Circulating tRNA-Derived Small RNAs as Novel Radiation Biomarkers of Heavy Ion, Proton and X-ray Exposure

The effective and minimally invasive radiation biomarkers are valuable for exposure scenarios in nuclear accidents or space missions. Recent studies have opened the new sight of circulating small non-coding RNA (sncRNA) as radiation biomarkers. The tRNA-derived small RNA (tsRNA) is a new class of sncRNA. It is more abundant than other kinds of sncRNAs in extracellular vesicles or blood, presenting great potential as promising biomarkers. However, the circulating tsRNAs in response to ionizing radiation have not been reported. In this research, Kunming mice were total-body exposed to 0.05–2 Gy of carbon ions, protons, or X-rays, and the RNA sequencing was performed to profile the expression of sncRNAs in serum. After conditional screening and validation, we firstly identified 5 tsRNAs including 4 tRNA-related fragments (tRFs) and 1 tRNA half (tiRNA) which showed a significant level decrease after exposure to three kinds of radiations. Moreover, the radiation responses of these 5 serum tsRNAs were reproduced in other mouse strains, and the sequences of them could be detected in serum of humans. Furthermore, we developed multi-factor models based on tsRNA biomarkers to indicate the degree of radiation exposure with high sensitivity and specificity. These findings suggest that the circulating tsRNAs can serve as new minimally invasive biomarkers and can make a triage or dose assessment from blood sample collection within 4 h in exposure scenarios.

A specific tRNA half, 5’tiRNA-His-GTG, responds to hypoxia via the HIF1α/ANG axis and promotes colorectal cancer progression by regulating LATS2

Background Currently, tRNA-derived small RNAs (tsRNAs) are recognized as a novel and potential type of non-coding RNAs (ncRNAs), which participate in various cellular processes and play an essential role in cancer progression. However, tsRNAs involvement in colorectal cancer (CRC) progression remains unclear. Methods Sequencing analyses were performed to explore the tsRNAs with differential expression in CRC. Gain- and loss-of functions of 5’tiRNA-His-GTG were performed in CRC cells and xenograft tumor to discover its role in the progression of CRC. Hypoxia culture and hypoxia inducible factor 1 subunit alpha (HIF1α) inhibitors were performed to uncover the biogenesis of 5’tiRNA-His-GTG. The regulation of 5’tiRNA-His-GTG for large tumor suppressor kinase 2 (LATS2) were identified by luciferase reporter assay, western blot, and rescue experiments. Results Here, our study uncovered the profile of tsRNAs in human CRC tissues and confirmed a specific tRNA half, 5’tiRNA-His-GTG, is upregulated in CRC tissues. Then, in vitro and in vivo experiments revealed the oncogenic role of 5’tiRNA-His-GTG in CRC and found that targeting 5’tiRNA-His-GTG can induce cell apoptosis. Mechanistically, the generation of 5’tiRNA-His-GTG seems to be a responsive process of tumor hypoxic microenvironment, and it is regulated via the HIF1α/angiogenin (ANG) axis. Remarkably, LATS2 was found to be an important and major target of 5’tiRNA-His-GTG, which renders 5’tiRNA-His-GTG to “turn off” hippo signaling pathway and finally promotes the expression of pro-proliferation and anti-apoptosis related genes. Conclusions In summary, the findings revealed a specific 5’tiRNA-His-GTG-engaged pathway in CRC progression and provided clues to design a novel therapeutic target in CRC.

Infection-induced 5′-half molecules of tRNAHisGUG activate Toll-like receptor 7

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5′-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5′-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5′-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5′-tRNAHisGUG half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5′-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5′-tRNA half species into EVs. The EV-5′-tRNAHisGUG half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5′-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of “immune activators” to 5′-tRNA half molecules.

Allergen-induced tRNA halves repress focal adhesion of airway smooth muscle cells

In the pathogenesis of asthma, inflammatory mediators cause structural and functional changes in resident airway cells including airway smooth muscle (ASM) cells. Here, we report that intranasal treatment of mice with house dust mite (HDM) highly upregulated the expression of tRNA half molecules in asthmatic lungs. The 5′-tRNA haves contain a 2′,3′-cyclic phosphate (cP) and thus belong to cP-containing RNAs (cP-RNAs). Capturing the whole cP-RNA transcriptome using cP-RNA-seq revealed the global upregulation of not only tRNA halves but also the cP-RNAs derived from mRNAs and rRNAs. Our investigation of the biological significance of the major upregulated 5′-tRNA half species in human primary ASM cells revealed that the 5′-tRNA halves repress autophosphorylation at Tyr416 of Src protein kinase, a key regulator of the cellular focal adhesion pathway, leading to reduction of ASM cellular focal adhesion. These results assign a novel role as a protein modification modulator to tRNA half molecules and unveil a tRNA-engaged pathway in the molecular pathogenesis of asthma.

Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5’-tRNA halves

The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EV) into the maternal circulation throughout gestation. STB-EV dependent signalling is believed to contribute to the widespread maternal adaptive physiological changes seen in pregnancy. Transfer RNA (tRNA) halves have been identified in vesicles released from other human and murine organ systems, which alter gene expression in target cells. Here, we characterise tRNA-half expression in STB-EV and demonstrate biological activity of a highly abundant tRNA-half. Short RNA from ex-vivo, dual-lobe placental perfusion STB-EV was sequenced, showing that most (>95%) comprised tRNA species. Whole placental tissue contained <50% tRNA species, suggesting selective packaging and export of tRNA into STB-EV. Most tRNA within STB-EV were 5’-tRNA halves cleaved at 30-32 nucleotides. The pattern of tRNA expression differed depending on the size/origin of the STB-EV; this was confirmed by qPCR. Protein synthesis was suppressed in human fibroblasts when they were cultured with a 5’-tRNA half identified from STB-EV sequencing. This study is the first to evaluate tRNA species in STB-EV. The presence of biologically active 5’-tRNA halves, specific to a vesicular origin, suggests a novel mechanism for maternal-fetal signalling in normal pregnancy.

Deep Sequencing of Serum Small RNAs Identifies Patterns of 5′ tRNA Half and YRNA Fragment Expression Associated with Breast Cancer

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5′ tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5′ tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5′ tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.