Search

The impact of environmental factors on epigenetic changes is well established, and cellular function is determined not only by the genome but also by interacting partners such as metabolites. Given the significant impact of metabolism on disease progression, exploring the interaction between the metabolome and epigenome may offer new insights into Huntington's disease (HD) diagnosis and treatment. Using fourteen post-mortem HD cases and fourteen control subjects, we performed metabolomic profiling of human postmortem brain tissue (striatum and frontal lobe), and we performed DNA methylome profiling using the same frontal lobe tissue. Along with finding several perturbed metabolites and differentially methylated loci, Aminoacyl-tRNA biosynthesis (adj p -value = 0.0098) was the most significantly perturbed metabolic pathway with which two CpGs of the SEPSECS gene were correlated. This study improves our understanding of molecular biomarker connections and, importantly, increases our knowledge of metabolic alterations driving HD progression.

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway., Competing Interests: The authors have declared that no competing interests exist.

When encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H 2 O 2 ) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3' end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H 2 O 2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H 2 O 2 -treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H 2 O 2 -mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H 2 O 2 -treated S. oligofermentans . Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H 2 O 2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H 2 O 2 -suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H 2 O 2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans . IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H 2 O 2 . This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H 2 O 2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H 2 O 2 challenge. So-RNaseZ orthologs and H 2 O 2 -sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H 2 O 2 .

RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3'-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3'-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3'-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3'-cleavage activity increases growth rate but is nonessential., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides, which greatly increase the complexity and utility of RNAs. Transfer RNAs (tRNAs) are the most heavily modified cellular RNA molecules and contain the largest variety of modifications. Modification of tRNAs is pivotal for protein synthesis and also precisely regulates the noncanonical functions of tRNAs. Defects in tRNA modifications lead to numerous human diseases. Up to now, more than 100 types of modifications have been found in tRNAs. Intriguingly, some modifications occur widely on all tRNAs, while others only occur on a subgroup of tRNAs or even only a specific tRNA. The modification frequency of each tRNA is approximately 7% to 25%, with 5-20 modification sites present on each tRNA. The occurrence and modulation of tRNA modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered. In particular, tRNA modifications are responsive to environmental changes, indicating their dynamic and highly organized nature. In this review, we summarized the known occurrence order, cross-talk, and cooperativity of tRNA modifications., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Processing of mature transfer RNAs (tRNAs) produces complex populations of tRNA-derived fragments (tRFs). Emerging evidence shows that tRFs have important functions in bacteria, animals, and plants. Here, we review recent advances in understanding plant tRFs, focusing on their biological and cellular functions, such as regulating stress responses, mediating plant-pathogen interactions, and modulating post-transcriptional gene silencing and translation. We also review sequencing strategies and bioinformatics resources for studying tRFs in plants. Finally, we discuss future directions for plant tRF research, which will expand our knowledge of plant non-coding RNAs., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery., Competing Interests: The authors have declared that no competing financial interests exist.

Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro . Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models. Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly. Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development., Competing Interests: Competing Interests: A.-M.Y., M.-J.T. and P.Y.H. are inventors on US Patent Application No. 62/674,939 filed on May 22, 2018 as well as US Patent No. 10,619,156 issued on April 14, 2020 and European Patent No. 3150980 granted on May 20, 2020 that are related to this work. A.-M.Y. is a founder of AimRNA, Inc. that intends to license the intellectual property., (© The author(s).)

Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast Rpc160 to single Pol III leukodystrophy mutations in the pore domain can be overcome by a second mutation in the domain., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

RNA motifs guide the interaction with specific proteins leading to the assembly of ribonucleoprotein complexes that perform key functions in cellular processes. Internal ribosome entry site (IRES) elements are organized in structural domains that determine internal initiation of translation. In this chapter we describe a pull-down assay using streptavidin-aptamer tagged RNAs that combines RNA structure-dependent protein isolation with proteomic analysis to identify novel interactors recognizing RNA structural domains. This approach takes advantage of tRNA-scaffold guided expression, allowing the identification of factors belonging to networks involved in RNA and protein metabolism.

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).

