Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

BIOGENESIS OF RNA-CONTAINING EXTRACELLULAR VESICLES AT ER MEMBRANE CONTACT SITES

Alissa M. Weaver 1 ; 1 Vanderbilt University School of Medicine, Cell and Developmental Biology, Nashville, TN, USA

Extracellular RNA is a novel mechanism for cell-to-cell communication and drives many physiological and pathological processes including cancer. Extracellular vesicles (EVs) are a major vehicle for transmitting these RNAs between cells. However, the underlying mechanisms by which RNA-containing EVs are generated are poorly understood. Due to the association of many RNA processing granules with the endoplasmic reticulum, we investigated the role of endoplasmic reticulum membrane contact sites (ER MCS) as key subcellular locations for the biogenesis of RNA-containing EVs. We found that inhibition or overexpression of key molecules that control ER MCS (VAP-A-KD, VAP-A OE) and ceramide transport at ER MCS (CERT-KD) greatly affected the small RNA content of both small and large EVs. Density gradient sub-fractionation of EV pellets revealed that VAP-A regulates a select subpopulation of small EVs that are enriched with RNA. Confocal microscopy data revealed that key RNA and RNA binding proteins are altered in multivesicular endosomes in VAP-A KD cells. Experiments testing EV function indicated that this VAP-A-controlled small EV population is critical for both in vitro transfer of miR-100 to recipient cells and for in vivo growth of xenograft mouse tumors. Altogether, these and additional data suggest a model in which ceramide transfer at ER MCS drives biogenesis of a select subpopulation of EVs containing RNA-RBP complexes. We are now actively investigating regulatory mechanisms that control this key sorting event, as well as the functional consequences.

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