Nontemplated Nucleotide Additions Distinguish the Small RNA Composition in Cells from Exosomes

• Small ncRNAs are nonrandomly distributed in B cells and their released exosomes
• 3′-end adenylated miRNA isoforms are relatively enriched in cells
• 3′-end uridylated miRNA isoforms are overrepresented in exosomes
• Nucleotide addition may affect the relative abundance of ncRNAs in cells and exosomes

SummaryFunctional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3′ end adenylated miRNAs are relatively enriched in cells, whereas 3′ end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3′ end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.

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