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gastric cancer (GC) is the third most common cause of cancer death worldwide, and the incidence of GC is higher in males than females. To investigate the gastric cellular response to hormone therapy, we developed a cell pseudotargeted metabolomics method based on liquid chromatography-hybrid triple quadrupole linear ion trap mass spectrometry (LC-QTRAP MS). Chromatographic separation, sample analysis and metabolite extraction were optimized in an integrated manner. The established pseudotargeted method, which combined nontargeted and targeted analyses, exhibited high selectivity, good repeatability and wide metabolome coverage. The method was then applied to discover differential metabolites from hormone-stimulated gastric cancer cells compared with the controls for the first time. The results demonstrated that hormone had subtle but phenotypically important alterations in nucleotide metabolism, amino acid metabolism, glycolysis, tricarboxylic acid (TCA) cycle, aminoacyl-tRNA biosynthesis and so on, which indicate that the developed method is a powerful tool for effective screening of endogenous polar metabolites in cell samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1's function in tRNA repression is inhibited by mTOR-dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Purpose : Choroidal neovascularization (CNV) is one of the major clinical characteristics of neovascular age-related macular degeneration (AMD). Small non-coding RNAs, such as microRNAs (miRNAs) and transfer RNA-derived small RNAs (tsRNAs) play key roles in diverse biological functions. The purpose of the study was to investigate the roles and possible functions of the miRNAs and tsRNAs in CNV. Methods : The mouse model of laser-induced CNV was conducted by laser photocoagulation. The expression profiles of miRNAs and tsRNAs were accessed by small RNA sequencing (RNA-Seq) in RPE-choroid-sclera complexes of mice in CNV group and control group. Selected altered miRNAs and tsRNAs were validated by qRT-PCR. Target genes were predicted by informatics analysis and intersected with the previous microarray study of altered mRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to reveal the biological functions and signaling pathways with which these target genes are most enriched. Results : The results revealed that 79 miRNAs and 72 tsRNAs in total were significantly altered in the RPE-choroid-sclera complexes of CNV mice. GO analysis revealed that the altered target genes of the selected miRNAs most enriched in immune response, integral component of membrane and peptide binding, while the altered target genes of tsRNAs most enriched in regulation of immune system process, extracellular region, and core promoter binding. Moreover, KEGG pathway analysis demonstrated that altered target genes of miRNAs and tsRNAs most enriched in hematopoietic cell lineage and nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, respectively. Conclusions : Our study identified differential expressions of miRNAs and tsRNAs in CNV model, and these altered miRNAs and tsRNAs might be novel potential targets in treating CNVs in patients with neovascular AMD.

PreQ 1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ 1 . Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ 1 -bound states of the preQ 1 -II riboswitch from Lactobacillus rhamnosus . We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ 1  stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ 1 -II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.

Metastasis of colorectal cancer (CRC) is the leading cause of CRC-associated mortality. Angiogenin (ANG), a member of the ribonuclease A superfamily, not only activates endothelial cells to induce tumor angiogenesis, but also targets tumor cells to promote cell survival, proliferation and/or migration. However, its clinical significance and underlying mechanism in CRC metastasis are still largely unknown. Here, we reported that ANG was upregulated in CRC tissues and associated with metastasis in CRC patients. We then revealed that ANG enhanced CRC growth and metastasis in both in vitro and in vivo systems. Intriguingly, we characterized a bunch of tRNA-derived stress-induced small RNAs (tiRNAs), produced through ANG cleavage, that was enriched in both CRC tumor tissues and highly metastatic cells, and functioned in ANG-promoted CRC metastasis. Moreover, higher level of a 5'-tiRNA from mature tRNA-Val (5'-tiRNA-Val) was observed in CRC patients and was correlated with tumor metastasis. Taken together, we propose that a novel ANG-tiRNAs-cell migration and invasion regulatory axis promotes CRC metastasis, which might be of potential target for CRC diagnosis and treatment., (© 2019 UICC.)

