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RNA Modifications
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RNA Modifications

A topical collection in Biomolecules (ISSN 2218-273X). This collection belongs to the section "Molecular Genetics".

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Editors

Prof. Dr. Valérie De Crécy-Lagard

E-Mail Website
Collection Editor
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA
Interests: comparative genomics; gene annotation; vitamin metabolism; tRNA modification pathways; 7-deazaguanosine derivatives; translation accuracy; tRNA metabolism; metabolite repair; bacterial genetics; t6A modification
Prof. Dr. Juan Alfonzo

E-Mail Website
Collection Editor
Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA
Interests: intracellular RNA transport and its impact on modifications; biochemistry of RNA modifications; enzymology

Topical Collection Information

Dear Colleagues,

The field of RNA modification, that started in 1957 with the discovery of the fifth nucleotide pseudouridine has recently gone through a revival or, as recently discussed by one of the pioneers of the field, Henri Grosjean, a new golden era (RNA. 2015 Apr; 21(4): 625–626). Over 100 different modifications are found in RNAs and in the last 20 years a combination of biochemical, genetic and bioinformatic studies has allowed the identification of many of the enzymes responsible for their synthesis that had been missing for decades. It is now clear that every type of nucleic acid in cells undergoes some type of modification, including, but not limited to, DNA, rRNA, snRNA, miRNA and tRNA. This has led to the discovery of new enzyme families, has revealed a great diversity of RNA recognition modes and allowed to address the functional roles of many modifications. RNA modification detection and quantification methods derived from new generation sequencing techniques as well as new developments in advanced mass spectrometry have recently revolutionized the field.  We are just starting to appreciate the multiple roles that RNA modifications have in the cell and how these fundamental processes are linked to human diseases. These recent studies have also shown the strong parallels between RNA editing and RNA modification, between DNA and RNA modifications and between the modification of tRNAs and modifications of other types of RNAs with the different fields cross-fertilizing each other.
This is a very exciting time in the field and we encourage scientists of diverse backgrounds (biochemical, genetic, systems biology) and who use different model systems to contribute original research or review articles that focus on the synthesis or function of RNA modifications.

Prof. Dr. Valérie de Crécy-Lagard
Prof. Dr. Juan Alfonzo
Guest Editors

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Keywords

  • miscoding
  • translation accuracy
  • translational control
  • tRNA metabolism
  • tRNA modification
  • tRNA quality control
  • tRNA recognition
  • modification tunable transcripts
  • codon usage
  • tRNA fragments
  • rRNA modification
  • ribosome
  • mRNA modification
  • mRNA stability
  • mRNA splicing
  • RNA modification regulation

Published Papers (33 papers)

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2023

Jump to: 2020, 2019, 2018, 2017, 2016

15 pages, 1228 KiB  
Review
The Regulatory Network of METTL3 in the Nervous System: Diagnostic Biomarkers and Therapeutic Targets
by Xiaojuan Su, Yi Qu and Dezhi Mu
Biomolecules 2023, 13(4), 664; https://doi.org/10.3390/biom13040664 - 11 Apr 2023
Cited by 6 | Viewed by 1797
Abstract
Methyltransferase-like 3 (METTL3) is a typical component of N6-methyladenosine writers that exhibits methyltransferase activity and deposits methyl groups on RNA. Currently, accumulating studies have demonstrated the involvement of METTL3 in the regulation of neuro-physiological and pathological events. However, no reviews have comprehensively summarized [...] Read more.
Methyltransferase-like 3 (METTL3) is a typical component of N6-methyladenosine writers that exhibits methyltransferase activity and deposits methyl groups on RNA. Currently, accumulating studies have demonstrated the involvement of METTL3 in the regulation of neuro-physiological and pathological events. However, no reviews have comprehensively summarized and analyzed the roles and mechanisms of METTL3 in these events. Herein, we are focused on reviewing the roles of METTL3 in regulating normal neurophysiological (Neurogenesis, Synaptic Plasticity and Glial Plasticity, Neurodevelopment, Learning and Memory,) and neuropathological (Autism Spectrum Disorder, Major Depressive Disorder, Neurodegenerative disorders, Brain Tumors, Brain Injuries, and Other Brain Disorders) events. Our review found that although the down-regulated levels of METTL3 function through different roles and mechanisms in the nervous system, it primarily inactivates neuro-physiological events and triggers or worsens neuropathological events. In addition, our review suggests that METTL3 could be used as a diagnostic biomarker and therapeutic target in the nervous system. Collectively, our review has provided an up-to-date research outline of METTL3 in the nervous system. In addition, the regulatory network for METTL3 in the nervous system has been mapped, which could provide directions for future research, biomarkers for clinical diagnosis, and targets for disease treatment. Furthermore, this review has provided a comprehensive view, which could improve our understanding of METTL3 functions in the nervous system. Full article
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23 pages, 3473 KiB  
Article
The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells
by Moses Olalekan Ogunkola, Gaelle Guiraudie-Capraz, Francois Feron and Silke Leimkühler
Biomolecules 2023, 13(1), 144; https://doi.org/10.3390/biom13010144 - 10 Jan 2023
Cited by 2 | Viewed by 1767
Abstract
Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA [...] Read more.
Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics. Full article
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2020

