tRF & tiRNA : Why and How to Study Them?

Overview

Transfer RNA (tRNA) is the adaptor molecule that decodes mRNA codons and delivers amino acids for protein synthesis. Recent work shows that tRNAs are also a major source of small non-coding RNAs with distinct, varied functions [1]. These tRNA-derived ncRNAs are not random degradation products; they are generated through precise biogenesis processes (Fig. 1). They fall into two broad groups: tiRNAs (tRNA halves) and tRFs (tRNA-derived fragments), each with characteristic sizes, nucleotide compositions, functions, and biogenesis [1-3].

tRNA halves (tiRNAs) arise from specific cleavage in the anticodon loop of mature tRNA by angiogenin under stress conditions, producing 29-50 nucleotide 5-prime and 3-prime halves.

tRFs are 16-28 nt fragments derived from tRNA or pre-tRNA, classified by their site of origin: (i) tRF-5 from the 5-prime part of mature tRNA by D-loop cleavage; (ii) tRF-3 from the 3-prime part by T-loop cleavage, ending with a 3-prime CCA terminus; (iii) tRF-1 from the 3-prime trailer of pre-tRNA, carrying terminal poly-U residues; (iv) i-tRF, from the internal region of mature tRNA, belonging to none of the above.

Fig. 1. Biogenesis of tRFs/tiRNAs. tRF-1 arises from the 3-prime trailer of primary tRNA; tRF-5, i-tRF, and tRF-3 are produced from the 5-prime, internal, and 3-prime portions of mature tRNA. Cleavage within the anticodon loop generates 5-prime and 3-prime tiRNA halves.

Biological Functions

tRFs and tiRNAs act as small non-coding RNAs with many functions (Fig. 7). They can behave like microRNAs in RNA interference (Fig. 2); directly inhibit protein synthesis by displacing the translation initiation factor eIF4G from mRNA on ribosomes [9-10]; bind protein factors such as CBX1 to control target mRNA stability (Fig. 3); interact with cytochrome c to modulate apoptosis [13]; assemble stress granules in response to stress (Fig. 4); sensitize cells to oxidative-stress-induced p53 activation and p53-dependent death [19]; and alter transcriptional cascades in intergenerational inheritance as paternal epigenetic factors (Fig. 6).

Fig. 2. tRFs share many miRNA features: Dicer-dependent biogenesis, RISC complex formation with Argonaute proteins, and RNA silencing. Some catalogued miRNAs map directly to tRFs [8].

Fig. 3. tRFs or their mimetics displace the oncogenic RNA-binding protein YBX1 and destabilize many cancer-driver mRNAs, markedly diminishing cancer metastasis [11].

Fig. 4. tRNA halves (tiRNAs) are generated by angiogenin cleavage in response to cellular stress, promoting stress granule assembly, translational inhibition, cell repair, and survival [12].

Human Diseases

tRFs/tiRNAs are associated with, or causally contribute to, cancers, neurodegeneration, and metabolic disorders (Fig. 5).

Fig. 5. tRF/tiRNA molecular functions and diseases.

Cancers

Differential expression of tRFs/tiRNAs is observed across cancer cell lines, including the prostate cancer lines LNCaP and C4-2, and their levels rise under cellular stress, particularly hypoxia [16]. A 3-prime-derived tRF in B-cell lymphoma cells acts like a guide RNA, suppressing proliferation and modulating the DNA damage response in a miRNA fashion [7]. By competing for YBX1 binding and destabilizing oncogenic transcripts, tRFs/tiRNAs act as tumor suppressors [11] (Fig. 3). tRF-1001, a 3-prime tRF from pre-tRNA-Ser, is highly expressed in several cancer cell lines and is required for prostate cancer cell proliferation [21]. ANG-produced tiRNAs promote stress granule assembly that helps cells survive adverse conditions, and may contribute directly to ANG-mediated angiogenesis and cancer cell proliferation. tiRNAs can also help cancer cells evade apoptosis by binding cytochrome c [13]. Collectively, these findings point to a functional role of tRFs/tiRNAs in tumorigenesis.

