Staying together after the breakup: tRNA halves in extracellular fluids

February 13, 2023
120 (8) e2300300120
Research Article
Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids
Bruno Costa, Marco Li Calzi [...] Juan Pablo Tosar
Transfer RNAs (tRNAs) are essential interpreters of the genetic code and are required for translation, yet over the past two decades, it has become clear that they are subject to nuclease cleavage under a variety of stress conditions. Once thought to serve simply to deplete tRNAs, there is increasing evidence that the resulting tRNA-derived RNAs may exhibit biological functions of their own (1, 2). Yet, understanding the functions of tRNA fragments is complicated by the range of possible molecular species that could in principle exist following tRNA cleavage. In this issue of PNAS, Costa et al. explore the biochemistry of tRNA halves in several biological fluids, showing that tRNA halves predominantly exist as stable nicked tRNA complexes between 5′ and 3′ halves (3). These findings have major implications for the biological functions of tRNA fragments in systems from oncogenesis to reproductive biology.
Nucleolytic cleavage of tRNAs is well known to occur in the context of interspecific conflict, as seen in bacterial cleavage of tRNAs following phage infection (4). Surprisingly, this process can also occur in the absence of pathogens; tRNA cleavage has been documented in response to transient stresses, such as starvation or oxidative stress in a range of organisms (57), and is implicated in the translational repression that occurs following such stressors. Curiously, although depleting tRNAs would provide a simple way to reduce protein translation, only a small fraction—a few percent—of tRNAs are typically subject to stress-induced cleavage, leaving sufficient intact tRNAs to support translation. Moreover, it is increasingly evident that cleaved tRNAs—particularly tRNA halves—are also widespread in extracellular fluids in the absence of any clear stressor (810). Together, these findings suggest that tRNA fragments may exhibit new functions as regulatory species in their own right, motivating a substantial community to explore tRNA fragment function in various biological contexts.
Understanding the biological functions of tRNA fragments is complicated by two features of these small RNAs. The first is the diversity of tRNA cleavage products, which include ~20 nucleotide (nt) products produced by cleavage of tRNAs in the T or D loops, as well as longer ~30- to 35-nt tRNA halves (Fig. 1), along with various other less-studied cleavage products. The ~19- to 22-nt RNAs are comparable in size to microRNAs and siRNAs and have been implicated in similar Argonaute-guided gene regulation (11). However, even for these shorter tRNA fragments, idiosyncratic functions have been reported, as for example, a 22-nt 3′ fragment of tRNA-Leu-CAG supports ribosome biogenesis by relieving an inhibitory RNA stem loop in the 5′ UTR of ribosomal protein-coding genes (12), while 18-nt 5′ fragments of tRNA-Val/Ala/Cys (tRNAs with a “miniTOG” 5′ G-rich sequence) bind to PABPC1 and displace eIF4A/G to repress eIF4E-dependent protein translation (13, 14). Compared to short tRNA fragments, longer tRNA halves are implicated in a wider range of disparate functions through a diverse array of mechanisms. As with shorter tRNA fragments, tRNA halves have been linked to translational control (8), but other tRNA halves are reported to protect cells from apoptosis (15), or control noncoding RNA production in Cajal bodies (16), among a range of proposed functions.
Fig. 1.
Possible implications of nicked tRNAs for tRNA fragment function. tRNA cleavage generates 5′ and 3′ halves, often thought to serve independent functional roles. Here, we consider a 5′ tRNA fragment with some arbitrary regulatory function (shown binding to an “effector” protein or RNA)—hypotheses below are the same whether the 3′ half has an independent function or not. We consider three possible implications of data from Costa et al. showing that tRNA halves often remain associated after cleavage. (A) Most simply, tight association between tRNA halves could prevent effector binding by the 5′ tRNA fragment, with the 3′ half acting as an inhibitor. (B) Nicked tRNAs could serve as a stable reservoir of functional tRNA halves; Costa et al. speculate that nicked tRNAs in circulation could allow long-range delivery of tRNA fragments to recipient cells. (C) Nicked tRNA halves could function together as a complex, for instance, by nonproductive binding to ribosomes or other tRNA-binding proteins.
Costa et al. explore the biochemistry of tRNA halves in several biological fluids, showing that tRNA halves predominantly exist as stable nicked tRNA complexes between 5′ and 3′ halves.
The second complicating feature of tRNA fragments arises from our limited understanding of their molecular nature; most small RNA cloning methods only capture a handful of 5′ tRNA halves (Glu-CTC, Gly-GCC, and Val-CAC typically being most prominent) due to difficulties in tRNA cloning caused by various nucleotide modifications. However, it is clear from northern blots and updated cloning protocols that additional tRNA halves, including 3′ halves, are also present in many biological samples (17). This raises the question of whether individual tRNA halves—e.g., the 5′ half of Glu-CTC—act in isolation or whether they exist in complex with the corresponding 3′ half as a nicked tRNA.
Here, Costa et al. explore the molecular nature of tRNA halves in multiple biological fluids. They show, first, that several tested tRNA fragments (tRFs) are surprisingly stable to RNase1 treatment in vitro even in the absence of RNA-binding proteins that might protect the RNAs from degradation. This is specific for some tRNA-derived fragments (tRNA-Gly-GCC) but not for all RNAs, such as rRNA and rRNA fragments. tRNA fragments were similarly stable in biofluids including serum and cerebrospinal fluid.