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16 pages, 3700 KiB  
Article
Impact of Pus1 Pseudouridine Synthase on Specific Decoding Events in Saccharomyces cerevisiae
by Bahar Khonsari and Roland Klassen
Biomolecules 2020, 10(5), 729; https://doi.org/10.3390/biom10050729 - 07 May 2020
Cited by 9 | Viewed by 3781
Abstract
Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNAGlnCUG in yeast. Synthetic lethality [...] Read more.
Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNAGlnCUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNAGlnCUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNAGlnCUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNAGlnCAG variants. We also determined the misreading of CGC (Arg) codons by tRNAHisGUG, where the CGC decoder tRNAArgICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNAHis. We found that the absence of Pus1 increased CGC misreading by tRNAHis, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in pus1 mutants and strains carrying destabilized tRNAGlnCAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant MET22, suggesting the involvement of RTD-mediated tRNA destabilization. Full article
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19 pages, 4530 KiB  
Article
Reductive Evolution and Diversification of C5-Uracil Methylation in the Nucleic Acids of Mollicutes
by Pascal Sirand-Pugnet, Damien Brégeon, Laure Béven, Catherine Goyenvalle, Alain Blanchard, Simon Rose, Henri Grosjean, Stephen Douthwaite, Djemel Hamdane and Valérie de Crécy-Lagard
Biomolecules 2020, 10(4), 587; https://doi.org/10.3390/biom10040587 - 10 Apr 2020
Cited by 6 | Viewed by 3512
Abstract
The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, [...] Read more.
The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U-modifying enzymes that is highly dynamic and has undergone horizontal transfer. Full article
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24 pages, 7685 KiB  
Article
Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast
by Leticia Pollo-Oliveira, Roland Klassen, Nick Davis, Akif Ciftci, Jo Marie Bacusmo, Maria Martinelli, Michael S. DeMott, Thomas J. Begley, Peter C. Dedon, Raffael Schaffrath and Valérie de Crécy-Lagard
Biomolecules 2020, 10(2), 322; https://doi.org/10.3390/biom10020322 - 18 Feb 2020
Cited by 17 | Viewed by 5729
Abstract
Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human [...] Read more.
Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications. Full article
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2019

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10 pages, 851 KiB  
Communication
Small, Smaller, Smallest: Minimal Structural Requirements for a Fully Functional Box C/D Modification Guide RNA
by Svetlana Deryusheva and Joseph G. Gall
Biomolecules 2019, 9(9), 457; https://doi.org/10.3390/biom9090457 - 07 Sep 2019
Cited by 4 | Viewed by 3902
Abstract
Site-specific 2’-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, [...] Read more.
Site-specific 2’-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes. Full article
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2018

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18 pages, 4890 KiB  
Article
Oxidation of 5-methylaminomethyl uridine (mnm5U) by Oxone Leads to Aldonitrone Derivatives
by Qishun Zhou, Bao Tram Vu Ngoc, Grazyna Leszczynska, Jean-Luc Stigliani and Geneviève Pratviel
Biomolecules 2018, 8(4), 145; https://doi.org/10.3390/biom8040145 - 14 Nov 2018
Cited by 4 | Viewed by 4552
Abstract
Oxidative RNA damage is linked to cell dysfunction and diseases. The present work focuses on the in vitro oxidation of 5-methylaminomethyl uridine (mnm5U), which belongs to the numerous post-transcriptional modifications that are found in tRNA. The reaction of oxone with mnm [...] Read more.
Oxidative RNA damage is linked to cell dysfunction and diseases. The present work focuses on the in vitro oxidation of 5-methylaminomethyl uridine (mnm5U), which belongs to the numerous post-transcriptional modifications that are found in tRNA. The reaction of oxone with mnm5U in water at pH 7.5 leads to two aldonitrone derivatives. They form by two oxidation steps and one dehydration step. Therefore, the potential oxidation products of mnm5U in vivo may not be only aldonitrones, but also hydroxylamine and imine derivatives (which may be chemically more reactive). Irradiation of aldonitrone leads to unstable oxaziridine derivatives that are susceptible to isomerization to amide or to hydrolysis to aldehyde derivative. Full article
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16 pages, 3137 KiB  
Article
Crystal Structure of the Human tRNA Guanine Transglycosylase Catalytic Subunit QTRT1
by Sven Johannsson, Piotr Neumann and Ralf Ficner
Biomolecules 2018, 8(3), 81; https://doi.org/10.3390/biom8030081 - 24 Aug 2018
Cited by 13 | Viewed by 4644
Abstract
RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at [...] Read more.
RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at position 34. This exchange is catalyzed by members of the enzyme class of tRNA guanine transglycosylases (TGTs). These enzymes incorporate guanine substituents into tRNAAsp, tRNAAsn tRNAHis, and tRNATyr in all kingdoms of life. In contrast to the homodimeric bacterial TGT, the active eukaryotic TGT is a heterodimer in solution, comprised of a catalytic QTRT1 subunit and a noncatalytic QTRT2 subunit. Bacterial TGT enzymes, that incorporate a queuine precursor, have been identified or proposed as virulence factors for infections by pathogens in humans and therefore are valuable targets for drug design. To date no structure of a eukaryotic catalytic subunit is reported, and differences to its bacterial counterpart have to be deducted from sequence analysis and models. Here we report the first crystal structure of a eukaryotic QTRT1 subunit and compare it to known structures of the bacterial TGT and murine QTRT2. Furthermore, we were able to determine the crystal structure of QTRT1 in complex with the queuine substrate. Full article
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12 pages, 4118 KiB  
Article
Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification
by Bruno Leonardo Bozaquel-Morais, Leonie Vogt, Valentina D’Angelo, Raffael Schaffrath, Roland Klassen and Mónica Montero-Lomelí
Biomolecules 2018, 8(3), 49; https://doi.org/10.3390/biom8030049 - 11 Jul 2018
Cited by 4 | Viewed by 3806
Abstract
The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance [...] Read more.
The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone. Full article
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2017

