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1.
Annu Rev Microbiol ; 77: 111-129, 2023 09 15.
Article in English | MEDLINE | ID: mdl-37018842

ABSTRACT

Infections caused by malaria parasites place an enormous burden on the world's poorest communities. Breakthrough drugs with novel mechanisms of action are urgently needed. As an organism that undergoes rapid growth and division, the malaria parasite Plasmodium falciparum is highly reliant on protein synthesis, which in turn requires aminoacyl-tRNA synthetases (aaRSs) to charge tRNAs with their corresponding amino acid. Protein translation is required at all stages of the parasite life cycle; thus, aaRS inhibitors have the potential for whole-of-life-cycle antimalarial activity. This review focuses on efforts to identify potent plasmodium-specific aaRS inhibitors using phenotypic screening, target validation, and structure-guided drug design. Recent work reveals that aaRSs are susceptible targets for a class of AMP-mimicking nucleoside sulfamates that target the enzymes via a novel reaction hijacking mechanism. This finding opens up the possibility of generating bespoke inhibitors of different aaRSs, providing new drug leads.


Subject(s)
Amino Acyl-tRNA Synthetases , Antimalarials , Malaria , Humans , Antimalarials/pharmacology , Antimalarials/therapeutic use , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Plasmodium falciparum/genetics , Malaria/drug therapy , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA, Transfer/therapeutic use
2.
Nat Rev Mol Cell Biol ; 21(7): 361, 2020 07.
Article in English | MEDLINE | ID: mdl-32001807
3.
Cell ; 149(1): 202-13, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22464330

ABSTRACT

Transfer RNA (tRNA) gene content is a differentiating feature of genomes that contributes to the efficiency of the translational apparatus, but the principles shaping tRNA gene copy number and codon composition are poorly understood. Here, we report that the emergence of two specific tRNA modifications shaped the structure and composition of all extant genomes. Through the analysis of more than 500 genomes, we identify two kingdom-specific tRNA modifications as major contributors that separated archaeal, bacterial, and eukaryal genomes in terms of their tRNA gene composition. We show that, contrary to prior observations, genomic codon usage and tRNA gene frequencies correlate in all kingdoms if these two modifications are taken into account and that presence or absence of these modifications explains patterns of gene expression observed in previous studies. Finally, we experimentally demonstrate that human gene expression levels correlate well with genomic codon composition if these identified modifications are considered.


Subject(s)
Biological Evolution , Codon , RNA Processing, Post-Transcriptional , RNA, Transfer/metabolism , Animals , Archaea/genetics , Archaea/metabolism , Bacteria/genetics , Bacteria/metabolism , Eukaryota/genetics , Eukaryota/metabolism , Genome , Humans , Phylogeny , tRNA Methyltransferases/metabolism
4.
Nucleic Acids Res ; 52(18): 11158-11176, 2024 10 14.
Article in English | MEDLINE | ID: mdl-39268577

ABSTRACT

RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer's disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.


Subject(s)
Alzheimer Disease , Hippocampus , Transcription Factors , X-Box Binding Protein 1 , Animals , Humans , Male , Mice , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Disease Models, Animal , HEK293 Cells , Hippocampus/metabolism , Neurons/metabolism , RNA Ligase (ATP)/metabolism , RNA Ligase (ATP)/genetics , RNA Splicing/genetics , RNA, Transfer/metabolism , RNA, Transfer/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Unfolded Protein Response/genetics , X-Box Binding Protein 1/metabolism , X-Box Binding Protein 1/genetics
5.
Nucleic Acids Res ; 51(18): 10001-10010, 2023 Oct 13.
Article in English | MEDLINE | ID: mdl-37638745

ABSTRACT

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.


Subject(s)
Serine-tRNA Ligase , Animals , Amino Acyl-tRNA Synthetases/metabolism , Catalytic Domain , Serine-tRNA Ligase/chemistry , Serine-tRNA Ligase/metabolism
6.
J Biol Chem ; 299(1): 102755, 2023 01.
Article in English | MEDLINE | ID: mdl-36455626

ABSTRACT

Engineering new protein functionalities through the addition of noncoded amino acids is a major biotechnological endeavor that needs to overcome the natural firewalls that prevent misincorporation during protein synthesis. This field is in constant evolution driven by the discovery or design of new tools, many of which are based on archeal biology. In a recent article published in JBC, one such tool is characterized and its evolution studied, revealing unexpected details regarding the emergence of the universal genetic code machinery.


