Identification of tRNA-derived Fragments and Their Potential Roles in Atherosclerosis.

OBJECTIVE: Atherosclerosis (AS), a chronic inflammatory disease, is the basis of cardiovascular disease (CVD). Although the treatment has been greatly improved, AS still imposes a large burden on human health and the medical system, and we still need to further study its pathogenesis. As a novel biomolecule, transfer RNA-derived fragments (tRFs) play a key role in the progression of various disease. However, whether tRFs contribute to atherosclerosis pathogenesis remains unexplored. METHODS: With deep sequencing technology, the change of tRFs expression profiles in patients with AS compared to healthy control group was identified. The accuracy of the sequencing data was validated using RT qPCR. Subsequently, we predicted the potential target genes of tRFs by online miRNA target prediction algorithms. The potential functions of tRFs were evaluated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. RESULTS: There were 13 tRFs differentially expressed between patients with AS and healthy controls, of which 2 were up-regulated and 11 were down-regulated. Validation by RT-qPCR analysis confirmed the sequencing results, and tRF-Gly-GCC-009 was highly up-regulated in the AS group based on the results of sequencing which was confirmed by RT-qPCR analysis. Furthermore, GO enrichment and KEGG pathway analyses indicated that 10 signaling pathways were related to tRF-Gly-GCC-009. These pathways might be physiopathological fundamentals of AS, mainly involving in Apelin signaling, Notch signaling and calcium signaling. CONCLUSION: The results of our study provide important novel insight into the underlying pathogenesis and demonstrate that tRFs might be potential biomarkers and therapeutic targets for AS in the future.

Using tRNA Scaffold to Assist RNA Crystallization.

Recent studies have solidified RNA's regulatory and catalytic roles in all life forms. Understanding such functions necessarily requires high-resolution understanding of the molecular structure of RNA. Whereas proteins tend to fold into a globular structure and gain most of the folding energy from tertiary interactions, RNAs behave the opposite. Their tertiary structure tends to be irregular and porous, and they gain the majority of their folding free energy from secondary structure formation. These properties lead to higher conformational dynamics in RNA structure. As a result, structure determination proves more difficult for RNA using X-ray crystallography and other structural biology tools. Despite the painstaking effort to obtain large quantities of chemically pure RNA molecules, many still fail to crystallize due to the presence of conformational impurity. To overcome the challenge, we developed a new method to crystallize the RNA of interest as a tRNA chimera. In most cases, tRNA fusion significantly increased the conformational purity of our RNA target, improved the success rate of obtaining RNA crystals, and made the subsequent structure determination process much easier. Here in this chapter we describe our protocol to design, stabilize, express, and purify an RNA target as a tRNA chimera. While this method continues a series of work utilizing well-behaving macromolecules/motifs as "crystallization tags" (Ke and Wolberger. Protein Sci 12:306-312, 2003; Ferre-D'Amare and Doudna. J Mol Biol 295:541-556, 2000; Koldobskaya et al . Nat Struct Mol Biol 18:100-106, 2011; Ferre-D'Amare et al. J Mol Biol 279:621-631, 1998), it was inspired by the work of Ponchon and Dardel to utilize tRNA scaffold to express, stabilize, and purify RNA of interest in vivo (Ponchon and Dardel. Nat Methods 4:571-576, 2007). The "tRNA scaffold," where the target RNA is inserted into a normal tRNA, replacing the anticodon sequence, can effectively help the RNA fold, express in various sources and even assist crystallization and phase determination. This approach applies to any generic RNA whose 5' and 3' ends join and form a helix.

Expression and Purification of tRNA/ pre-miRNA-Based Recombinant Noncoding RNAs.

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).

RNA Precipitation.

Precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt, alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA recovery.

Mass Spectrometric Analysis of Mitochondrial RNA Modifications.

Mitochondrial RNAs are modified posttranscriptionally. These modifications are required for proper functioning of RNA molecules, and thereby contribute to essential mitochondrial processes. Herein, we describe our latest mass spectrometry-based platform for analysis of posttranscriptional modifications of mitochondrial tRNAs, and measuring the in vitro activity of mitochondrial RNA-modifying enzymes.

Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins.

Post-translational addition of amino acids to proteins by enzymes using aminoacyl-tRNA is an emerging regulatory mechanism. Examples include Arg transfer in eukaryotes, Leu/Phe transfer in bacteria, and tRNA-synthetase-mediated addition of amino acids to Lys side chains. Here, we present a method of purification and use of tRNA for such reactions, focusing on tRNAArg and its use for arginylation. This method can also be used for other tRNA-mediated reactions. For complete details on the use and execution of this protocol, please refer to Avcilar-Kucukgoze et al. (2020).

Complete chemical structures of human mitochondrial tRNAs.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

A low-bias and sensitive small RNA library preparation method using randomized splint ligation.

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.

LC-MS/MS Profiling of Post-Transcriptional Modifications in Ginseng tRNA Purified by a Polysaccharase-Aided Extraction Method.

