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A broad role for YBX1 in defining the small non-coding RNA composition of exosomes

By Matthew J. Shurtleff, Jun Yao, Yidan Qin, Ryan M Nottingham, Morayma Temoche-Diaz, Randy Schekman, Alan M Lambowitz

Posted 07 Jul 2017
bioRxiv DOI: 10.1101/160556 (published DOI: 10.1073/pnas.1712108114)

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their co-isolation with contaminants, a lack of knowledge of the mechanisms of RNA sorting into EVs and limitations of conventional RNA-seq methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspannin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature tRNAs and other small non-coding RNAs encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long non-coding, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5′ terminal oligopyrimidine (5′ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which we showed previously is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small non-coding RNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for post-transcriptional modification in the sorting of some RNA species into EVs. The identification of full-length small non-coding RNAs within EVs suggests a role for EVs in the export and possible intercellular functional transfer of abundant cellular transcripts.

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