Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

Helena Sork,Janne Lehtiö,Oscar P B Wiklander,Oscar P B Wiklander,Jakub Orzechowski Westholm,Giulia Corso,Joel Z Nordin,Samir El Andaloussi,Samir El Andaloussi,Henrik J Johansson,Joel Z Nordin,Henrik J Johansson,Kaarel Krjutskov,Matthew J A Wood,Imre Mäger,Samir El Andaloussi,Yi Xin Fiona Lee,Matthew J A Wood,Imre Mäger,Yi Xin Fiona Lee,Janne Lehtiö,Jakub Orzechowski Westholm

doi:10.1038/s41598-018-28485-9

Abstract

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49–96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.

Highlights

Extracellular vesicles (EVs) mediate their native biological effects by transferring or displaying their cargo to target cells, whereas the effects are either related to their overall complex cargo signature, or to certain individual biologically active macromolecules
EVs were characterized by Nanoparticle Tracking Analysis, transmission electron microscopy and western blot, confirming their size around 100 nm, presence of cup-shape morphology and the enrichment of ALIX and TSG101 throughout the tested samples (Fig. 1, Supplementary Figure S1)
Among the commonly overrepresented Gene Ontology (GO) classes for both vesicle types we found overrepresentation of entries related to ribosomes, mRNA catabolism, translation initiation, protein targeting to membranes, unfolded protein binding, extracellular matrix, focal adhesion, blood microparticles and extracellular exosomes (Fig. 5C)

Summary

Introduction

EVs mediate their native biological effects by transferring or displaying their cargo to target cells, whereas the effects are either related to their overall complex cargo signature, or to certain individual biologically active macromolecules. Some EV effects have been attributed to their specific miRNA content (reviewed in5), which can either reflect that of their cell of origin or display enrichment of certain miRNA subsets[6]. Independence, presence of specific nucleotide sequences or -interaction with certain RNA binding proteins seem to promote the sorting of a subset of miRNAs into EVs (reviewed in 7). To shed light onto the interplay of bioactive macromolecules, we analysed the small RNA transcriptome of cells and EVs by generation sequencing as well as investigated RNA/miRNA binding proteins in EVs with respect to the discovered EV RNA species. A thorough understanding of the synergy between these different EV bioactive macromolecules aids to develop novel EV RNA therapeutics by taking advantage of underlying cargo sorting mechanisms

Methods

Results

Conclusion