tRNA Halves Research Articles

A Comprehensive Repertoire of Transfer RNA-Derived Fragments and Their Regulatory Networks in Colorectal Cancer.

To provide systematic insight into the composition and expression of transfer RNA (tRNA) derivatives transcriptome in colorectal cancer (CRC). tRNA derivatives expression profiles in three pairs of CRC and adjacent normal colon tissues were performed by tRNA-derived small RNA fragments (tRFs) and tRNA halves (tiRNA) sequencing, and microarray data of transcriptomes from CRC and paired controls were retrieved from Gene Expression Omnibus database. The differentially expressed tRFs and tiRNAs and differentially expressed genes between CRC and paired normal samples were screened. The functional regulations between tRF and tiRNA and gene were identified. A total of 60 upregulated and 48 downregulated tRNA derivatives and 7373 upregulated and 12,138 downregulated messenger RNA (mRNA) were identified. The tRF and tiRNA-gene regulatory modules were constructed by analyzing computational tRF and tiRNA-target predictions and inverse expression relationships between tRF and tiRNAs and mRNA. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway annotation showed that the function of targets of tiRNA-Tyr-GTA was mainly enriched in negative regulation of epithelial cell apoptotic process and peroxisome proliferator activated-receptors (PPAR) signaling pathway. Cellular response to monoamine stimulus and inflammatory bowel disease was enriched in function of tiRNA-Val-CAC. Two functions, including negative regulation of c-Jun N-terminal kinase (JNK) cascade and choline metabolism in cancer, were enriched in tRF-Gln-CTG. The function of mesenchymal to epithelial transition was enriched in tRF-Leu-TAG. For the first time to our knowledge, our study provided a landscape of tRNA derivatives expression profiles in CRC. Further tRF and tiRNA-gene regulatory modules construction explored the potential functions related to these tRNA derivatives in the pathogenesis of CRC.

Modulation of mammalian translation by a ribosome-associated tRNA half

ABSTRACT Originally considered futile degradation products, tRNA-derived RNA fragments (tdRs) have been shown over the recent past to be crucial players in orchestrating various cellular functions. Unlike other small non-coding RNA (ncRNA) classes, tdRs possess a multifaceted functional repertoire ranging from regulating transcription, apoptosis, RNA interference, ribosome biogenesis to controlling translation efficiency. A subset of the latter tdRs has been shown to directly target the ribosome, the central molecular machine of protein biosynthesis. Here we describe the function of the mammalian tRNAPro 5ʹ half, a 35 residue long ncRNA associated with ribosomes and polysomes in several mammalian cell lines. Addition of tRNAPro halves to mammalian in vitro translation systems results in global translation inhibition and concomitantly causes the upregulation of a specific low molecular weight translational product. This tRNAPro 5ʹ half-dependent translation product consists of both RNA and amino acids. Transfection of the tRNAPro half into HeLa cells leads to the formation of the same product in vivo. The migration of this product in acidic gels, the insensitivity to copper sulphate treatment, the resistance to 3ʹ polyadenylation, and the association with 80S monosomes indicate that the accumulated product is peptidyl-tRNA. Our data thus suggest that binding of the tRNAPro 5ʹ half to the ribosome leads to ribosome stalling and to the formation of peptidyl-tRNA. Our findings revealed a so far unknown functional role of a tdR thus further enlarging the functional heterogeneity of this emerging class of ribo-regulators.

Stress Induced tRNA Halves (tiRNAs) as Biomarkers for Stroke and Stroke Therapy; Pre-clinical Study

tiRNAs are small non-coding RNAs generated by angiogenin-mediated tRNA cleavage during cellular stress. Some tiRNAs were shown to be cytoprotective, while other reports indicate that the generation of tiRNAs is cytotoxic. We used rat model of focal cerebral ischemia–reperfusion (I/R) injury to study the generation and regulation of tiRNAs following in vivo I/R and the impact of neuroprotective therapy on their generation. tiRNAs were induced after I/R and Minocycline therapy reduced global tiRNA levels. Our results showed that tRNA cleavage is tRNA species specific, and neuroprotective treatment does not affect all tiRNA species. We also evaluated the temporal changes in several tRNA modifying enzymes and showed a correlation between their expression and tRNA cleavage. In conclusion, we show that tiRNAs can serve as biomarkers for stroke and stroke therapy, further adding them to the repertoire of tools that can be used to monitor and treat stroke.