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2307 KiB  
Article
tRNA Modification Detection Using Graphene Nanopores: A Simulation Study
by Khadijah Onanuga, Thomas J. Begley, Alan A. Chen and Srivathsan V. Ranganathan
Biomolecules 2017, 7(3), 65; https://doi.org/10.3390/biom7030065 - 25 Aug 2017
Cited by 1 | Viewed by 5262
Abstract
There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection [...] Read more.
There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection of RNA modifications requires working with nucleosides in bulk samples. Nanopore detection technology uses a single-molecule approach that has the potential to detect tRNA modifications. To evaluate the feasibility of this approach, we have performed all-atom molecular dynamics (MD) simulation studies of a five-layered graphene nanopore by localizing canonical and modified uridine nucleosides. We found that in a 1 M KCl solution with applied positive and negative biases not exceeding 2 V, nanopores can distinguish U from 5-carbonylmethyluridine (cm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um) based on changes in the resistance of the nanopore. Specifically, we observed that in nanopores with dimensions less than 3 nm diameter, a localized mcm5Um and mcm5U modifications could be clearly distinguished from the canonical uridine, while the other modifications showed a modest yet detectable decrease in their respective nanopore conductance. We have compared the results between nanopores of various sizes to aid in the design, optimization, and fabrication of graphene nanopores devices for tRNA modification detection. Full article
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Article
TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli
by Joseph I. Aubee, Morenike Olu and Karl M. Thompson
Biomolecules 2017, 7(2), 39; https://doi.org/10.3390/biom7020039 - 04 May 2017
Cited by 13 | Viewed by 6323
Abstract
Previous work demonstrated that efficient RNA Polymerase sigma S-subunit (RpoS) translation requires the N6-isopentenyladenosine i6A37 transfer RNA (tRNA) modification for UUX-Leu decoding. Here we investigate the effect of two additional tRNA modification systems on RpoS translation; the analysis was also extended to another [...] Read more.
Previous work demonstrated that efficient RNA Polymerase sigma S-subunit (RpoS) translation requires the N6-isopentenyladenosine i6A37 transfer RNA (tRNA) modification for UUX-Leu decoding. Here we investigate the effect of two additional tRNA modification systems on RpoS translation; the analysis was also extended to another High UUX-leucine codon (HULC) protein, Host Factor for phage Qβ (Hfq). One tRNA modification, the addition of the 2’-O-methylcytidine/uridine 34 (C/U34m) tRNA modification by tRNA (cytidine/uridine-2’O)-ribose methyltransferase L (TrmL), requires the presence of the N6-isopentenyladenosine 37 (i6A37) and therefore it seemed possible that the defect in RpoS translation in the absence of i6A37 prenyl transferase (MiaA) was in fact due to the inability to add the C/U34m modification to UUX-Leu tRNAs. The second modification, addition of 2-thiouridine (s2U), part of (mnm5s2U34), is dependent on tRNA 2-thiouridine synthesizing protein A (TusA), previously shown to affect RpoS levels. We compared expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons were changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolished PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppressed the trmL requirement for PBAD-rpoS990-lacZ expression. Thus, it is likely that the C/U34m and s2U34 tRNA modifications are necessary for full rpoS translation. We also measured PBAD-hfq306-lacZ translational fusion activity in the absence of C/U34m (trmL) or i6A37 (miaA). The absence of i6A37 resulted in decreased PBAD-hfq306-lacZ expression, consistent with a role for i6A37 tRNA modification for hfq translation. Full article
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2333 KiB  
Article
QueF-Like, a Non-Homologous Archaeosine Synthase from the Crenarchaeota
by Adriana Bon Ramos, Lide Bao, Ben Turner, Valérie De Crécy-Lagard and Dirk Iwata-Reuyl
Biomolecules 2017, 7(2), 36; https://doi.org/10.3390/biom7020036 - 06 Apr 2017
Cited by 9 | Viewed by 5523
Abstract
Archaeosine (G+) is a structurally complex modified nucleoside ubiquitous to the Archaea, where it is found in the D-loop of virtually all archaeal transfer RNA (tRNA). Its unique structure, which includes a formamidine group that carries a formal positive charge, and [...] Read more.
