Exosomal tRF-Leu-AAG-001 Derived from Mast Cell as a Potential Non-Invasive Diagnostic Biomarker for Endometriosis

Background: The diagnosis of endometriosis (EMs) is still based on laparoscopic observation. This study tries to verify whether exosomal tRNA-derived fragments (tRFs) in leucorrhea can be used as non-invasive diagnostic markers.Methods: Endometrial tissues and leucorrhea were sampled from women hospitalized in Ningbo University Affiliated Hospital from January 2021 to July 2021 with (n=26) and without endometriosis (n=25). Exosomes were isolated from samples by differential centrifugation. The small RNA sequencing was performed to detect the exosomal tRNA halves (tiRNAs)&tRFs. RNA probe and immunofluorescence antibody were used to localize the origin of tRFs. From mast cell lines infected with tRF-Leu-AAG-001 siRNA, we observed the change in vascular capacity and expression of inflammatory factors. The specificity and sensitivity tRF were determined by receiver operating characteristic analyses.Results: 63 up-regulated and 45 down-regulated tRFs&tiRNAs were identified in ectopic exosomes. We selected tRF-Leu-AAG-001 as a candidate marker through KEGG pathway enrichment and PCR verification. We found that mast cells highly expressed tRF-Leu-AAG-001 in ectopic foci by immunofluorescence staining. We used siRNA to silenced tRF-Leu-AAG-001 expression in luva, qPCR analysis showed IL-6, IL-10, IL-1β, and TNF-α were significantly decreased. Meanwhile, tRF-Leu-AAG-001 siRNA dramatically reduced the angiogenic ability of luva. Finally, we examined the expression of exosomal tRF-Leu-AAG-001 in the leucorrhea. It was found exosomal tRF-Leu-AAG-001 had high specificity and sensitivity for predicting the occurrence of ectopic disease. Conclusions: Exosomal tRF-Leu-AAG-001 derived from mast cells in ectopic foci might promote inflammation and angiogenesis. Meanwhile，leucorrhea exosomal tRF-Leu-AAG-001 could be a potential diagnostic biomarker for endometriosis.

Stress response in Entamoeba histolytica is associated with robust processing of tRNA to tRNA-halves

AbstracttRNA-derived fragments have been reported in many different organisms and have diverse cellular roles such as regulating gene expression, inhibiting protein translation, silencing transposable elements and modulating cell proliferation. In particular tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here we report the presence of tRNA-derived fragments in Entamoeba with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite to cyst conversion with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments are associated with Entamoeba Argonaute proteins, EhAgo2-2, and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoeba. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses including encystation suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba.ImportancetRNA-derived fragments are small RNAs formed by the cleavage of tRNAs at specific positions. These have been reported in many organisms to modulate gene expression and thus regulate various cell functions. In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small RNA sequencing datasets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway which mediates robust gene silencing in Entamoeba. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

Large-Scale Computational Discovery of Binding Motifs in tRNA Fragments

Accumulating evidence has suggested that tRNA-derived fragments (tRFs) could be loaded to Argonaute proteins and function as regulatory small RNAs. However, their mode of action remains largely unknown, and investigations of their binding mechanisms have been limited, revealing little more than microRNA-like seed regions in a handful of tRFs and a few targets. Here, we identified such regions of potential interaction on a larger scale, using in vivo formed hybrids of guides and targets in crosslinked chimeric reads in two orientations. We considered “forward pairs” (with guides located on the 5′ ends and targets on the 3′ ends of hybrids) and “reverse pairs” (opposite orientation) and compared them as independent sets of biological constructs. We observed intriguing differences between the two chimera orientations, including the paucity of tRNA halves and abundance of polyT-containing targets in forward pairs. We found a total of 197 quality-ranked motifs supported by ∼120,000 tRF–mRNA chimeras, with 103 interacting motifs common in forward and reverse pairs. By analyzing T→C conversions in human and mouse PAR-CLIP datasets, we detected Argonaute crosslinking sites in tRFs, conserved across species. We proposed a novel model connecting the formation of asymmetric pairs in two sets to the potential binding mechanisms of tRFs, involving the identified interaction motifs and crosslinking sites to Argonaute proteins. Our results suggest the way forward for further experimental elucidation of tRF-binding mechanisms.

