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

Bruno Costa, Marco Li Calzi, Mauricio Castellano, Valentina Blanco, Ernesto Cuevasanta, Irene Litvan, Pavel Ivanov, Kenneth Witwer, Alfonso Cayota, Juan Pablo Tosar

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article numbere2216330120
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number4
StatePublished - Jan 24 2023


  • RNA stability
  • extracellular RNA
  • liquid biopsies
  • tRF
  • tRNA halves

ASJC Scopus subject areas

  • General


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