Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner.

Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.