Global survey of miRNAs and tRNA-derived small RNAs from the human parasitic protist Trichomonas vaginalis

Background Small non-coding RNAs play critical regulatory roles in post-transcription. However, their characteristics in Trichomonas vaginalis, the causative agent of human sexually transmitted trichomoniasis, still remain to be determined. Methods Small RNA transcriptomes from Trichomonas trophozoites were deep sequenced using the Illumina NextSeq 500 system and comprehensively analyzed to identify Trichomonas microRNAs (miRNAs) and transfer RNA (tRNA)-derived small RNAs (tsRNAs). The tsRNA candidates were confirmed by stem-loop quantitative reverse transcription-PCR, and motifs to guide the cleavage of tsRNAs were predicted using the GLAM2 algorithm. Results The miRNAs were found to be present in T. vaginalis but at an extremely low abundance (0.0046%). Three categories of endogenous Trichomonas tsRNAs were identified, namely 5′tritsRNAs, mid-tritsRNAs and 3′tritsRNAs, with the 5′tritsRNAs constituting the dominant category (67.63%) of tsRNAs. Interestingly, the cleavage site analysis verified both conventional classes of tRNA-derived fragments (tRFs) and tRNA-halves in tritsRNAs, indicating the expression of tRNA-halves in the non-stress condition. A total of 25 tritsRNAs were experimentally confirmed, accounting for 78.1% of all tested candidates. Three motifs were predicted to guide the production of tritsRNAs. The results prove the expression of tRFs and tRNA-halves in the T. vaginalis transcriptome. Conclusions This is the first report of genome-wide investigation of small RNAs, particularly tsRNAs and miRNAs, from Trichomonas parasites. Our findings demonstrate the expression profile of tsRNAs in T. vaginalis, while miRNA was barely detected. These results may promote further research aimed at gaining a better understanding of the evolution of small non-coding RNA in T. vaginalis and their functions in the pathogenesis of trichomoniasis.

Circulating Non-Coding RNAs in Head and Neck Cancer: Roles in Diagnosis, Prognosis, and Therapy Monitoring

Head and neck cancer (HNC), the seventh most common form of cancer worldwide, is a group of epithelial malignancies affecting sites in the upper aerodigestive tract. The 5-year overall survival for patients with HNC has stayed around 40–50% for decades, with mortality being attributable mainly to late diagnosis and recurrence. Recently, non-coding RNAs, including tRNA halves, YRNA fragments, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), have been identified in the blood and saliva of patients diagnosed with HNC. These observations have recently fueled the study of their potential use in early detection, diagnosis, and risk assessment. The present review focuses on recent insights and the potential impact that circulating non-coding RNA evaluation may have on clinical decision-making in the management of HNC.

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.

Global Survey of miRNAs and tRNA-derived Small RNAs From Human Parasitic Protist Trichomonas Vaginalis

BackgroundSmall non-coding RNAs play critical regulatory roles in post-transcription. However, their characteristics in Trichomonas vaginalis (T. vaginalis), the causative agent of human sexually transmitted trichomoniasis, still remain to be unveiled. MethodsSmall RNA transcriptomes from Trichomonas trophozoites were deeply sequenced through Illumina NextSeq 500 system and comprehensively analyzed to identify Trichomonas miRNAs and tRNA-derived small RNAs (tsRNAs). The tsRNAs candidates were confirmed by stem-loop RT-PCR and motifs to guide the cleavage of tsRNAs were predicted by performing GLAM2 algorithm. ResultsThe miRNAs were found at extremely low abundance (0.0046%) in T. vaginalis. Three categories of endogenous Trichomonas tsRNAs were identified as 5'tritsRNAs, mid-tritsRNAs and 3'tritsRNAs, with 5'tritsRNAs dominating (67.63%) in tsRNAs. Interestingly, the cleavage site analysis verified both conventional classes of tRFs and tRNA-halves in tritsRNAs, indicating the expression of tRNA-halves in non-stress condition. A total of 25 tritsRNAs were experimentally confirmed, accounting for 78.1% of all tested candidates. Three motifs were predicted to guide the production of tritsRNAs. This proved the expression of tRFs and tRNA-halves in T. vaginalis transcriptome. ConclusionsThis is the first report of genome-wide investigation of small RNAs, particularly tsRNAs and miRNAs, from Trichomonas parasites. Our findings demonstrate the expression profile of tsRNAs in T. vaginalis, while miRNA was hardly discovered. These results might promote the further research to better understand the evolution of small non-coding RNA in T. vaginalis and their functions in pathogenesis of trichomoniasis.

Mechanisms of tRNA-derived fragments and tRNA halves in cancer treatment resistance

The tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) are newly discovered noncoding RNAs in recent years. They are derived from specific cleavage of mature and pre-tRNAs and expressed in various cancers. They enhance cell proliferation and metastasis or inhibit cancer progression. Many studies have investigated their roles in the diagnosis, progression, metastasis, and prognosis of various cancers, but the mechanisms through which they are involved in resistance to cancer treatment are unclear. This review outlines the classification of tRFs and tiRNAs and their mechanisms in cancer drug resistance, thus providing new ideas for cancer treatment.

Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment

Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our in vitro assay. These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.