Archaeosine (G+) is a structurally complex modified nucleoside ubiquitous to the Archaea, where it is found in the D-loop of virtually all archaeal transfer RNA (tRNA). Its unique structure, which includes a formamidine group that carries a formal positive charge, and location in the tRNA, led to the proposal that it serves a key role in stabilizing tRNA structure. Although G+ is limited to the Archaea, it is structurally related to the bacterial modified nucleoside queuosine, and the two share homologous enzymes for the early steps of their biosynthesis. In the Euryarchaeota, the last step of the archaeosine biosynthetic pathway involves the amidation of a nitrile group on an archaeosine precursor to give formamidine, a reaction catalyzed by the enzyme Archaeosine Synthase (ArcS). Most Crenarchaeota lack ArcS, but possess two proteins that inversely distribute with ArcS and each other, and are implicated in G+ biosynthesis. Here, we describe biochemical studies of one of these, the protein QueF-like (QueF-L) from Pyrobaculum calidifontis, that demonstrate the catalytic activity of QueF-L, establish where in the pathway QueF-L acts, and identify the source of ammonia in the reaction. Full article
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6523 KiB  
Review
tRNA Modifications: Impact on Structure and Thermal Adaptation
by Christian Lorenz, Christina E. Lünse and Mario Mörl
Biomolecules 2017, 7(2), 35; https://doi.org/10.3390/biom7020035 - 04 Apr 2017
Cited by 218 | Viewed by 26739
Abstract
Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all [...] Read more.
Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation. Full article
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2108 KiB  
Review
Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions
by Chenkang Zheng, Katherine A. Black and Patricia C. Dos Santos
Biomolecules 2017, 7(1), 33; https://doi.org/10.3390/biom7010033 - 22 Mar 2017
Cited by 25 | Viewed by 7271
Abstract
Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all [...] Read more.
Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes. Full article
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3515 KiB  
Review
Trm5 and TrmD: Two Enzymes from Distinct Origins Catalyze the Identical tRNA Modification, m1G37
by Sakurako Goto-Ito, Takuhiro Ito and Shigeyuki Yokoyama
Biomolecules 2017, 7(1), 32; https://doi.org/10.3390/biom7010032 - 21 Mar 2017
Cited by 32 | Viewed by 4810
Abstract
The N1-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m1G37 positively regulates the aminoacylation [...] Read more.
The N1-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m1G37 positively regulates the aminoacylation of the tRNA, and simultaneously functions to prevent the +1 frameshift on the ribosome. Interestingly, Trm5 and TrmD have completely distinct origins, and therefore bear different tertiary folds. In this review, we describe the different strategies utilized by Trm5 and TrmD to recognize their substrate tRNAs, mainly based on their crystal structures complexed with substrate tRNAs. Full article
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6107 KiB  
Article
Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide
by Adeba Mohammad, Adriana Bon Ramos, Bobby W. K. Lee, Spencer W. Cohen, Maryam K. Kiani, Dirk Iwata-Reuyl, Boguslaw Stec and Manal A. Swairjo
Biomolecules 2017, 7(1), 30; https://doi.org/10.3390/biom7010030 - 16 Mar 2017
Cited by 8 | Viewed by 7142
Abstract
QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide [...] Read more.
QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin-like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo. Full article
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3670 KiB  
Review
Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function
by Ville Y. P. Väre, Emily R. Eruysal, Amithi Narendran, Kathryn L. Sarachan and Paul F. Agris
Biomolecules 2017, 7(1), 29; https://doi.org/10.3390/biom7010029 - 16 Mar 2017
Cited by 107 | Viewed by 10898
Abstract
RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability [...] Read more.
RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA’s cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs. Full article
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Graphical abstract