5' tRNA halves are highly expressed in the primate hippocampus and might sequence-specifically regulate gene expression.

Fragments of mature tRNAs have long been considered as mere degradation products without physiological function. However, recent reports show that tRNA-derived small RNAs (tsRNAs) play prominent roles in diverse cellular processes across a wide spectrum of species. Contrasting the situation in other small RNA pathways the mechanisms behind these effects appear more diverse, more complex, and are generally less well understood. In addition, surprisingly little is known about the expression profiles of tsRNAs across different tissues and species. Here, we provide an initial overview of tsRNA expression in different species and tissues, revealing very high levels of 5′ tRNA halves (5′ tRHs) particularly in the primate hippocampus. We further modulated the regulation capacity of selected 5′ tRHs in human cells by transfecting synthetic tsRNA mimics (“overexpression”) or antisense-RNAs (“inhibition”) and identified differentially expressed transcripts based on RNA-seq. We then used a novel k-mer mapping approach to dissect the underlying targeting rules, suggesting that 5′ tRHs silence genes in a sequence-specific manner, while the most efficient target sites align to the mid-region of the 5′ tRH and are located within the CDS or 3′ UTR of the target. This amends previous observations that tsRNAs guide Argonaute proteins to silence their targets via a miRNA-like 5′ seed match and suggests a yet unknown mechanism of regulation. Finally, our data suggest that some 5′ tRHs that are also able to sequence-specifically stabilize mRNAs as up-regulated mRNAs are also significantly enriched for 5′ tRH target sites.

TRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

ABSTRACT tRNA-derived small fragments (tRFs) and tRNA halves have emerging functions in different biological pathways, such as regulating gene expression, protein translation, retrotransposon activity, transgenerational epigenetic changes and response to environmental stress. However, small RNAs like tRFs and microRNAs in the maternal-fetal interface during gestation have not been studied extensively. Here we investigated the small RNA composition of mouse placenta/decidua, which represents the interface where the mother communicates with the foetus, to determine whether there are specific differences in tRFs and microRNAs during fetal development and in response to maternal immune activation (MIA). Global tRF expression pattern, just like microRNAs, can distinguish tissue types among placenta/decidua, fetal brain and fetal liver. In particular, 5ʹ tRNA halves from tRNAGly, tRNAGlu, tRNAVal and tRNALys are abundantly expressed in the normal mouse placenta/decidua. Moreover, tRF and microRNA levels in the maternal-fetal interface change dynamically over the course of embryonic development. To see if stress alters non-coding RNA expression at the maternal-fetal interface, we treated pregnant mice with a viral infection mimetic, which has been shown to promote autism-related phenotypes in the offspring. Acute changes in the levels of specific tRFs and microRNAs were observed 3–6 h after MIA and are suppressed thereafter. A group of 5ʹ tRNA halves is down-regulated by MIA, whereas a group of 18-nucleotide tRF-3a is up-regulated. In conclusion, tRFs show tissue-specificity, developmental changes and acute response to environmental stress, opening the possibility of them having a role in the fetal response to MIA.

The role of Transfer RNA-Derived Small RNAs (tsRNAs) in Digestive System Tumors.

Transfer RNA-derived small RNA(tsRNA) is a type of non-coding tRNA undergoing cleavage by specific nucleases such as Dicer. TsRNAs comprise of tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs). Based on the splicing site within the tRNA, tRFs can be classified into tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF. TiRNAs can be classified into 5′-tiRNA and 3′-tiRNA. Both tRFs and tiRNAs have important roles in carcinogenesis, especially cancer of digestive system. TRFs and tiRNAs can promote cell proliferation and cell cycle progression by regulating the expression of oncogenes, combining with RNA binding proteins such as Y-box binding protein 1 (YBX1) to prevent transcription. Despite many reviews on the basic biological function of tRFs and tiRNAs, few have described their correlation with tumors especially gastrointestinal tumor. This review focused on the relationship of tRFs and tiRNAs with the biological behavior, clinicopathological characteristics, diagnosis, treatment and prognosis of digestive system tumors, and would provide novel insights for the early detection and treatment of digestive system tumors.

Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner

ABSTRACT Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5ʹ tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5ʹ tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.

A Molecular Signature of Three tRNA-Derived RNA Fragments May Discriminate Smoldering from Symptomatic Multiple Myeloma Patients

Introduction: Multiple myeloma (MM) is the second most common hematological malignancy arising from terminally differentiated plasma cells. The diagnosis of symptomatic ΜΜ is usually based on the presence of end-organ damage, as defined by the CRAB criteria. ΜΜ is part of a spectrum of disorders characterized as monoclonal gammopathies. Smoldering multiple myeloma (SMM) is a plasma cell dyscrasia preceding MM. The risk of sMM progression to active MM is determined by risk stratification models, such as the Mayo Clinic and the Spanish models. The clinical course of MM is quite heterogenous; patients survival is ranging from months to more than a decade. MM risk is determined using the international staging system (ISS) and the revised-ISS (R-ISS), which incorporates LDH and cytogenetics to ISS. tRNA-derived RNA fragments suggest a class of small non-coding RNAs, which derive from either pre- or mature tRNAs. tRNA fragments have only recently emerged and therefore have not been broadly studied. These fragments are not random degradation products, yet occur through specific enzymatic activity. Two large groups of these molecules are currently known and are distinguished based on their length; tiRNAs or tRNA halves consist of 30-50 nucleotides, while tRNA-derived fragments (tRFs) consist of 16-28 nucleotides. Both categories are further subgrouped according to their site of origin, in 5′-, 3′-, and internal fragments. Although their role is still under evaluation, studies have linked them to pathological situations, such as neurodegenerative diseases, various cancer types and hematological malignancies. In this study, the clinical significance of 6 of these fragments was evaluated in MM, and three of them showed interesting results. These molecules are 3′-tRFs or internal tRFs (i-tRFs), which occur from the tRNAs bearing leucine, glutamic acid and proline anticondons, namely 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG, respectively. Methods: CD138+ plasma cells were collected from 80 patients at the time of diagnosis: 65 with MM and 15 with SMM. Total RNA was extracted from CD138+ cells using TRIzol and thereafter was polyadenylated by Escherichia coli poly(A) polymerase. First-strand cDNA synthesis was performed by MMLV transcriptase, using an oligo-dT-adaptor primer. Subsequently, we used and in-house real-time quantitative PCR assay, based on SYBR Green chemistry, in order to quantify tRFs in all samples. Specific forward primers were designed for all tested molecules, along with a common reverse primer, complementary to the adaptor used during reverse transcription. The small nucleolar RNAs RNU43 and RNU48 were used as reference genes, in order to normalize qPCR data. Biostatistical analysis was carried out to assess these results. Results: Out of 65 MM patients 13 were classified as having ISS I stage, 22 as ISS II stage, and 30 as ISS III stage. Similarly, 13 of them were grouped in R-ISS I stage, 35 as R-ISS II, and 17 as R-ISS III. The Mann Whitney U test revealed that the levels of 3′-tRF-LeuAAG/TAG, itRF-GluCTC, and i-tRF-ProTGG differed significantly between MM and SMM cases (P=0.001, P=0.047 and P=0.033, respectively). Specifically, these molecules had higher levels in the CD138+ cells of the SMM cases. These results were validated by logistic regression analysis, which showed that patients with lower expression levels of these molecules were at a higher risk of suffering from MM (P=0.003 for 3′-tRF-LeuAAG/TAG , P=0.036 for itRF-GluCTC, and P=0.032 for i-tRF-ProTGG). Furthermore, 3′-tRF-LeuAAG/TAG was related with translocation t(4;14). Conclusions: 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG may represent novel molecular biomarkers for the differential diagnosis of MM from SMM in ambiguous cases, in which the diagnostic work-up does not provide a clear diagnosis. These novel biomarkers could also play a role in the prediction of sMM cases at high risk of evolution to active multiple myeloma given their overexpression in asymptomatic cases. A possible correlation of these biomarkers to prognosis in MM patients may be indicated by their relationship with the t(4;14) translocation. Disclosures Gavriatopoulou: Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Prothena: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Terpos:Genesis: Honoraria, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Medison: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding.

Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3–mediated gene silencing

tRNA fragments (tRFs) and tRNA halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more. Overexpressed angiogenin (ANG) cleaves tRNA anticodons and produces tRNA halves similar to those produced in response to stress. However, it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRFs are generated from the tRNA halves. Here, using global short RNA-Seq approach, we found that ANG overexpression selectively cleaves a subset of tRNAs, including tRNAGlu, tRNAGly, tRNALys, tRNAVal, tRNAHis, tRNAAsp, and tRNASeC to produce tRNA halves and tRF-5s that are 26-30 bases long. Surprisingly, ANG knockout revealed that the majority of stress-induced tRNA halves, except for the 5' half from tRNAHisGTG and the 3' half from tRNAAspGTC, are ANG independent, suggesting there are other RNases that produce tRNA halves. We also found that the 17-25 bases-long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from tRNA halves. Consistent with this, ANG knockout did not decrease tRF-3 levels or gene-silencing activity. We conclude that ANG cleaves specific tRNAs and is not the only RNase that creates tRNA halves and that the shorter tRFs are not generated from the tRNA halves or from independent tRNA cleavage by ANG.

Profiling of tRNA Halves and YRNA Fragments in Serum and Tissue From Oral Squamous Cell Carcinoma Patients Identify Key Role of 5' tRNA-Val-CAC-2-1 Half.

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer and, as indicated by The Oral Cancer Foundation, kills at an alarming rate of roughly one person per hour. With this study, we aimed at better understanding disease mechanisms and identifying minimally invasive disease biomarkers by profiling novel small non-coding RNAs (specifically, tRNA halves and YRNA fragments) in both serum and tumor tissue from humans. Small RNA-Sequencing identified multiple 5′ tRNA halves and 5′ YRNA fragments that displayed significant differential expression levels in circulation and/or tumor tissue, as compared to control counterparts. In addition, by implementing a modification of weighted gene coexpression network analysis, we identified an upregulated genetic module comprised of 5′ tRNA halves and miRNAs (miRNAs were described in previous study using the same samples) with significant association with the cancer trait. By consequently implementing miRNA-overtargeting network analysis, the biological function of the module (and by “guilt by association,” the function of the 5′ tRNA-Val-CAC-2-1 half) was found to involve the transcriptional targeting of specific genes involved in the negative regulation of the G1/S transition of the mitotic cell cycle. These findings suggest that 5′ tRNA-Val-CAC-2-1 half (reduced in serum of OSCC patients and elevated in the tumor tissue) could potentially serve as an OSCC circulating biomarker and/or target for novel anticancer therapies. To our knowledge, this is the first time that the specific molecular function of a 5′-tRNA half is specifically pinpointed in OSCC.

TRNA cleavage: a new insight.

Over the past decades, tRNA was found to be a rich hub of RNA modifications such as 1-methyladenosine and 5-methycytosine modifications and others, holding more than half of all modifications occurring in RNA molecules. Moreover, tRNA was discovered to be a source of various small noncoding RNA species, such as the stress induced angiogenin cleaved tRNA halves (tiRNA) or the miRNA like tRNA derived fragments. tRNA cleavage under stress was fist discovered in bacteria and later was found to be conserved across different species, including mammals. Under cellular stress conditions, tRNA undergoes conformational changes and angiogenin cleaves it into 3′ and 5′ halves. 5′tiRNA halves were shown to repress protein translations. tRNA cleavage is thought of to be a cytoprotective mechanism by which cells evade apoptosis, however some data hints to the opposite; that tiRNA are cytotoxic or at least related to apoptosis initiation. tRNA cleavage also was shown to be affected by tRNA modifications via different enzymes in the cytosol and mitochondria. In this review, we will highlight the biology of tRNA cleavage, show the evidence of it being cytoprotective or a marker of cell death and shed a light on its role in disease models and human diseases as well as possible future directions in this field of RNA research.