Transfer RNAs (tRNAs) are the most heavily modified RNA species in life entities. Post-transcriptional modifications severely impact the structure and function of tRNAs. To date, hundreds of modifications have been identified in tRNAs, mainly from microorganisms and animals. However, tRNAs in plant roots or tubers that have been widely used for food and medical purpose for centuries are rarely studied because isolation of RNA from plants still remains a challenge. In this paper, a polysaccharase-aided RNA isolation (PARI) method for extraction of high-quality RNA from plants containing large quantities of polysaccharides is developed. This method presents a new strategy of "digesting" polysaccharides that is completely different from the conventional method of "dissolving" the contaminants. By using this method, RNA of high integrity and purity were successfully extracted from ginseng roots because polysaccharide contaminations were removed efficiently with α-amylase digestion. Ginseng tRNAs were first sequenced by NGS and a total of 41 iso acceptors were identified. ChloroplastictRNAGly(GCC) in ginseng root was purified and four modified nucleosides, including m7G, D, T, and Ψ, were identified by LC-MS/MS. The results also revealed that the m7G occurs at a novel position 18, which may be related to the deformation of D-loop. PARI is the first enzyme-assisted technique for RNA isolation from plants, which could fundamentally solve the problem of polysaccharide contaminations. By using the PARI method, more individual tRNAs could be isolated easily from polysaccharide-rich plant tissues, which would have a positive impact on the feasibility of research on structure and function of tRNA in plants.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry.

Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.

Complete Genome Sequence of Penicillium oxalicum Strain SGAir0226 Isolated from Outdoor Tropical Air in Singapore.

Penicillium oxalicum strain SGAir0226 was isolated from a tropical air sample collected in Singapore. The complete genome was assembled from long reads obtained from single-molecule real-time sequencing and was further polished and error corrected using short read sequencing data. The assembly comprises 20 contigs with a total length of 30.7 Mb. The genome was predicted to contain 8310 protein-coding genes, 237 tRNAs and 83 rRNAs.

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

Absence of AfuXpot, the yeast Los1 homologue, limits Aspergillus fumigatus growth under amino acid deprived condition.

In Saccharomyces cerevisiae, los1 encodes a nuclear tRNA exporter. Despite the non-essentiality, the deletion of los1 has been shown to extend replicative life span in yeast. Here, we characterized AfuXpot, the los1 homologue in human pathogen Aspergillus fumigatus and found that it is continuously expressed during fungal growth. Microscopic examination of an AfuXpot-GFP-expressing transformant confirmed the nuclear localization of the fusion protein. The targeted gene deletion affirmed the non-essential role of AfuXpot in hyphal growth and sporulation. However, the growth of the deletion mutant was affected by amino acid, but not glucose, deprivation. The susceptibility of the deletant strain to protein and DNA/RNA synthesis inhibitors was also altered. Using bioinformatics tools, some transcription factor binding sites were predicted in AfuXpot promoter. Expression analyses of potential AfuXpot-interacting genes showed a marked down-regulation of sfp1 and mtr10 homologues in ΔAfuXpot strain. Our data demonstrates some conserved aspects of AfuXpot as a tRNA exporter in A. fumigatus.

tRNA­derived small RNAs: A novel class of small RNAs in human hypertrophic scar fibroblasts.

tRNA­derived small RNAs (tsRNAs) have been shown to play regulatory roles in many physiological and pathological processes. However, their roles in hypertrophic scars remain unclear. The present study investigated differentially expressed tsRNAs in human hypertrophic scar fibroblasts and normal skin fibroblasts via high­throughput sequencing. Several dysregulated tsRNAs were validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, target prediction, coexpression networks and competing endogenous RNA (ceRNA) networks were evaluated to discover the principal functions of significantly differentially expressed tsRNAs. In total, 67 differentially expressed tsRNAs were detected, of which 27 were upregulated and 40 downregulated in hypertrophic scar fibroblasts. The GO analysis indicated that the dysregulated tsRNAs are associated with numerous biological functions, including 'nervous system development', 'cell adhesion', 'focal adhesion', 'protein binding', 'angiogenesis' and 'actin binding'. KEGG pathway analysis revealed that the most altered pathways include 'Ras signaling pathway', 'Rap1 signaling pathway' and 'cGMP­PKG signaling pathway'. The target genes of the differentially expressed tsRNAs participate in several signaling pathways important for scar formation. The results of RT­qPCR were consistent with those of sequencing. MicroRNA (miR)­29b­1­5p was identified as a target of tsRNA­23678 and was downregulated in hypertrophic scar fibroblasts, constituting a negative regulatory factor for scar formation. Furthermore, tsRNA­23761 acted as a ceRNA and bound to miR­3135b to regulate the expression of miR­3135b targets, including angiotensin­converting enzyme. Collectively, these findings reveal that tsRNAs are differentially expressed in human hypertrophic scar fibroblasts, and may contribute to the molecular mechanism and treatment of hypertrophic scars.

tRNA-derived fragments (tRFs) contribute to podocyte differentiation.

