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

Abstract

Cells can release ribosomes and full-length tRNAs into the extracellular space, but these species are rapidly processed by extracellular RNases into stable rRNA and tRNA-derived fragments (tDRs). These nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their function or biomarker potential. The high extracellular stability of most of these fragments is also mechanistically unclear. To interrogate this, we measured the stability of several naked RNAs when incubated in human serum, urine and CSF. Interestingly, we identified extracellularly-produced tDRs with half-lives of several hours in human biofluids. Contrary to widespread assumptions, these intrinsically stable tRNA halves are mostly not in fragment form. On the contrary, they are present as full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs), as supported by native electrophoresis. Standard molecular biology techniques, including phenol-base RNA extraction, induce the denaturation of nicked tRNAs and the release of tDRs. To demonstrate this by an alternative approach, we performed enzymatic repair of nicked tRNAs with either RtcB or T4 PNK and Rnl1, obtaining a single tRNA-sized northern blot band that was not produced if samples were heat-denatured before enzymatic repair. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions. These protocols were used to identify nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Additionally, we showed that dissociation of nicked tRNAs produces single-stranded tRNA halves that can be spontaneously taken up by human epithelial cells. Overall, this study builds a framework for investigating potential intercellular communication pathways mediated by stable nv-exRNAs. Moreover, we provide useful methods to uncover a hidden layer of the extracellular RNAome composed of intrinsically stable, nick-containing, highly structured RNAs.

Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon

“…Additionally, 5' tRNA Gly(GCC) fragments in mouse sperm were reported to repress transcription of endogenous retroelements in embryos (Sharma et al 2016). Our finding that most 5' halves of this tRNA exist as nicked tRNA in HEK293A cells, coupled with an independent report in human U-2 OS cells (Costa et al 2023), suggests that, for these and other tRNA halves proposed to have novel functions, it will be important to determine the fraction that is unpaired in the cells under study.…”

Ribonuclease activity undermines immune sensing of naked extracellular RNA

“…However, several RNA species are stable in the extracellular space even when not associated with EVs. These resilient extracellular RNAs include ribonucleoprotein particles (RNPs) such as Ago2/miRNA complexes (Turchinovich et al 2011;Arroyo et al 2011;Geekiyanage et al 2020), and protein-protected RNA fragments (LaPlante et al 2023) derived from the ribosome (Tosar et al 2020(Tosar et al , 2022Costa et al 2023), U2 snRNPs (Tosar et al 2022), and possibly Xist RNPs (Dou et al 2024). Additionally, a population of intrinsically stable nicked tRNAs circulates in human biofluids, probably unprotected or naked (Costa et al 2023).…”

“…Because cell culture experiments are typically done in the presence of FBS, a rich source of RNases, we hypothesized that this could explain the apparent lack of naked exRNA bioactivity (Tosar et al 2020(Tosar et al , 2021Costa et al 2023). To study this, we worked with primary cultures of murine bone marrow-derived cells, differentiated into antigen presenting cells (Helft et al 2015;Na et al 2016).…”

“…We have recently shown that some RNAs are intrinsically stable in the extracellular space and act as stable reservoirs of shorter RNA fragments that can enter cells spontaneously (Costa et al 2023).…”

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs

“…For instance, the tRNA cleavage and increased generation of tRNA-derived RNAs (tDRs) in response to cellular stress is a well-known phenomenon [Thompson et al, 2008]. However, the Tosar group has recently demonstrated that tRNA halves or tDRs can in fact be produced during RNA isolation from denaturation of full-length “nicked” tRNAs [Costa et al, 2022]. In any case, the absence of full-length tRNAs in libraries from both untreated and SARS-CoV-2-infected cells indicates that the utilized small RNA-seq kit failed to incorporate them into the final NGS library.…”