Broad role for YBX1 in defining the small noncoding RNA composition of exosomes

Proceedings of the National Academy of Sciences of the United States of America
Matthew J ShurtleffAlan M Lambowitz

Abstract

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing 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 tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, 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 is required for the sorting of se...Continue Reading

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Methods Mentioned

BETA
RNA-seq
chip
Illumina sequencing
TGIRT-seq
affinity purification
reverse transcription PCR

Software Mentioned

DESeq2
Ensemble GRCh38
Integrative Genomics Viewer ( IGV
Ensembl
IGV
ExoQuick

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