2508 KiB  
Review
Biosynthesis of Sulfur-Containing tRNA Modifications: A Comparison of Bacterial, Archaeal, and Eukaryotic Pathways
by Mirela Čavužić and Yuchen Liu
Biomolecules 2017, 7(1), 27; https://doi.org/10.3390/biom7010027 - 11 Mar 2017
Cited by 49 | Viewed by 8726
Abstract
Post-translational tRNA modifications have very broad diversity and are present in all domains of life. They are important for proper tRNA functions. In this review, we emphasize the recent advances on the biosynthesis of sulfur-containing tRNA nucleosides including the 2-thiouridine (s2U) [...] Read more.
Post-translational tRNA modifications have very broad diversity and are present in all domains of life. They are important for proper tRNA functions. In this review, we emphasize the recent advances on the biosynthesis of sulfur-containing tRNA nucleosides including the 2-thiouridine (s2U) derivatives, 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A). Their biosynthetic pathways have two major types depending on the requirement of iron–sulfur (Fe–S) clusters. In all cases, the first step in bacteria and eukaryotes is to activate the sulfur atom of free l-cysteine by cysteine desulfurases, generating a persulfide (R-S-SH) group. In some archaea, a cysteine desulfurase is missing. The following steps of the bacterial s2U and s4U formation are Fe–S cluster independent, and the activated sulfur is transferred by persulfide-carrier proteins. By contrast, the biosynthesis of bacterial s2C and ms2A require Fe–S cluster dependent enzymes. A recent study shows that the archaeal s4U synthetase (ThiI) and the eukaryotic cytosolic 2-thiouridine synthetase (Ncs6) are Fe–S enzymes; this expands the role of Fe–S enzymes in tRNA thiolation to the Archaea and Eukarya domains. The detailed reaction mechanisms of Fe–S cluster depend s2U and s4U formation await further investigations. Full article
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2327 KiB  
Review
Factors That Shape Eukaryotic tRNAomes:  Processing, Modification and Anticodon–Codon Use
by Richard J. Maraia and Aneeshkumar G. Arimbasseri
Biomolecules 2017, 7(1), 26; https://doi.org/10.3390/biom7010026 - 08 Mar 2017
Cited by 29 | Viewed by 10134
Abstract
Transfer RNAs (tRNAs) contain sequence diversity beyond their anticodons and the large variety of nucleotide modifications found in all kingdoms of life. Some modifications stabilize structure and fit in the ribosome whereas those to the anticodon loop modulate messenger RNA (mRNA) decoding activity [...] Read more.
Transfer RNAs (tRNAs) contain sequence diversity beyond their anticodons and the large variety of nucleotide modifications found in all kingdoms of life. Some modifications stabilize structure and fit in the ribosome whereas those to the anticodon loop modulate messenger RNA (mRNA) decoding activity more directly. The identities of tRNAs with some universal anticodon loop modifications vary among distant and parallel species, likely to accommodate fine tuning for their translation systems. This plasticity in positions 34 (wobble) and 37 is reflected in codon use bias. Here, we review convergent evidence that suggest that expansion of the eukaryotic tRNAome was supported by its dedicated RNA polymerase III transcription system and coupling to the precursor‐tRNA chaperone, La protein. We also review aspects of eukaryotic tRNAome evolution involving G34/A34 anticodon‐sparing, relation to A34 modification to inosine, biased codon use and regulatory information in the redundancy (synonymous) component of the genetic code. We then review interdependent anticodon loop modifications involving position 37 in eukaryotes. This includes the eukaryote‐specific tRNA modification, 3‐methylcytidine‐32 (m3C32) and the responsible gene, TRM140 and homologs which were duplicated and subspecialized for isoacceptor‐specific substrates and dependence on i6A37 or t6A37. The genetics of tRNA function is relevant to health directly and as disease modifiers. Full article
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5999 KiB  
Article
Modulation of the Proteostasis Machinery to  Overcome Stress Caused by Diminished Levels of  t6A‐Modified tRNAs in Drosophila
by Diego Rojas‐Benítez, Cristián Eggers and Alvaro Glavic
Biomolecules 2017, 7(1), 25; https://doi.org/10.3390/biom7010025 - 06 Mar 2017
Cited by 15 | Viewed by 5742
Abstract
Transfer RNAs (tRNAs) harbor a subset of post‐transcriptional modifications required for structural stability or decoding function. N6‐threonylcarbamoyladenosine (t6A) is a universally conserved modification found at position 37 in tRNA that pair A‐starting codons (ANN) and is required for proper translation initiation and to [...] Read more.
Transfer RNAs (tRNAs) harbor a subset of post‐transcriptional modifications required for structural stability or decoding function. N6‐threonylcarbamoyladenosine (t6A) is a universally conserved modification found at position 37 in tRNA that pair A‐starting codons (ANN) and is required for proper translation initiation and to prevent frame shift during elongation. In its absence, the synthesis of aberrant proteins is likely, evidenced by the formation of protein aggregates. In this work, our aim was to study the relationship between t6A‐modified tRNAs and protein synthesis homeostasis machinery using Drosophila melanogaster. We used the Gal4/UAS system to knockdown genes required for t6A synthesis in a tissue and time specific manner and in vivo reporters of unfolded protein response (UPR) activation. Our results suggest that t6A‐modified tRNAs, synthetized by the threonyl‐carbamoyl transferase complex (TCTC), are required for organismal growth and imaginal cell survival, and is most likely to support proper protein synthesis. Full article
