Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Vanderbilt University Cell Imaging Shared Resource, Nashville, TN, USA.
Dev Cell. 2022 Apr 25;57(8):974-994.e8. doi: 10.1016/j.devcel.2022.03.012. Epub 2022 Apr 13.
RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.
RNA 通过细胞外囊泡 (EV) 的转移会影响细胞表型；然而，由于缺乏有关含 RNA 的 EV 生物发生的信息，该领域的进展受到了限制。在这里，我们将内质网膜接触位点 (ER MCS) 鉴定为生成含 RNA 的 EV 的平台。我们鉴定了一小部分富含 RNA 的小 EV 亚群，该亚群受 ER MCS 连接蛋白 VAP-A 调控。功能上，VAP-A 调控的 EV 对于细胞间 miR-100 的转移和体内肿瘤形成至关重要。VAP-A 敲低 EV 的脂质分析显示，EV 生物发生脂质神经酰胺减少。VAP-A 结合的神经酰胺转移蛋白 CERT 的敲低导致 EV RNA 含量出现类似缺陷。成像实验表明，VAP-A 促进多泡体 (MVB) 的腔填充，CERT 定位于 MVB，而产生神经酰胺的酶中性鞘氨醇酶 2 与 VAP-A 阳性 ER 共定位。我们提出，通过 VAP-A-CERT 连接的神经酰胺转移驱动了特定 RNA 含量的 EV 群体的生物发生。