Subject(s)
Amino Acyl-tRNA Synthetases , Vaccines , Archaea/genetics , Lysine/metabolism , Genetic Code , RNA, Transfer/genetics , RNA, Transfer/metabolism , Amino Acyl-tRNA Synthetases/metabolism
7.
IUBMB Life ; 2024 Sep 09.
Article in English | MEDLINE | ID: mdl-39247978

ABSTRACT

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non-translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the 22 coded amino acids are handled by 16 evolutionary families of Class I aaRS and 21 families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia-like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non-experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS.

8.
Bioinformatics ; 38(10): 2934-2936, 2022 05 13.
Article in English | MEDLINE | ID: mdl-35561195

ABSTRACT

SUMMARY: High-throughput sequencing of transfer RNAs (tRNA-Seq) is a powerful approach to characterize the cellular tRNA pool. Currently, however, analyzing tRNA-Seq datasets requires strong bioinformatics and programming skills. tRNAstudio facilitates the analysis of tRNA-Seq datasets and extracts information on tRNA gene expression, post-transcriptional tRNA modification levels, and tRNA processing steps. Users need only running a few simple bash commands to activate a graphical user interface that allows the easy processing of tRNA-Seq datasets in local mode. Output files include extensive graphical representations and associated numerical tables, and an interactive html summary report to help interpret the data. We have validated tRNAstudio using datasets generated by different experimental methods and derived from human cell lines and tissues that present distinct patterns of tRNA expression, modification and processing. AVAILABILITY AND IMPLEMENTATION: Freely available at https://github.com/GeneTranslationLab-IRB/tRNAstudio under an open-source GNU GPL v3.0 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
RNA, Transfer , Software , High-Throughput Nucleotide Sequencing/methods , Humans , RNA Processing, Post-Transcriptional , RNA, Transfer/genetics , Sequence Analysis, RNA/methods
9.
Nucleic Acids Res ; 49(12): 7011-7034, 2021 07 09.
Article in English | MEDLINE | ID: mdl-34125917

ABSTRACT

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.


Subject(s)
Inosine/metabolism , Protein Biosynthesis , RNA, Transfer/metabolism , Adenosine Deaminase/genetics , Cell Adhesion , Cell Growth Processes , Cell Line , Codon , Eukaryota/genetics , Female , HEK293 Cells , Humans , Protein Domains/genetics , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , RNA, Transfer/chemistry , Ribosomes/metabolism
10.
Proc Natl Acad Sci U S A ; 116(17): 8451-8456, 2019 04 23.
Article in English | MEDLINE | ID: mdl-30962382

ABSTRACT

The human genome encodes hundreds of transfer RNA (tRNA) genes but their individual contribution to the tRNA pool is not fully understood. Deep sequencing of tRNA transcripts (tRNA-Seq) can estimate tRNA abundance at single gene resolution, but tRNA structures and posttranscriptional modifications impair these analyses. Here we present a bioinformatics strategy to investigate differential tRNA gene expression and use it to compare tRNA-Seq datasets from cultured human cells and human brain. We find that sequencing caveats affect quantitation of only a subset of human tRNA genes. Unexpectedly, we detect several cases where the differences in tRNA expression among samples do not involve variations at the level of isoacceptor tRNA sets (tRNAs charged with the same amino acid but using different anticodons), but rather among tRNA genes within the same isodecoder set (tRNAs having the same anticodon sequence). Because isodecoder tRNAs are functionally equal in terms of genetic translation, their differential expression may be related to noncanonical tRNA functions. We show that several instances of differential tRNA gene expression result in changes in the abundance of tRNA-derived fragments (tRFs) but not of mature tRNAs. Examples of differentially expressed tRFs include PIWI-associated RNAs, tRFs present in tissue samples but not in cells cultured in vitro, and somatic tissue-specific tRFs. Our data support that differential expression of tRNA genes regulate noncanonical tRNA functions performed by tRFs.


Subject(s)
Organ Specificity/genetics , RNA, Transfer , Transcriptome/genetics , Anticodon/genetics , Brain/metabolism , Cells, Cultured , Computational Biology , Gene Expression Profiling , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , RNA, Small Interfering/analysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Transfer/analysis , RNA, Transfer/genetics , RNA, Transfer/metabolism , Sequence Analysis, RNA
11.
RNA ; 25(5): 607-619, 2019 05.
Article in English | MEDLINE | ID: mdl-30737359

ABSTRACT

Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNAArg Here we investigate the recognition mechanisms of tRNAArg and tRNAAla by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNAArg, while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.