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form., Competing Interests: AG, GR, CK, SW and MM are employees of Medimmune LLC/AstraZeneca. The authors confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dysregulation of protein translation is emerging as a unifying mechanism in the pathogenesis of many neuronal disorders. Ribosomal RNA (rRNA) and transfer RNA (tRNA) are structural molecules that have complementary and coordinated functions in protein synthesis. Defects in both rRNAs and tRNAs have been described in mammalian brain development, neurological syndromes, and neurodegeneration. In this review, we present the molecular mechanisms that link aberrant rRNA and tRNA transcription, processing and modifications to translation deficits, and neuropathogenesis. We also discuss the interdependence of rRNA and tRNA biosynthesis and how their metabolism brings together proteotoxic stress and impaired neuronal homeostasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major .

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.

The synthesis of transfer RNA (tRNA) is directed by RNA polymerase III (Pol III) specialized in high-level transcription of short DNA templates. Pol III recruitment to tRNA genes is controlled by two general initiation factors, TFIIIB and TFIIIC. They are multi-protein complexes regulated at the level of expression of individual subunits, as well as through phosphorylation and interaction with partner proteins. Here, we describe particular aspects of TFIIIB and TFIIIC control in yeast and human cells. Under stress conditions, tRNA synthesis is negatively regulated by the MAF1 protein, which interacts directly with Pol III. Sequence and function of MAF1 are conserved among eukaryotic organisms from yeast to humans. MAF1 is a phosphoprotein which mediates diverse regulatory signals to Pol III. Interestingly, there is a subset of housekeeping tRNA genes, both in the yeast and human genome, which are less sensitive to MAF1-dependent repression. The possible mechanisms responsible for this differential regulation of tRNA synthesis by MAF1 are discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

RNA polymerase III catalyses the synthesis of tRNAs in eukaryotic organisms. Through combined biochemical and structural characterisation, multiple auxiliary factors have been identified alongside RNA Polymerase III as critical in both facilitating and regulating transcription. Together, this machinery forms dynamic multi-protein complexes at tRNA genes which are required for polymerase recruitment, DNA opening and initiation and elongation of the tRNA transcripts. Central to the function of these complexes is their ability to undergo multiple conformational changes and rearrangements that regulate each step. Here, we discuss the available biochemical and structural data on the structural plasticity of multi-protein complexes involved in RNA Polymerase III transcriptional initiation and facilitated re-initiation during tRNA synthesis. Increasingly, structural information is becoming available for RNA polymerase III and its functional complexes, allowing for a deeper understanding of tRNA transcriptional initiation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

RNA polymerase III (RNAPIII) transcribes tRNA genes, 5S RNA as well as a number of other non-coding RNAs. Because transcription by RNAPIII is an energy-demanding process, its activity is tightly linked to the stress levels and nutrient status of the cell. Multiple signaling pathways control RNAPIII activity in response to environmental cues, but exactly how these pathways regulate RNAPIII is still poorly understood. One major target of these pathways is the transcriptional repressor Maf1, which inhibits RNAPIII activity under conditions that are detrimental to cell growth. However, recent studies have found that the cell can also directly regulate the RNAPIII machinery through phosphorylation and sumoylation of RNAPIII subunits. In this review we summarize post-translational modifications of RNAPIII subunits that mainly have been identified in large-scale proteomics studies, and we highlight several examples to discuss their relevance for regulation of RNAPIII., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The field of tRNA biology, encompassing the functional and structural complexity of tRNAs, has fascinated scientists over the years and is continuously growing. Besides their fundamental role in protein translation, new evidence indicates that tRNA-derived molecules also regulate gene expression and protein synthesis in all domains of life. This review highlights some of the recent findings linking tRNA transcription and modification with plant cell growth and response to pathogens. In fact, mutations in proteins directly involved in tRNA synthesis and modification most often lead to pleiotropic effects on plant growth and immunity. As plants need to optimize and balance their energy and nutrient resources towards growth and defense, regulatory pathways that play a central role in integrating tRNA transcription and protein translation with cell growth control and organ development, such as the auxin-TOR signaling pathway, also influence the plant immune response against pathogens. As a consequence, distinct pathogens employ an array of effector molecules including tRNA fragments to target such regulatory pathways to exploit the plant's translational capacity, gain access to nutrients and evade defenses. An example includes the RNA polymerase III repressor MAF1, a conserved component of the TOR signaling pathway that controls ribosome biogenesis and tRNA synthesis required for plant growth and which is targeted by a pathogen effector molecule to promote disease. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The short, non-coding genes transcribed by the RNA polymerase (pol) III, necessary for survival of a cell, need to be repressed under the stress conditions in vivo. The pol III-transcribed genes have adopted several novel chromatin-based regulatory mechanisms to their advantage. In the budding yeast, the sub-nucleosomal size tRNA genes are found in the nucleosome-free regions, flanked by positioned nucleosomes at both the ends. With their chromosomes-wide distribution, all tRNA genes have a different chromatin context. A single nucleosome dynamics controls the accessibility of the genes for transcription. This dynamics operates under the influence of several chromatin modifiers in a gene-specific manner, giving the scope for differential regulation of even the isogenes within a tRNA gene family. The chromatin structure around the pol III-transcribed genes provides a context conducive for steady-state transcription as well as gene-specific transcriptional regulation upon signaling from the environmental cues. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. However, when high G analogue:GTP ratios are used to bias G analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine ( th G):GTP increased the percent incorporation and yield of 5'- th G-initiated precursor tRNA for a net ≈threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'- th G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of [Formula: see text]. Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N 4 -acetylcytidine (ac 4 C) in tRNA and Bud13 as a factor controlling the levels of ac 4 C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) [Formula: see text]. Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered [Formula: see text]. These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence [Formula: see text] biogenesis.

