Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Extracellular RNAs (exRNAs) are implicated in intercellular communication pathways and show promising use as disease biomarkers in noninvasive liquid biopsies. To date, most research has focused in RNAs encapsulated inside extracellular vesicles (EVs) or in total unfractionated biofluids. It is known that exRNAs also exist outside vesicles or lipoprotein particles. However, nonvesicular exRNAs remain widely uncharacterized. Our interest in nonvesicular exRNAs arises from the observation that some small RNAs, such as specific tRNA-derived fragments, have much higher relative representation in this extracellular fraction. At least in part, this enrichment seems to be a consequence of their differential extracellular stability. To get a representative picture of the whole set of RNAs released by cultured human cells, we inhibited enzymatic RNA degradation in the extracellular space. This revealed the presence of extracellular tRNAs and ribosomes in a variety of malignant and nonmalignant cell lines. We also demonstrated the extracellular biogenesis of certain ncRNA-derived fragments, some of which are highly stable and can be detected in biofluids. We also highlight the immunomodulatory potential of purified RNA-containing extracellular complexes, whose release to the extracellular space follows certain forms of cell damage or death. Overall, we provide evidence showing that exRNA profiles are dynamic and nonvesicular RNAs are highly biased toward stable extracellularly-generated ncRNA fragments.