Identified Hybrid tRNA Structure Genes in Archaeal Genome.

Background: In Archaea, previous studies have revealed the presence of multiple intron-containing tRNAs and split tRNAs. The full unexpurgated analysis of archaeal tRNA genes remains a challenging task in the field of bioinformatics, because of the presence of various types of hidden tRNA genes in archaea. Here, we suggested a computational method that searched for widely separated genes encoding tRNA halves to generate suppressive variants of missing tRNAs.Objectives: The exploration of tRNA genes from a genome with varying hypotheses, among all three domain of life (eukaryotes, bacteria and archaea), has been rapidly identified in different ways in the field of bioinformatics. Like eukaryotic tRNA genes, it has been established that two separated regions of the coding sequence of a tRNA gene are essential and sufficient for promotion of transcription. Our objective is to find out the two essential regions in the genome sequence which comprises two halves of the hidden tRNAs.Material and Methods: Considering the existence of split tRNA genes widely separated throughout the genome, we developed our tRNA search algorithm to predict such separated tRNA genes by searching both a conserved terminal 5'- and 3'-motif of tRNA in agreement with the split hypothesis on the basis of cloverleaf prediction and precise insilico determination of bulge-helix-bulge secondary structure at the splice sites.Results: By a comprehensive search for all kinds of missing tRNA genes, we have constructed hybrid tRNA genes containing one essential region from tDNA (XYZ) and the other from tDNA (ABC), both from same species in the archaea. We have also found, this type of hybrid tRNA genes are identified in the different species of the archaea (XYZ ASN, ARG and MET; ABC ASP,SER, ARG and PRO).These hybrid split tRNA share a common structural motif called bulge-helix-bulge (BHB) a more relaxed bulge-helix loop (BHL), at the leader exon boundary and suggested to be evolutionary interrelated.Conclusions: Analysis of the complete genome sequences of Metallosphaera sedula DSM 5348, Desulfurococcus kamchatkensis 1221n and Ignicoccus hospitalis KIN4/I in archaea by our algorithm revealed that a number of hybrid tRNAs are constructed from different tDNAs . Asymmetric combination of 5’ and 3’ tRNA halves may have generated the diversity of tRNA molecules. Our study of hybrid tRNA genes will provide a new molecular basis for upcoming tRNA studies.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage

ABSTRACTWith the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

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.

The Uroboros Theory of Life's Origin: 22-Nucleotide Theoretical Minimal RNA Rings Reflect Evolution of Genetic Code and tRNA-rRNA Translation Machineries.

Theoretical minimal RNA rings attempt to mimick life's primitive RNAs. At most 25 22-nucleotide-long RNA rings code once for each biotic amino acid, a start and a stop codon and form a stem-loop hairpin, resembling consensus tRNAs. We calculated, for each RNA ring's 22 potential splicing positions, similarities of predicted secondary structures with tRNA vs. rRNA secondary structures. Assuming rRNAs partly derived from tRNA accretions, we predict positive associations between relative secondary structure similarities with rRNAs over tRNAs and genetic code integration orders of RNA ring anticodon cognate amino acids. Analyses consider for each secondary structure all nucleotide triplets as potential anticodon. Anticodons for ancient, chemically inert cognate amino acids are most frequent in the 25 RNA rings. For RNA rings with primordial cognate amino acids according to tRNA-homology-derived anticodons, tRNA-homology and coding sequences coincide, these are separate for predicted cognate amino acids that presumably integrated late the genetic code. RNA ring secondary structure similarity with rRNA over tRNA secondary structures associates best with genetic code integration orders of anticodon cognate amino acids when assuming split anticodons (one and two nucleotides at the spliced RNA ring 5' and 3' extremities, respectively), and at predicted anticodon location in the spliced RNA ring's midst. Results confirm RNA ring homologies with tRNAs and CDs, ancestral status of tRNA half genes split at anticodons, the tRNA-rRNA axis of RNA evolution, and that single theoretical minimal RNA rings potentially produce near-complete proto-tRNA sets. Hence genetic code pre-existence determines 25 short circular gene- and tRNA-like RNAs. Accounting for each potential splicing position, each RNA ring potentially translates most amino acids, realistically mimicks evolution of the tRNA-rRNA translation machinery. These RNA rings 'of creation' remind the uroboros' (snake biting its tail) symbolism for creative regeneration.

