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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 120; no. 4; p. e2216330120
• Main Authors: Costa, Bruno, Li Calzi, Marco, Castellano, Mauricio, Blanco, Valentina, Cuevasanta, Ernesto, Litvan, Irene, Ivanov, Pavel, Witwer, Kenneth, Cayota, Alfonso, Tosar, Juan Pablo
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24-01-2023
• Subjects: Amplification; Bacteria - metabolism; Biological Sciences; Biomarkers; Cell interactions; Cerebrospinal fluid; Chromatography; Denaturation; Epithelial cells; Epithelial Cells - metabolism; Epithelium; Humans; Molecular biology; Phages; Phenols; Phosphodiester bonds; Ribonuclease; RNA; RNA, Transfer - metabolism; Stability; Transfer RNA; tRNA; RNA stability; tRF; liquid biopsies; tRNA halves; extracellular RNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2216330120

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.