Inflammatory cytokine-regulated tRNA-derived fragment tRF-21 suppresses pancreatic ductal adenocarcinoma progression.

The tumorigenic mechanism for pancreatic ductal adenocarcinoma (PDAC) is not clear, although chronic inflammation is implicated. Here, we identified an inflammatory cytokine-regulated transfer RNA-derived (tRNA-derived) fragment, tRF-21-VBY9PYKHD (tRF-21), as a tumor suppressor in PDAC progression. We found that the biogenesis of tRF-21 could be inhibited by leukemia inhibitory factor and IL-6 via the splicing factor SRSF5. Reduced tRF-21 promoted AKT2/1-mediated heterogeneous nuclear ribonucleoprotein L (hnRNP L) phosphorylation, enhancing hnRNP L to interact with dead-box helicase 17 (DDX17) to form an alternative splicing complex. The provoked hnRNP L-DDX17 activity preferentially spliced Caspase 9 and mH2A1 pre-mRNAs to form Caspase 9b and mH2A1.2, promoting PDAC cell malignant phenotypes. The tRF-21 levels were significantly lower in PDACs than in normal tissues, and patients with low tRF-21 levels had a poor prognosis. Treatment of mouse PDAC xenografts or patient-derived xenografts (PDXs) with tRF-21 mimics repressed tumor growth and metastasis. These results demonstrate that tRF-21 has a tumor-suppressive effect and is a potential therapeutic agent for PDAC.

Transfer- or 'transmission'-RNA fragments? The roles of tsRNAs in the reproductive system.

Transfer-RNAs (tRNAs) help ribosomes decode mRNAs and synthesize proteins; however, tRNA fragments produced under certain conditions, known as tRNA-derived small RNAs (tsRNAs), have been found to play important roles in pathophysiological processes. In the reproductive system, tsRNAs are abundant in gametes and embryos and at the maternal-fetal interface, as well as in microvesicles like epididymosomes, seminal plasma exosomes, and syncytiotrophoblast-derived extracellular vesicles. tsRNAs can affect gamete cell maturation, zygote activation, and early embryonic development. tsRNAs can transmit epigenetic information to later generations. In particular, exposure to environmental factors such as nutrition, isoproterenol, and poly(I:C) may allow tsRNAs to transfer information to the gametes or placenta to alter offspring phenotype. The underlying mechanisms of tsRNAs action include transposon silencing, translation regulation, and target mRNA degradation. Herein, we review the currently reported tsRNAs in the reproductive system, their validated functions, and potential roles. A better understanding of this field may help to provide useful recommendations or develop strategies to increase fertility and conception of healthy babies.

Genes on the circular code alphabet.

The X motifs, motifs from the circular code X, are enriched in the (protein coding) genes of bacteria, archaea, eukaryotes, plasmids and viruses, moreover, in the minimal gene set belonging to the three domains of life, as well as in tRNA and rRNA sequences. They allow to retrieve, maintain and synchronize the reading frame in genes, and contribute to the regulation of gene expression. These results lead here to a theoretical study of genes based on the circular code alphabet. A new occurrence relation of the circular code X under the hypothesis of an equiprobable (balanced) strand pairing is given. Surprisingly, a statistical analysis of a large set of bacterial genes retrieves this relation on the circular code alphabet, but not on the DNA alphabet. Furthermore, the circular code X has the strongest balanced circular code pairing among 216 maximal C3 self-complementary trinucleotide circular codes, a new property of this circular code X. As an application of this theory, different tRNAs studied on the circular code alphabet reveal an unexpected stem structure. Thus, the circular code X would have constructed a coding stem in tRNAs as an outline of the future gene structure and the future DNA double helix.

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).

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid.

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

A Natural Variation in PLEIOTROPIC DEVELOPMENTAL DEFECTS Uncovers a Crucial Role for Chloroplast tRNA Modification in Translation and Plant Development.