Acquired Metabolic Disorder

Growing evidence links offspring metabolic disorders to paternal diet. In a paternal high-fat diet (HFD) mouse model, a subset of sperm tRFs/tiRNAs, mainly 5-prime halves of 30-34 nucleotides, changed expression and RNA modification profiles under HFD. Injecting sperm tRFs/tiRNAs from HFD males into normal zygotes produced metabolic disorders in F1 offspring and altered expression of metabolic pathway genes in early embryos and islets, unrelated to DNA methylation at CpG-enriched regions. Sperm tRFs/tiRNAs thus represent a paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorder [14] (Fig. 6). Protein restriction also alters small RNA levels in mature sperm, decreasing let-7 and increasing 5-prime tRFs/tiRNAs of glycine tRNAs, and tRFs/tiRNAs regulate transcripts driven by endogenous retroelements active in the preimplantation embryo [22].

Fig. 6. tRNA-derived small RNAs (tsRNAs, mostly tRNA halves) show altered expression and RNA modifications in sperm of high-fat-diet-fed mice. Sperm tsRNAs confer metabolic disorder phenotypes in offspring by oocyte injection, mediating intergenerational inheritance by modulating embryonic gene transcription that cascades into adulthood [14].

Neurological Disorders

Several neurological disorders stem from defects in tRNA metabolism and processing enzymes such as ANG. Angiogenin mutants with reduced RNase activity are implicated in amyotrophic lateral sclerosis (ALS) [23], and a subset of ALS-associated ANG mutants also occurs in Parkinson's disease (PD) patients [24]. Further links connect ANG-induced tRNA halves, cellular stress, and neurodevelopmental disorders [25]. Loss of the RNA kinase CLP1 accumulates novel small RNA fragments from aberrant processing of tyrosine pre-tRNA; these tRFs/tiRNAs sensitize cells to oxidative-stress-induced p53 activation and p53-dependent neuronal death, causing motor neuron loss, muscle denervation, and respiratory failure [19]. Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities: loss of cytosine-5 tRNA methylation increases ANG binding and cleavage, accumulating 5-prime halves that reduce translation rates and activate stress pathways, leading to smaller cells and increased apoptosis of cortical, hippocampal, and striatal neurons [25].

Pathological Stress Injuries

Hypoxia, nutrient deprivation, oxidative conditions, and metabolic imbalance damage cells and promote disease, and these stresses stimulate tRNA half production. In animal models of tissue damage, such as toxic injury, irradiation, and ischemic reperfusion, tRNA half production correlates with the degree of damage, for example through tRNA conformational changes that promote ANG-mediated cleavage [1]. Increased 5-prime halves are associated with viral and rickettsial infections and may prevent apoptosis and promote survival. tRFs/tiRNAs, specifically 30-35 nt 5-prime halves, are abundant in non-malignant liver and significantly increase with chronic viral hepatitis [27].

Biomarker Potentials

The composition and abundance of tRFs/tiRNAs depend strongly on cell type and disease condition [15]. tRNA and tRF/tiRNA populations are highly enriched in biofluids, sometimes more so than microRNAs [16-18] (Fig. 7A). Although miRNAs remain the main focus of biofluid biomarker work, the high stability and abundance of tRFs/tiRNAs in body fluids, their involvement in pathology, their differential expression in solid tumors and hematological malignancies, and their power to discriminate cancer patients from healthy controls open the door to minimally invasive tRF/tiRNA-based biomarker tests. tRF profiles, for example, discriminate triple-negative and triple-positive breast cancer cells from normal controls by unsupervised clustering [15] (Fig. 7B,C), and the tRF/tiRNA ratio is a candidate prognostic indicator of cancer progression-free survival [16].

Fig. 7. Abundant tRNA-derived fragments in serum [17] (A). tRF profiles discriminate triple-negative (B) and triple-positive (C) breast cancer cells from normal controls by unsupervised clustering.

How to Study tRFs/tiRNAs

The nrStar tRF&tiRNA PCR Array systematically profiles the abundance and expression of tRFs/tiRNAs. Differentially expressed tRFs and tiRNAs can then be followed up with established approaches (Fig. 8). Many methodologies are shared with miRNA research, including qPCR confirmation, knockdown by LNA oligos, and gain-of-function studies with synthetic small RNA mimics.

Fig. 8. Roadmap for next-step tRF/tiRNA studies.

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