et al. VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites Dev Cell (2022) 57: 1-21. doi: 10.1016/j.devcel.2022.03.012 PMID: 35421371.
Silverman DA, Calin GA, Myers JN & Amit M. Neural reprogramming via microRNAs: the new kid on the p53-deficient block. (2020) Mol Cell Oncol 7:1756723. doi: 10.1080/23723556.2020.1756723 PMID: 32944617.