Cicada Endosymbionts Have tRNAs That Are Correctly Processed Despite Having Genomes That Do Not Encode All of the tRNA Processing Machinery.

Gene loss and genome reduction are defining characteristics of endosymbiotic bacteria. The most highly reduced endosymbiont genomes have lost numerous essential genes related to core cellular processes such as replication, transcription, and translation. Computational gene predictions performed for the genomes of the two bacterial symbionts of the cicada Diceroprocta semicincta, "Candidatus Hodgkinia cicadicola" (Alphaproteobacteria) and "Ca Sulcia muelleri" (Bacteroidetes), have found only 26 and 16 tRNA genes and 15 and 10 aminoacyl tRNA synthetase genes, respectively. Furthermore, the original "Ca Hodgkinia cicadicola" genome annotation was missing several essential genes involved in tRNA processing, such as those encoding RNase P and CCA tRNA nucleotidyltransferase as well as several RNA editing enzymes required for tRNA maturation. How these cicada endosymbionts perform basic translation-related processes remains unknown. Here, by sequencing eukaryotic mRNAs and total small RNAs, we show that the limited tRNA set predicted by computational annotation of "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" is likely correct. Furthermore, we show that despite the absence of genes encoding tRNA processing activities in the symbiont genomes, symbiont tRNAs have correctly processed 5' and 3' ends and seem to undergo nucleotide modification. Surprisingly, we found that most "Ca Hodgkinia cicadicola" and "Ca Sulcia muelleri" tRNAs exist as tRNA halves. We hypothesize that "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" tRNAs function in bacterial translation but require host-encoded enzymes to do so.IMPORTANCE The smallest bacterial genomes, in the range of about 0.1 to 0.5 million base pairs, are commonly found in the nutritional endosymbionts of insects. These tiny genomes are missing genes that encode proteins and RNAs required for the translation of mRNAs, one of the most highly conserved and important cellular processes. In this study, we found that the bacterial endosymbionts of cicadas have genomes which encode incomplete tRNA sets and lack genes required for tRNA processing. Nevertheless, we found that endosymbiont tRNAs are correctly processed at their 5' and 3' ends and, surprisingly, that mostly exist as tRNA halves. We hypothesize that the cicada host must supply its symbionts with these missing tRNA processing activities.

Using tRNA halves as novel biomarkers for the diagnosis of gastric cancer.

tRNA halves (tiRNAs) are produced from mature tRNAs. They have important roles both with in normal cells and cancer cells. However, the diagnostic value of tiRNAs in cancers have not yet been elucidated. To explore the diagnostic value of tiRNA-5034-GluTTC-2 in gastric cancer. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression levels of tiRNA-5034-GluTTC-2 in paired gastric cancer tissues and adjacent normal tissues, plasmas from patients with gastric cancer and healthy people, and gastric cancer cell lines. Then, the relationship between its levels and clinicopathological factors of patients with gastric cancer was analyzed. A receiver operating characteristic (ROC) curve was established to predict the diagnostic value. tiRNA-5034-GluTTC-2 was first found to be down-regulated in gastric cancer tissues and plasmas. Its levels were significantly associated with tumor size. The area under the ROC curve (AUC) was 0.779 and 0.835 in tissue and plasma, respectively. The sensitivity, specificity and AUC were 84.7%, 92.8%, and 0.915 when tissues and plasmas were used in combination, respectively. The overall survival rate of patients with a lower expression of tiRNA-5034-GluTTC-2 was significantly lower than those with a higher expression. These results indicated that tiRNA-5034-GluTTC-2 may be a novel biomarker for the diagnosis of gastric cancer.

Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host

The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the M. tuberculosis TA systems belong to the VapBC (virulence associated protein) family. In this work, we first identified the RNA targets for the M. tuberculosis VapC-mt11 (VapC11, Rv1561) toxin in vitro to learn more about the general function of this family of toxins. Recombinant VapC-mt11 cleaved 15 of the 45 M. tuberculosis tRNAs at a single site within their anticodon stem loop (ASL) to generate tRNA halves. Cleavage was dependent on the presence of a GG consensus sequence immediately before the cut site and a structurally intact ASL. However, in striking contrast to the broad enzyme activity exhibited in vitro, we used a specialized RNA-seq method to demonstrate that tRNA cleavage was highly specific in vivo. Expression of VapC-mt11 in M. tuberculosis resulted in cleavage of only two tRNA isoacceptors containing the GG consensus sequence, tRNAGln32-CUG and tRNALeu3-CAG. Therefore, our results indicate that although in vitro studies are useful for identification of the class of RNA cleaved and consensus sequences required for accurate substrate recognition by endoribonuclease toxins, definitive RNA target identification requires toxin expression in their native host. The restricted in vivo specificity of VapC-mt11 suggests that it may be enlisted to surgically manipulate pathogen physiology in response to stress.

Identification of tRNA-Derived Fragments Expression Profile in Breast Cancer Tissues.

Background:In recent years, tRFs(transfer RNA-Derived Fragments) and transfer RNADerived Stress-induced RNAs (or tRNA halves) have been shown to have vital roles in cancer biology.We aimed to reveal the expression profile of tRNA-derived fragments in breast cancer tissues in the study, and to explore their potential as biomarkers of breast cancer.Methods:We characterized the tRNA-derived fragments expression profile from 6 paired clinical breast cancer tissues and adjacent normal samples. Then we selected 6 significantly expressed tRNAderived fragments and screened the genes for validation by using Quantitative Real-time PCR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biological pathway were finally analyzed.Results:We found 30 differentially expressed tRNA-derived fragments across our dataset, out of which 17 were up-regulated, and 13 were down-regulated. Compared with 16 clinical breast cancer tissues and adjacent normal tissues by qPCR, the results demonstrated that tRF-32-Q99P9P9NH57SJ(FC = -2.6476, p = 0.0189), tRF-17-79MP9PP (FC = -4.8984, p = 0.0276) and tRF-32-XSXMSL73VL4YK (FC = 6.5781, p = 0.0226) were significantly expressed in breast cancer tissues(p < 0.001). tRF-32-XSXMSL73VL4YK was significantly up-regulated, and tRF-32-Q99P9P9NH57SJ and tRF-17-79MP9PP were significantly down-regulated in which the expressionpatterns were similar to the sequencing results. The top ten significant results of GO and KEGG pathways enrichment analysis were presented.Conclusion:Our studies have demonstrated that there were significantly expressed tRNA-derived fragments in breast cancer tissues. They are hopefully to become biomarkers and would be valuable researches in this area.

TRNA-derived fragments and tRNA halves: The new players in cancers.

tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) are small non-coding RNAs derived from precursor tRNAs or mature tRNAs. Depending on the sources, tRFs can be divided into tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF; tiRNAs can be divided into 5'tiRNA and 3'tiRNA. Both tRFs and tiRNAs play important roles in tumorigenesis. Some tRFs and tiRNAs promote cell proliferation and cell cycle progression by regulating the expression of oncogenes. Other tRFs and tiRNAs inhibit cancer progression. Mechanism studies have shown that tRFs and tiRNAs may bind to RNA binding proteins such as Y-box binding protein 1 (YBX1) and prevent transcription, inactivate initiation factor eIF4G/A, promote translation of ribosomal proteins, or activate aurora kinase A, the regulator of mitosis. Therefore, tRFs and tiRNAs regulate the occurrence and development of cancers, including lung cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, B cell lymphoma, chronic lymphocytic leukemia, etc. This article reviews the classification of tRFs and tiRNAs, their biological functions in the occurrence of cancers, and their relationships with some common cancers. It will provide new ideas for the diagnosis and treatment of cancers.