Inhibition of acid or neutral sphingomyelinases differentially impacts RNA and protein cargo sorting to extracellular vesicles

Extracellular vesicles (EVs) form through regulated biogenesis processes involving sphingomyelinases (SMases), enzymes that metabolize sphingomyelin to produce ceramide-a lipid influencing membrane rigidity and essential for EV generation. This study explores the impact of inhibiting neutral SMase (NSM) and acid SMase (ASM) on the sorting of EV protein and RNA cargoes in human MCF7 cells. Our results revealed that NSM inhibition reduces EV nanoparticles and diminishes RNA and protein cargoes, including endosomal, spliceosomal, and translation-related proteins. Conversely, ASM inhibition increased RNA-binding proteins within and enhanced the expression of ribonucleoprotein complex-associated RNA in released EVs, including several snRNAs and 7SL RNA. Intriguingly, ASM-inhibited EVs enhanced the migration and translational activity of recipient MCF10A cells. These findings suggest an important role for SMase-dependent vesiculation in governing RNA and protein trafficking to the extracellular space, unveiling potential implications for cellular communication and function. To explore sphingomyelinases' impact on cargo loading in extracellular vesicles, MCF7 cells were treated with sphingomyelinase inhibitors GW4869 or FTY720. Total RNA was isolated from both MCF7 cells and extracellular vesicles (EVs) under control and drug treatment conditions. After ribosomal RNA depletion, library preparation utilized the KAPA RNA HyperPrep Kit with RiboErase (HMR) from Roche. For cellular samples, 1 µg of total RNA was utilized, while EV samples under drug-induced conditions used 2-6 ng of total RNA. Libraries underwent 7 and 16 PCR cycles for amplification. Quantification using qPCR was followed by loading at equimolar concentrations for sequencing on the Novaseq platform with the Novaseq S4 kit, aiming for a coverage of 35 million paired-end reads per sample.