To interrogate the basis for this unexpected stability, the authors report improved methods for isolating and characterizing RNAs without disrupting RNA–RNA interactions. By eliminating phenol-based RNA extraction, coupled with solid phase extraction-based methods or RNA purification using guanidine salts, the authors were able to avoid artifactually dissociating complexes between tRNA halves. They leveraged these methods to show that many tRNA halves remain associated following their production and thus exist as nicked tRNAs. Nicked tRNAs could be repaired by T4 PNK and T4 RNA ligase 1, or by the tRNA repair enzyme RtcB, to regenerate an almost full-length tRNA. Denaturing nicked tRNAs with heat, or by phenol-based RNA purification, leads to dissociation of the two tRNA halves and prevented subsequent tRNA repair, emphasizing the importance of the technical advances in RNA purification used throughout the manuscript. Properly purified nicked tRNAs were shown to coelute with intact tRNAs in size exclusion chromatography, whereas heat- or phenol-dissociated tRNA halves coeluted with microRNAs. Intriguingly, nicked tRNAs—but not heat-denatured tRNA halves—were stable in the presence of RNAse1, suggesting that they could serve as a stable reservoir of tRNA halves. The authors then briefly explore the biology of nicked tRNAs in biological fluids, showing that cells can take up tRNA halves from culture media and suggesting that nicked tRNAs could act as stable carriers of tRNA fragments to enable RNA trafficking through the circulation.
These findings have wide-ranging implications for understanding the regulatory functions of tRNA fragments. Most importantly, they motivate a renewed focus on the biochemical nature of tRNA halves in other systems, where the majority of functional studies make the implicit assumption that a particular tRNA half acts in isolation. Functions of tRNA halves are most commonly interrogated using two approaches—transfection (or microinjection) of the tRNA half in question (whether synthetic or purified) for gain-of-function studies, contrasted with transfection of antisense inhibitors for loss-of-function studies. For instance, multiple studies have linked tRNA halves in mammalian sperm with transmission of paternal environmental cues to modulate phenotypes in offspring. In several of these studies, purified sperm tRNA halves (1820) or synthetic tRNA fragments (10) were microinjected into control zygotes and shown to modulate phenotypic outcomes in resulting embryos or in progeny. The findings reported by Costa et al. raise the question of whether sperm carry nicked tRNAs or separated tRNA halves; in the former scenario, typical RNA purification methods are likely to radically alter the nature of the RNA species delivered to zygotes. These considerations motivate a more detailed exploration of whether individual tRNA halves are the appropriate physiologically relevant candidates for injection studies.
More broadly, Costa et al. raise the question of whether nicked tRNAs serve any biological function beyond providing substrates for rapid tRNA repair following the resolution of stressful conditions. We consider three hypotheses (Fig. 1). In the first, although tRNA halves are capable of serving biological functions when separated from their partners, binding to their other half might prevent them from forming effector complexes. In this scenario, small RNA cloning studies will need to determine not only the absolute levels of a given tRNA half but also the abundance of free vs. complexed tRNA halves. In other words, it might not matter that 5′ tRF-Gly-GCC levels increase 10-fold in some biological system if the corresponding 3′ tRF levels increase similarly; conversely, the regulatory output of a 5′ tRF could increase even in the face of unchanged levels if the 3′ inhibitor were degraded to free up previously inhibited 5′ fragments. A second, related, hypothesis would be that nicked tRNAs serve as a stable reservoir for long-range delivery of functional tRNA halves. Consistent with this, Costa et al. show that tRNA halves are taken up by cells in vitro, and propose that nicked tRNAs might stabilize tRNA halves long enough to enable their delivery to distant cells as signaling molecules. Finally, rather than simply masking or stabilizing functional tRNA halves, nicked tRNAs could serve independent functions that cannot be accessed by either fragment in isolation. Some evidence for this idea exists as pumpkin phloem tRNA fragments inhibit translation, but this activity is lost following denaturation, implicating nicked tRNAs in this process (8).
Together, the findings reported by Costa et al. deepen our understanding of a mysterious class of regulatory RNAs. They provide technical guidance for working with tRNA fragments and update our understanding of these species as they exist in many biological systems. These results have wide-ranging implications for tRNA fragment biology in cancer, host–pathogen interactions, early development, and countless other biological contexts.

Acknowledgments

Author contributions

E.K. and O.J.R. wrote the paper.

Competing interests

The authors have research support to disclose: O.J.R. and E.K. were funded by NIH grant R01HD099816.

References

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J. M. Dhahbi et al., 5’ tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction. BMC Genom. 14, 298 (2013).
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U. Sharma et al., Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals. Science. 351, 391–396 (2016).
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Q. Chen et al., Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science. 351, 397–400 (2016).
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G. Sarker et al., Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs. Proc. Natl. Acad. Sci. U.S.A. 116, 10547–10556 (2019).
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Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 120 | No. 8
February 21, 2023
PubMed: 36780520

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Published online: February 13, 2023
Published in issue: February 21, 2023

Acknowledgments

Author Contributions
E.K. and O.J.R. wrote the paper.
Competing Interests
The authors have research support to disclose: O.J.R. and E.K. were funded by NIH grant R01HD099816.

Notes

See companion article, “Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids,” https://doi.org/10.1073/pnas.2216330120.

Authors

Affiliations

Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605
Oliver J. Rando1 [email protected]
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605

Notes

1
To whom correspondence may be addressed. Email: [email protected] or [email protected].

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Staying together after the breakup: tRNA halves in extracellular fluids
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