Loss of glomerular podocytes is the crucial event in the progression of chronic kidney disease (CKD). tRNA-derived fragments (tRFs), a newfangled branch of small non-coding RNA (sncRNA), recently reported to play a vital part in several diseases. In present study, we aimed to detect and reveal the role of tRFs in podocyte differentiation. The expression levels of tRFs between undifferentiated and differentiated podocytes were sequenced by illumina nextseq 500, and further verified by quantitative RT-PCR. 69 upregulated and 70 downregulated tRFs in total were singled out (Fold change > 2, P < 0.05). Gene ontology (GO) analysis indicated they are involved in the biological processes of transcription, DNA-templated, positive regulation of transcription from RNA polymerase II promoter, angiogenesis, cell adhesion. Besides, KEGG analysis suggested that these differentially tRFs are associated with PI3K-Akt signaling pathway, Rap1 signaling pathway, Ras signaling pathway, MAPK signaling pathway, and Wnt signaling pathway. Therefore, the differentially tRFs might regulate the differentiation of podocyte and the process of CKD. The functions and mechanisms of tRFs in podocytes are needed to be further explored.

Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli.

Transfer RNAs (tRNAs) are the most abundant class in small non-coding RNAs which have been proved to be pharmacologically active. In the present study, we evaluated the potential anticancer activities of tRNAs from Escherichia coli MRE 600 to investigate the relationship between non-pathogenic Escherichia coli strain and colorectal cancer. To purify individual tRNAs, we firstly developed a two-dimensional liquid chromatography (2D-LC) and successfully obtained two pure tRNAs. Nuclease mediated base-specific digestions coupled with UHPLC-MS/MS techniques led to an identification of these two tRNAs as tRNA-Val(UAC) and tRNA-Leu(CAG) with typical cloverleaf-like secondary structure. MTT assay demonstrated that both tRNA-1 and tRNA-2 exhibit strong cytotoxicity with IC50 of 113.0 nM and 124.8 nM on HCT-8 cells in a dose-dependent manner. Further clonogenic assay revealed that the purified tRNAs exhibit significant inhibition in colony formation with survival percentage of 79.0 ±â€¯1.6 and 71.2 ±â€¯2.2 at the concentration of 100 nM. These findings provided evidences of anticancer activities of tRNAs from non-pathogenic Escherichia coli strain, indicating that the pharmacological effects of these neglected biomacromolecules from microorganisms should be emphasized. This study put new insights into the therapeutic effects of intestinal microorganism on human diseases, therefore broadened our knowledge of the biological functions of gut microbiota.

Profile analysis reveals transfer RNA fragments involved in mesangial cells proliferation.

Mesangial cell (MCs) proliferation is an essential component of glomerulonephritis. To find some bio-markers of mesangial cell proliferation, we investigate the relationship between transfer RNA fragments (tRFs) and proliferating mesangial cells. The model of proliferating mesangial cells was built by using transforming growth factor-1(TGF-ß1) treated mesangial cells. Then we analyzed the expression of tRFs in normal mesangial cells and mesangial cells treated by TGF-ß1 through high-throughput sequencing technique. qRT-PCR was conducted to validate the differently expressed tRFs in normal mesangial cells and mesangial cells treated by TGF-ß1. tDR-000064 and tDR-000103 were notably down-regulated in mesangial cells treated by TGF-ß1 compared with normal mesangial cells. Then we confirmed that tDR-000064 and tDR-000103 were correlated with proliferation of mesangial cells through receiver operating characteristic curve analysis. Furthermore, Gene ontology (GO) and pathway analysis demonstrated that the two dys-regulated tRFs were mostly involved in mesangial cells and TGF-ß1 receptor-mediated signaling pathway. Our research provides a comprehensive analysis of tRFs in proliferating mesangial cells. (Figure 1A).

Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax.

As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.

Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria.

Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNAPhe are used to prepare two key components of this assay: (1) Cy5-EF-Tu:GTP and (2) Cy3-Phe-tRNAPhe. When mixed and Cy3 is excited at 532 nm, increased Cy5 fluorescence intensity is observed at 665 nm due to ternary complex formation and FRET. If the same assay is carried out in presence of an inhibitor, such as GE2270A (a known inhibitor of the EF-Tu-tRNA interaction), fluorescence intensity is significantly diminished. To establish proof of principle and to show the adaptability of this assay to high throughput screening (HTS), we analyzed the effect of different classes of antibiotics, including beta-lactams, quinolone compounds, and protein synthesis inhibitors, on fluorescence. The assay was done in a 96-well microplate. We observed inhibition by GE2270A, and no effect of nineteen other tested antibiotics, confirming the ability of this FRET assay to serve as a screen for potential inhibitor molecules of ternary complex formation from libraries of compounds.