The modification of tRNA is important for accurate, efficient protein translation. A number of tRNA-modifying enzymes were found to influence various developmental processes in distinct organisms. However, few genetic or molecular studies have focused on genes encoding tRNA-modifying enzymes in green plant organelles. Here, we discovered that PDD OL , a natural variation allele of PLEIOTROPIC DEVELOPMENTAL DEFECTS (PDD), leads to pleiotropic developmental defects in a near-isogenic line (NIL) generated by introgressing the wild rice Oryza longistaminata into the rice (Oryza sativa) cv 187R. Map-based cloning revealed that PDD encodes an evolutionarily conserved tRNA-modifying GTPase belonging to the tRNA modification E family. The function of PDD was further confirmed by genetic complementation experiments and mutant analysis. PDD mRNA is primarily expressed in leaves, and PDD is localized to chloroplasts. Biochemical analyses indicated that PDD187R forms homodimers and has strong GTPase activity, whereas PDDOL fails to form homodimers and has weak GTPase activity. Liquid chromatography-coupled tandem quadrupole mass spectrometry revealed that PDD is associated with the 5-methylaminomethyl-2-thiouridine modification of chloroplast tRNA. Furthermore, compared to 187R, NIL-PDD OL has severely reduced levels of proteins involved in photosynthesis and ribosome biogenesis but increased levels of plastid-encoded RNA polymerase subunits. Finally, we demonstrate that the defect due to PDD OL alters chloroplast gene expression, thereby affecting communication between the chloroplast and the nucleus.

Accretion history of large ribosomal subunits deduced from theoretical minimal RNA rings is congruent with histories derived from phylogenetic and structural methods.

Accretions of tRNAs presumably formed the large complex ribosomal RNA structures. Similarities of tRNA secondary structures with rRNA secondary structures increase with the integration order of their cognate amino acid in the genetic code, indicating tRNA evolution towards rRNA-like structures. Here analyses rank secondary structure subelements of three large ribosomal RNAs (Prokaryota: Archaea: Thermus thermophilus; Bacteria: Escherichia coli; Eukaryota: Saccharomyces cerevisiae) in relation to their similarities with secondary structures formed by presumed proto-tRNAs, represented by 25 theoretical minimal RNA rings. These ranks are compared to those derived from two independent methods (ranks provide a relative evolutionary age to the rRNA substructure), (a) cladistic phylogenetic analyses and (b) 3D-crystallography where core subelements are presumed ancient and peripheral ones recent. Comparisons of rRNA secondary structure subelements with RNA ring secondary structures show congruence between ranks deduced by this method and both (a) and (b) (more with (a) than (b)), especially for RNA rings with predicted ancient cognate amino acid. Reconstruction of accretion histories of large rRNAs will gain from adequately integrating information from independent methods. Theoretical minimal RNA rings, sequences deterministically designed in silico according to specific coding constraints, might produce adequate scales for prebiotic and early life molecular evolution.

Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation.

Rhizobial infection and root nodule formation in legumes require recognition of signal molecules produced by the bacteria and their hosts. Here, we show that rhizobial transfer RNA (tRNA)-derived small RNA fragments (tRFs) are signal molecules that modulate host nodulation. Three families of rhizobial tRFs were confirmed to regulate host genes associated with nodule initiation and development through hijacking the host RNA-interference machinery that involves ARGONAUTE 1. Silencing individual tRFs with the use of short tandem target mimics or by overexpressing their targets represses root hair curling and nodule formation, whereas repressing these targets with artificial microRNAs identical to the respective tRFs or mutating these targets with CRISPR-Cas9 promotes nodulation. Our findings thus uncover a bacterial small RNA-mediated mechanism for prokaryote-eukaryote interaction and may pave the way for enhancing nodulation efficiency in legumes.

RNA phloem transport mediated by pre-miRNA and viral tRNA-like structures.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

The Role of tRNA in Establishing New Genetic Codes.

One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/L-shaped molecule must implement the first genetic code, via base pairing and wobble interactions, but is also largely responsible for the specificity of the second genetic code, the pairings between amino acids, tRNA synthetases, and tRNAs. Despite the overarching similarities between tRNAs, they must nonetheless be specifically recognized by cognate tRNA synthetases and largely rejected by noncognate synthetases. Conversely, despite the differences between tRNAs that allow such discrimination, they must be uniformly accepted by the ribosome, in part via the machinations of the translation elongation factors, which work with a diverse coterie of tRNA-amino acid conjugates to balance binding and loading. While it is easy to ascribe both discrimination and acceptance to the individual proteins (synthetases and EF-Tu/eEF-1) that recognize tRNAs, there is a large body of evidence that suggests that the sequences, structures, and dynamics of tRNAs are instrumental in the choices these proteins make.

tRNA modifications and islet function.