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4287 KiB  
Review
Dealing with an Unconventional Genetic Code in  Mitochondria: The Biogenesis and Pathogenic  Defects of the 5‐Formylcytosine Modification in  Mitochondrial tRNAMet
by Lindsey Van Haute, Christopher A. Powell and Michal Minczuk
Biomolecules 2017, 7(1), 24; https://doi.org/10.3390/biom7010024 - 02 Mar 2017
Cited by 22 | Viewed by 7068
Abstract
Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt‐tRNAMet) also recognises AUA during translation initiation and elongation. Post‐transcriptional modifications of tRNAs are important for structure, stability, [...] Read more.
Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt‐tRNAMet) also recognises AUA during translation initiation and elongation. Post‐transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5‐formylcytosine (f5C) modification of position 34 in mt‐tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease. Full article
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1714 KiB  
Review
Transfer RNA methyltransferases with a SpoU‐TrmD  (SPOUT) fold and their modified nucleosides in  tRNA
by Hiroyuki Hori
Biomolecules 2017, 7(1), 23; https://doi.org/10.3390/biom7010023 - 28 Feb 2017
Cited by 38 | Viewed by 7859
Abstract
The existence of SpoU‐TrmD (SPOUT) RNA methyltransferase superfamily was first predicted by bioinformatics. SpoU is the previous name of TrmH, which catalyzes the 2’‐Omethylation of ribose of G18 in tRNA; TrmD catalyzes the formation of N1‐methylguanosine at position 37 in tRNA. Although SpoU [...] Read more.
The existence of SpoU‐TrmD (SPOUT) RNA methyltransferase superfamily was first predicted by bioinformatics. SpoU is the previous name of TrmH, which catalyzes the 2’‐Omethylation of ribose of G18 in tRNA; TrmD catalyzes the formation of N1‐methylguanosine at position 37 in tRNA. Although SpoU (TrmH) and TrmD were originally considered to be unrelated, the bioinformatics study suggested that they might share a common evolution origin and form a single superfamily. The common feature of SPOUT RNA methyltransferases is the formation of a deep trefoil knot in the catalytic domain. In the past decade, the SPOUT RNA methyltransferase superfamily has grown; furthermore, knowledge concerning the functions of their modified nucleosides in tRNA has also increased. Some enzymes are potential targets in the design of antibacterial drugs. In humans, defects in some genes may be related to carcinogenesis. In this review, recent findings on the tRNA methyltransferases with a SPOUT fold and their methylated nucleosides in tRNA, including classification of tRNA methyltransferases with a SPOUT fold; knot structures, domain arrangements, subunit structures and reaction mechanisms; tRNA recognition mechanisms, and functions of modified nucleosides synthesized by this superfamily, are summarized. Lastly, the future perspective for studies on tRNA modification enzymes are considered. Full article
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1644 KiB  
Review
Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry
by Robert L. Ross, Xiaoyu Cao and Patrick A. Limbach
Biomolecules 2017, 7(1), 21; https://doi.org/10.3390/biom7010021 - 22 Feb 2017
Cited by 26 | Viewed by 6859
Abstract
Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post‐transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it [...] Read more.
Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post‐transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it has proven to be the gold standard for accurately assigning nucleobases to the sequence. For the past few decades many labs have worked to improve the analysis, contiguous to instrumentation manufacturers developing faster and more sensitive instruments. With biological discoveries relating to ribonucleic acid happening more frequently, mass spectrometry has been invaluable in helping to understand what is happening at the molecular level. Here we present a brief overview of the methods that have been developed and refined for the analysis of modified tRNAs by liquid chromatography tandem mass spectrometry. Full article
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3014 KiB  
Review
m1A Post‐Transcriptional Modification in tRNAs
by Stephanie Oerum, Clément Dégut, Pierre Barraud and Carine Tisné
Biomolecules 2017, 7(1), 20; https://doi.org/10.3390/biom7010020 - 21 Feb 2017
Cited by 103 | Viewed by 11482
Abstract
To date, about 90 post‐transcriptional modifications have been reported in tRNA expanding their chemical and functional diversity. Methylation is the most frequent post‐transcriptional tRNA modification that can occur on almost all nitrogen sites of the nucleobases, on the C5 atom of pyrimidines, on [...] Read more.
To date, about 90 post‐transcriptional modifications have been reported in tRNA expanding their chemical and functional diversity. Methylation is the most frequent post‐transcriptional tRNA modification that can occur on almost all nitrogen sites of the nucleobases, on the C5 atom of pyrimidines, on the C2 and C8 atoms of adenosine and, additionally, on the oxygen of the ribose 2′-OH. The methylation on the N1 atom of adenosine to form 1‐methyladenosine (m1A) has been identified at nucleotide position 9, 14, 22, 57, and 58 in different tRNAs. In some cases, these modifications have been shown to increase tRNA structural stability and induce correct tRNA folding. This review provides an overview of the currently known m1A modifications, the different m1A modification sites, the biological role of each modification, and the enzyme responsible for each methylation in different species. The review further describes, in detail, two enzyme families responsible for formation of m1A at nucleotide position 9 and 58 in tRNA with a focus on the tRNA binding, m1A mechanism, protein domain organisation and overall structures. Full article