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-'omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment., Competing Interests: The authors declare no conflict of interest.

Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions., Competing Interests: The authors declare no conflict of interest.

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species., Competing Interests: The authors have declared that no competing interests exist.

Small RNAs (sRNAs) derived from non-coding RNAs (ncRNAs), such as tRNAs, rRNAs and snoRNAs, have been identified in various organisms. Several observations have indicated that cleavage of tRNAs and rRNAs is induced by various stresses. To clarify whether sRNAs in wheat derived from tRNAs (stRNAs), rRNAs (srRNAs) and snoRNAs (sdRNAs) are produced specifically in association with heat stress responses, we carried out a bioinformatic analysis of sRNA libraries from wheat seedlings and performed comparisons between control and high-temperature-treated samples to measure the differential abundance of stRNAs, srRNAs and sdRNAs. We found that the production of sRNAs from tRNAs, 5.8S rRNAs, and 28S rRNAs was more specific than that from 5S rRNAs and 18S rRNAs, and more than 95% of the stRNAs were processed asymmetrically from the 3' or 5' ends of mature tRNAs. We identified 333 stRNAs and 8,822 srRNAs that were responsive to heat stress. Moreover, the expression of stRNAs derived from tRNA-Val-CAC, tRNA-Thr-UGU, tRNA-Tyr-GUA and tRNA-Ser-UGA was not only up-regulated under heat stress but also induced by osmotic stress, suggesting that the increased cleavage of tRNAs might be a mechanism that developed in wheat seedlings to help them cope with adverse environmental conditions.

Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant, readthrough tRNA transcripts. These transcripts are polyadenylated and targeted for degradation by the exosome. Similarly, other non-coding RNA molecules, such as snRNA, snoRNA and rRNA, are also enriched in the polyadenylate preparations in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway that affects Pol III-dependent transcripts., (Copyright © 2015. Published by Elsevier B.V.)

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.