Subject(s)
Adenosine Deaminase/metabolism , Anticodon/chemistry , RNA, Transfer, Ala/chemistry , RNA, Transfer, Arg/chemistry , Adenosine/metabolism , Adenosine Deaminase/genetics , Anticodon/genetics , Anticodon/metabolism , Base Sequence , Deamination , Evolution, Molecular , Gene Expression , Humans , Inosine/metabolism , Nucleic Acid Conformation , RNA, Transfer, Ala/genetics , RNA, Transfer, Ala/metabolism , RNA, Transfer, Arg/genetics , RNA, Transfer, Arg/metabolism , Sequence Alignment , Substrate Specificity
12.
RNA Biol ; 18(11): 1905-1919, 2021 11.
Article in English | MEDLINE | ID: mdl-33499731

ABSTRACT

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.


Subject(s)
Brain/metabolism , Cell Nucleus/metabolism , Neuroblastoma/pathology , Neurons/metabolism , Subcellular Fractions/metabolism , tRNA Methyltransferases/metabolism , Animals , Female , Mice , Mice, Knockout , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neurons/cytology , tRNA Methyltransferases/genetics
13.
Mol Biol Evol ; 36(4): 650-662, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30590541

ABSTRACT

The modification of adenosine to inosine at the first position of transfer RNA (tRNA) anticodons (I34) is widespread among bacteria and eukaryotes. In bacteria, the modification is found in tRNAArg and is catalyzed by tRNA adenosine deaminase A, a homodimeric enzyme. In eukaryotes, I34 is introduced in up to eight different tRNAs by the heterodimeric adenosine deaminase acting on tRNA. This substrate expansion significantly influenced the evolution of eukaryotic genomes in terms of codon usage and tRNA gene composition. However, the selective advantages driving this process remain unclear. Here, we have studied the evolution of I34, tRNA adenosine deaminase A, adenosine deaminase acting on tRNA, and their relevant codons in a large set of bacterial and eukaryotic species. We show that a functional expansion of I34 to tRNAs other than tRNAArg also occurred within bacteria, in a process likely initiated by the emergence of unmodified A34-containing tRNAs. In eukaryotes, we report on a large variability in the use of I34 in protists, in contrast to a more uniform presence in fungi, plans, and animals. Our data support that the eukaryotic expansion of I34-tRNAs was driven by the improvement brought by these tRNAs to the synthesis of proteins highly enriched in certain amino acids.


Subject(s)
Evolution, Molecular , Inosine , RNA, Transfer/genetics , Animals , Oenococcus/genetics , Phylogeny , Proteome , Tetrahymena thermophila/genetics
14.
J Cell Sci ; 131(10)2018 05 31.
Article in English | MEDLINE | ID: mdl-29700204

ABSTRACT

The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction.This article has an associated First Person interview with the first author of the paper.


Subject(s)
Fibroblasts/cytology , Flow Cytometry/methods , Peptide Chain Elongation, Translational , Single-Cell Analysis/methods , Animals , Elongation Factor 2 Kinase/genetics , Elongation Factor 2 Kinase/metabolism , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Biosynthesis , Proteins/genetics , Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism
15.
Bioconjug Chem ; 31(3): 933-938, 2020 03 18.
Article in English | MEDLINE | ID: mdl-32057238

ABSTRACT

3-Bromo-1,2,4,5-tetrazine has been synthesized in an oxidant- and metal-free method. The synthesis is scalable and relies on inexpensive starting materials. 3-Bromo-1,2,4,5-tetrazine can undergo nucleophilic aromatic substitutions with differently substituted heteroatoms under mild conditions. In particular, its excellent reactivity has been used to attain chemoselective protein labeling. The resulting labeled lysines can react with strained dienophiles to trigger fast click-to-release (CtR) biorthogonal reactions. The characterization of the CtR reaction in physiological conditions and a therapeutically relevant example with the monoclonal antibody Trastuzumab to showcase its application is presented. Finally, 3-bromo-1,2,4,5-tetrazine has been used to achieve site-selective protein labeling through the genetic incorporation of the first unnatural amino acid bearing an unsubstituted 1,2,4,5-tetrazin-3-yl functionality, which can also undergo CtR reactions.