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2716 KiB  
Review
Sulfur Modifications of the Wobble U34 in tRNAs and their Intracellular Localization in Eukaryotic Cells
by Yumi Nakai, Masato Nakai and Takato Yano
Biomolecules 2017, 7(1), 17; https://doi.org/10.3390/biom7010017 - 18 Feb 2017
Cited by 20 | Viewed by 8019
Abstract
The wobble uridine (U34) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG, harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of [...] Read more.
The wobble uridine (U34) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG, harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of U34, respectively. Both modifications are necessary to construct the proper anticodon loop structure and to enable them to exert their functions in translation. Thio-modification of U34 (s2U34) is found in both cytosolic tRNAs (cy-tRNAs) and mitochondrial tRNAs (mt-tRNAs). Although l-cysteine desulfurase is required in both cases, subsequent sulfur transfer pathways to cy-tRNAs and mt-tRNAs are different due to their distinct intracellular locations. The s2U34 formation in cy-tRNAs involves a sulfur delivery system required for the biosynthesis of iron-sulfur (Fe/S) clusters and certain resultant Fe/S proteins. This review addresses presumed sulfur delivery pathways for the s2U34 formation in distinct intracellular locations, especially that for cy-tRNAs in comparison with that for mt-tRNAs. Full article
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1081 KiB  
Review
Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification
by Ann E. Ehrenhofer-Murray
Biomolecules 2017, 7(1), 14; https://doi.org/10.3390/biom7010014 - 10 Feb 2017
Cited by 40 | Viewed by 8440
Abstract
Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 [...] Read more.
Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 methylation, foremost C38 (m5C38) of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP) by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed. Full article
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2429 KiB  
Article
Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation
by Virginie Marchand, Florian Pichot, Kathrin Thüring, Lilia Ayadi, Isabel Freund, Alexander Dalpke, Mark Helm and Yuri Motorin
Biomolecules 2017, 7(1), 13; https://doi.org/10.3390/biom7010013 - 09 Feb 2017
Cited by 48 | Viewed by 7666
Abstract
Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various [...] Read more.
Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing‐based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally‐important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′‐O‐methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species. Full article
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5687 KiB  
Article
The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ0) Transport
by Rémi Zallot, Yifeng Yuan and Valérie De Crécy-Lagard
Biomolecules 2017, 7(1), 12; https://doi.org/10.3390/biom7010012 - 08 Feb 2017
Cited by 25 | Viewed by 7091
Abstract
Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine [...] Read more.
Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ0), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ0 to 7-aminomethyl-7-deazaguanine (preQ1). PreQ1 is inserted into tRNAs by tRNA guanine(34) transglycosylase (TGT). The inserted base preQ1 is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ0 synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ0, preQ1 or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ0/preQ1 transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ0 transport. Full article
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5048 KiB  
Review
Trm112, a Protein Activator of Methyltransferases Modifying Actors of the Eukaryotic Translational Apparatus
by Gabrielle Bourgeois, Juliette Létoquart, Nhan Van Tran and Marc Graille
Biomolecules 2017, 7(1), 7; https://doi.org/10.3390/biom7010007 - 27 Jan 2017
Cited by 43 | Viewed by 6860
Abstract
Post-transcriptional and post-translational modifications are very important for the control and optimal efficiency of messenger RNA (mRNA) translation. Among these, methylation is the most widespread modification, as it is found in all domains of life. These methyl groups can be grafted either on [...] Read more.
Post-transcriptional and post-translational modifications are very important for the control and optimal efficiency of messenger RNA (mRNA) translation. Among these, methylation is the most widespread modification, as it is found in all domains of life. These methyl groups can be grafted either on nucleic acids (transfer RNA (tRNA), ribosomal RNA (rRNA), mRNA, etc.) or on protein translation factors. This review focuses on Trm112, a small protein interacting with and activating at least four different eukaryotic methyltransferase (MTase) enzymes modifying factors involved in translation. The Trm112-Trm9 and Trm112-Trm11 complexes modify tRNAs, while the Trm112-Mtq2 complex targets translation termination factor eRF1, which is a tRNA mimic. The last complex formed between Trm112 and Bud23 proteins modifies 18S rRNA and participates in the 40S biogenesis pathway. In this review, we present the functions of these eukaryotic Trm112-MTase complexes, the molecular bases responsible for complex formation and substrate recognition, as well as their implications in human diseases. Moreover, as Trm112 orthologs are found in bacterial and archaeal genomes, the conservation of this Trm112 network beyond eukaryotic organisms is also discussed. Full article