The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide, with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile, that actively transverses the gut wall and migrates to its final location in the liver. The regulation of the early developmental events in newly excysted juveniles is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the newly excysted juvenile fall into two classes, one represented by micro (mi)RNAs and a secondary group of larger (32-33 nucleotides) tRNA-derived sequences. We identified 40 different miRNAs, most of those belonging to ancient miRNAs conserved in protostomes and metazoans, notably with a highly predominant miR-125b variant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRnome reduction in parasitic flatworms. Additionally, a set of five novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent, suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA-derived sequences, mostly 5' tRNA halves of selected tRNAs in the small RNA population of newly excysted juveniles, raises the possibility that both miRNA and tRNA fragments participate in the regulation of gene expression in this parasite., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Transfer RNAs (tRNAs) are essential for mRNA translation. They are transcribed in the nucleus by RNA polymerase III and undergo many modifications before contributing to cytoplasmic protein synthesis. In this review I highlight our understanding of how tRNA biology may be linked to the regulation of mRNA translation, growth and tumorigenesis. First, I review how oncogenes and tumour suppressor signalling pathways, such as the PI3 kinase/TORC1, Ras/ERK, Myc, p53 and Rb pathways, regulate Pol III and tRNA synthesis. In several cases, this regulation contributes to cell, tissue and body growth, and has implications for our understanding of tumorigenesis. Second, I highlight some recent work, particularly in model organisms such as yeast and Drosophila, that shows how alterations in tRNA synthesis may be not only necessary, but also sufficient to drive changes in mRNA translation and growth. These effects may arise due to both absolute increases in total tRNA levels, but also changes in the relative levels of tRNAs in the overall pool. Finally, I review some recent studies that have revealed how tRNA modifications (amino acid acylation, base modifications, subcellular shuttling, and cleavage) can be regulated by growth and stress cues to selectively influence mRNA translation. Together these studies emphasize the importance of the regulation of tRNA synthesis and modification as critical control points in protein synthesis and growth. This article is part of a Special Issue entitled: Translation and Cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The essential and universal N(6)-threonylcarbamoyladenosine (t(6)A) modification at position 37 of ANN-decoding tRNAs plays a pivotal role in translational fidelity through enhancement of the cognate codon recognition and stabilization of the codon-anticodon interaction. In Escherichia coli, the YgjD (TsaD), YeaZ (TsaB), YjeE (TsaE) and YrdC (TsaC) proteins are necessary and sufficient for the in vitro biosynthesis of t(6)A, using tRNA, ATP, L-threonine and bicarbonate as substrates. YrdC synthesizes the short-lived L-threonylcarbamoyladenylate (TCA), and YgjD, YeaZ and YjeE cooperate to transfer the L-threonylcarbamoyl-moiety from TCA onto adenosine at position 37 of substrate tRNA. We determined the crystal structure of the heterodimer YgjD-YeaZ at 2.3 Å, revealing the presence of an unexpected molecule of ADP bound at an atypical site situated at the YgjD-YeaZ interface. We further showed that the ATPase activity of YjeE is strongly activated by the YgjD-YeaZ heterodimer. We established by binding experiments and SAXS data analysis that YgjD-YeaZ and YjeE form a compact ternary complex only in presence of ATP. The formation of the ternary YgjD-YeaZ-YjeE complex is required for the in vitro biosynthesis of t(6)A but not its ATPase activity., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s(2)U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s(2)U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s(2)U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms., (© 2015 Alings et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to Schizosaccharomyces pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5' and 3' processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3' oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3' oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a 'technical approaches' section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses., (Published by Elsevier B.V.)

tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies.

Rsp5 ubiquitin ligase is required for ubiquitination of a wide variety of proteins involved in essential processes. Rsp5 was shown to be involved in regulation of lipid biosynthesis, intracellular trafficking of proteins, response to various stresses, and many other processes. In this article, we provide a comprehensive review of the nuclear and cytoplasmic functions of Rsp5 with a focus on biogenesis of different RNAs. We also briefly describe the participation of Rsp5 in the regulation of the RNA polymerase II complex, and its potential role in the regulation of other RNA polymerases. Moreover, we emphasize the function of Rsp5 in the coordination of the different steps of rRNA, mRNA and tRNA metabolism in the context of protein biosynthesis. Finally, we highlight the involvement of Rsp5 in controlling diverse cellular mechanisms at multiple levels and in adaptation of the cell to changing growth conditions.

Escherichia coli tRNA N6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified from E. coli and crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space group P2₁, with unit-cell parameters a=65.4, b=96.8, c=83.3 Å, β=111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da(-1) and 42.1% solvent content.

Threonylcarbamoyladenosine (t(6)A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t(6)A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t(6)A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332-6346, 2013, http://dx.doi.org/10.1093/nar/gkt322). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t(6)A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t(6)A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli. The data revealed a potential for TC-AMP channeling in the t(6)A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t(6)A and bring additional advancement in our understanding of the reaction mechanism., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)