Efficient and accurate protein translation is essential to producing insulin in pancreatic ß-cells. Transfer RNA (tRNA) is known as the key component of the protein translational machinery. Interestingly, tRNA contains a wide variety of chemical modifications, which are posttranscriptionally catalysed by tRNA modifying enzymes. Recent advances in genome-sequencing technology have unveiled a number of genetic variations that are associated with the development of type 2 diabetes (T2D). Some of these mutations are located in the genes of tRNA modifying enzymes. Using cellular and animal models, it has been showed that dysregulation of tRNA modification impairs protein translation in pancreatic ß-cells and leads to aberrant insulin production. In this review, we discuss the recent findings in the molecular functions of tRNA modifications and their involvement in the development of T2D.

The T-Box Riboswitch: tRNA as an Effector to Modulate Gene Regulation.

The T-box riboswitch is a unique, RNA-based regulatory mechanism that modulates expression of a wide variety of amino acid-related genes, predominantly in Firmicutes. RNAs of this class selectively bind a specific cognate tRNA, utilizing recognition of the tRNA anticodon and other tRNA features. The riboswitch monitors the aminoacylation status of the tRNA to induce expression of the regulated downstream gene(s) at the level of transcription antitermination or derepression of translation initiation in response to reduced tRNA charging via stabilization of an antiterminator or antisequestrator. Recent biochemical and structural studies have revealed new features of tRNA recognition that extend beyond the initially identified Watson-Crick base-pairing of a codon-like sequence in the riboswitch with the tRNA anticodon, and residues in the antiterminator or antisequestrator with the tRNA acceptor end. These studies have revealed new tRNA contacts and new modes of riboswitch function and ligand recognition that expand our understanding of RNA-RNA recognition and the biological roles of tRNA.

Human tRNA-Derived Small RNAs Modulate Host-Oral Microbial Interactions.

Coevolution of the human host and its associated microbiota has led to sophisticated interactions to maintain a delicate homeostasis. Emerging evidence suggests that in addition to small molecules, peptides, and proteins, small regulatory noncoding RNAs (sRNAs) might play an important role in cross-domain interactions. In this study, we revealed the presence of diverse host transfer RNA-derived small RNAs (tsRNAs) among human salivary sRNAs. We selected 2 tsRNAs (tsRNA-000794 and tsRNA-020498) for further study based on their high sequence similarity to specific tRNAs from a group of Gram-negative oral bacteria, including Fusobacterium nucleatum, a key oral commensal and opportunistic pathogen. We showed that the presence of F. nucleatum triggers exosome-mediated release of tsRNA-000794 and tsRNA-020498 by human normal oral keratinocyte cells. Furthermore, both tsRNA candidates exerted a growth inhibition effect on F. nucleatum, likely through interference with bacterial protein biosynthesis, but did not affect the growth of Streptococcus mitis, a health-associated oral Gram-positive bacterium whose genome does not carry sequences bearing high similarity to either tsRNA. Our data provide the first line of evidence for the modulatory role of host-derived tsRNAs in the microbial-host interaction.

Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection

All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes.

Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.

The central role of tRNA in genetic code expansion.

BACKGROUND: The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation. SCOPE OF REVIEW: Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins. MAJOR CONCLUSIONS: The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio. GENERAL SIGNIFICANCE: Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Recurring RNA structural motifs underlie the mechanics of L1 stalk movement.

The L1 stalk of the large ribosomal subunit undergoes large-scale movements coupled to the translocation of deacylated tRNA during protein synthesis. We use quantitative comparative structural analysis to localize the origins of L1 stalk movement and to understand its dynamic interactions with tRNA and other structural elements of the ribosome. Besides its stacking interactions with the tRNA elbow, stalk movement is directly linked to intersubunit rotation, rotation of the 30S head domain and contact of the acceptor arm of deacylated tRNA with helix 68 of 23S rRNA. Movement originates from pivoting at stacked non-canonical base pairs in a Family A three-way junction and bending in an internal G-U-rich zone. Use of these same motifs as hinge points to enable such dynamic events as rotation of the 30S subunit head domain and in flexing of the anticodon arm of tRNA suggests that they represent general strategies for movement of functional RNAs.

Beyond the Triplet Code: Context Cues Transform Translation.

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.

tRNA fragments: novel players in intergenerational inheritance.

Non-genetic inheritance is an evocative topic; in the past few years, the debate around potential inheritance of life-time experiences independent of social factors in mammals has become highly prominent due to increasing evidence for phenotypes in the offspring after paternal environmental exposures. Strikingly, two independent studies published in Science newly implicate a special class of RNA, transfer RNA fragments, in the intergenerational effects of paternal dietary intervention.