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2085 KiB  
Article
Role of Pseudouridine Formation by Deg1 for Functionality of Two Glutamine Isoacceptor tRNAs
by Roland Klassen and Raffael Schaffrath
Biomolecules 2017, 7(1), 8; https://doi.org/10.3390/biom7010008 - 26 Jan 2017
Cited by 14 | Viewed by 5241
Abstract
Loss of Deg1/Pus3 and concomitant elimination of pseudouridine in tRNA at positions 38 and 39 (ψ38/39) was shown to specifically impair the function of tRNAGlnUUG under conditions of temperature-induced down-regulation of wobble uridine thiolation in budding yeast and is linked to [...] Read more.
Loss of Deg1/Pus3 and concomitant elimination of pseudouridine in tRNA at positions 38 and 39 (ψ38/39) was shown to specifically impair the function of tRNAGlnUUG under conditions of temperature-induced down-regulation of wobble uridine thiolation in budding yeast and is linked to intellectual disability in humans. To further characterize the differential importance of the frequent ψ38/39 modification for tRNAs in yeast, we analyzed the in vivo function of non-sense suppressor tRNAs SUP4 and sup70-65 in the absence of the modifier. In the tRNATyrGψA variant SUP4, UAA read-through is enabled due to an anticodon mutation (UψA), whereas sup70-65 is a mutant form of tRNAGlnCUG (SUP70) that mediates UAG decoding due to a mutation of the anticodon-loop closing base pair (G31:C39 to A31:C39). While SUP4 function is unaltered in deg1/pus3 mutants, the ability of sup70-65 to mediate non-sense suppression and to complement a genomic deletion of the essential SUP70 gene is severely compromised. These results and the differential suppression of growth defects in deg1 mutants by multi-copy SUP70 or tQ(UUG) are consistent with the interpretation that ψ38 is most important for tRNAGlnUUG function under heat stress but becomes crucial for tRNAGlnCUG as well when the anticodon loop is destabilized by the sup70-65 mutation. Thus, ψ38/39 may protect the anticodon loop configuration from disturbances by loss of other modifications or base changes. Full article
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981 KiB  
Review
Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis
by Juan Campos Guillen, George H. Jones, Carlos Saldaña Gutiérrez, José Luis Hernández-Flores, Julio Alfonso Cruz Medina, José Humberto Valenzuela Soto, Sergio Pacheco Hernández, Sergio Romero Gómez and Verónica Morales Tlalpan
Biomolecules 2017, 7(1), 6; https://doi.org/10.3390/biom7010006 - 20 Jan 2017
Cited by 9 | Viewed by 5601
Abstract
Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was [...] Read more.
Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3′ terminus of tRNAs may also be important in oxidative stress. The addition of the 3′ terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus. Full article
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5985 KiB  
Review
Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes
by Silke Leimkühler, Martin Bühning and Lena Beilschmidt
Biomolecules 2017, 7(1), 5; https://doi.org/10.3390/biom7010005 - 14 Jan 2017
Cited by 51 | Viewed by 10448
Abstract
Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the [...] Read more.
Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm5s2U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron–sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes. Full article
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2016

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Article
CoverageAnalyzer (CAn): A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles
by Ralf Hauenschild, Stephan Werner, Lyudmil Tserovski, Andreas Hildebrandt, Yuri Motorin and Mark Helm
Biomolecules 2016, 6(4), 42; https://doi.org/10.3390/biom6040042 - 10 Nov 2016
Cited by 16 | Viewed by 4999
Abstract
Combination of reverse transcription (RT) and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures [...] Read more.
Combination of reverse transcription (RT) and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures of modified ribonucleotides relying on both sequence-dependent mismatch patterns and reverse transcription arrests. Common alignment viewers lack specialized functionality, such as filtering, tailored visualization, image export and differential analysis. Consequently, the community will profit from a platform seamlessly connecting detailed visual inspection of RT signatures and automated screening for modification candidates. CoverageAnalyzer (CAn) was developed in response to the demand for a powerful inspection tool. It is freely available for all three main operating systems. With SAM file format as standard input, CAn is an intuitive and user-friendly tool that is generally applicable to the large community of biomedical users, starting from simple visualization of RNA sequencing (RNA-Seq) data, up to sophisticated modification analysis with significance-based modification candidate calling. Full article
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