Inhibition of acid or neutral sphingomyelinases differentially impacts RNA and protein cargo sorting to extracellular vesicles
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ABSTRACT:
PROVIDER: PRJNA1070310 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27756341_1.fastq.gz | Fastqsanger.gz | |||
| SRR27756341_2.fastq.gz | Fastqsanger.gz | |||
| SRR27756342_1.fastq.gz | Fastqsanger.gz | |||
| SRR27756342_2.fastq.gz | Fastqsanger.gz | |||
| SRR27756343_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:The biogenesis of extracellular vesicles (EVs) is a regulated process, driven by mechanisms at specific subcellular milieus. Sphingomyelinases (SMases), which metabolize sphingomyelin in membranes, play a role in EV biogenesis. Their metabolic product, ceramide, induces invaginations at the endosome or blebbing from the plasma membrane, both important in EV generation. Here, we sought to evaluate the impact of SMase inhibition on EV protein and RNA cargoes. For this, we treated human MCF7 cells with the neutral SMase (NSM) inhibitor GW4869 or the acid SMase (ASM) inhibitor FTY720. EVs were then purified from the conditioned media of control or inhibitor-treated cells and characterized by a variety of approaches, including LC-MS/MS and RNA-sequencing. SMase inhibition resulted in morphological and phenotypic changes in the heterogeneous EV population. Strikingly, NSM inhibition resulted in a depletion of nanoparticles, as well as a decrease in the RNA and protein content of EVs, with a marked reduction in endosomal, spliceosomal, and translation-related proteins. Furthermore, we observed a reduction in the overall RNA-binding proteins (RBPs) in EVs released by cells treated with the NSM-inhibitor. In contrast, the ASM-inhibitor treatment, which appears to reduce plasma membrane-derived vesicles, elicited an inverse response, leading to an increase in RBP and associated machineries within the released EV population. RNA sequencing of these EV revealed changes in the RNA biotypes composition, with an increase in protein coding transcripts. Interestingly, ASM-inhibitor resulting EVs induced increased cell migration and protein translation in recipient MCF10A cells. These results suggest that SMase-dependent vesiculation represents a major route of RBP and RNA trafficking outside the cell, via endosomal pathways.
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