Subject(s)
Aza Compounds/chemistry , Aza Compounds/chemical synthesis , Benzene Derivatives/chemistry , Benzene Derivatives/chemical synthesis , Proteins/chemistry , Staining and Labeling/methods , Click Chemistry , Drug Liberation , Kinetics , Models, Molecular , Protein Conformation , Ribonuclease, Pancreatic/chemistry
16.
J Biol Chem ; 293(49): 19157-19158, 2018 12 07.
Article in English | MEDLINE | ID: mdl-30530854

ABSTRACT

Since the origin of life, metabolism and protein synthesis have evolved together to balance the vast amounts of ATP and amino acids required for genetic translation with the rest of the cell's energy needs. A new study offers satisfying insights into a long-standing evolutionary mystery surrounding a fused, bifunctional aminoacyl-tRNA synthetase. To avoid depleting cells from an essential amino acid generated by the Krebs cycle, harvesting for Glu and Pro by the translation machinery was unified in animals, thus preventing a Pro-hungry translational apparatus from depleting the cell of essential Glu reserves.


Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Genetic Code , Amino Acids/genetics , Animals , Biological Evolution
17.
Biochemistry ; 57(39): 5641-5647, 2018 10 02.
Article in English | MEDLINE | ID: mdl-30199619

ABSTRACT

Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArgACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.


Subject(s)
Deoxyribonuclease IV (Phage T4-Induced)/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Inosine/analysis , Oligodeoxyribonucleotides/chemistry , RNA, Transfer, Arg/chemistry , RNA, Transfer, Val/chemistry , Animals , Base Sequence , HEK293 Cells , HeLa Cells , Humans , Inosine/genetics , Mice , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/genetics , RNA, Transfer, Arg/genetics , RNA, Transfer, Val/genetics
18.
RNA Biol ; 15(4-5): 500-507, 2018.
Article in English | MEDLINE | ID: mdl-28880718

ABSTRACT

The modification of adenosine to inosine at position 34 of tRNA anticodons has a profound impact upon codon-anticodon recognition. In bacteria, I34 is thought to exist only in tRNAArg, while in eukaryotes the modification is present in eight different tRNAs. In eukaryotes, the widespread use of I34 strongly influenced the evolution of genomes in terms of tRNA gene abundance and codon usage. In humans, codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Here we extend the analysis of distribution of codons that are recognized by I34 containing tRNAs to all phyla known to use this modification. We find that the preference for codons recognized by such tRNAs in genes with highly biased codon compositions is universal among eukaryotes, and we report that, unexpectedly, some bacterial phyla show a similar preference. We demonstrate that the genomes of these bacterial species contain previously undescribed tRNA genes that are potential substrates for deamination at position 34.


Subject(s)
Codon/chemistry , Cyanobacteria/genetics , Eukaryota/genetics , Firmicutes/genetics , Genetic Code , Inosine/metabolism , RNA, Transfer, Arg/genetics , Adenosine/genetics , Adenosine/metabolism , Amino Acids/genetics , Amino Acids/metabolism , Anticodon/chemistry , Anticodon/metabolism , Biological Evolution , Codon/metabolism , Cyanobacteria/metabolism , Eukaryota/metabolism , Firmicutes/metabolism , Humans , Inosine/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer, Arg/metabolism , Transcriptome
19.
Methods ; 113: 34-45, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27989759

ABSTRACT

Current biochemical methods available to monitor the activity of aminoacyl-tRNA synthetases (ARS) are ill-suited to high-throughput screening approaches for the identification of small-molecule inhibitors of these enzymes. In an attempt to improve the limitations of current assays we have developed a suite of new methods designed to streamline the discovery of new ARS antagonists. This set of assays includes approaches to monitor ARS activity in vitro, in human cells, and in bacteria. They are applicable to several ARSs from any given organism, can be easily adapted to very high-throughput set-ups, and allow for a multi-factorial selection of drug candidates.


Subject(s)
Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays , RNA, Transfer, Amino Acid-Specific/genetics , Small Molecule Libraries/pharmacology , Transfer RNA Aminoacylation , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Drug Discovery , Enzyme Assays , Escherichia coli/enzymology , Escherichia coli/genetics , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements/methods , Methicillin-Resistant Staphylococcus aureus/enzymology , Methicillin-Resistant Staphylococcus aureus/genetics , RNA, Transfer, Amino Acid-Specific/metabolism
20.
Proc Natl Acad Sci U S A ; 112(19): 6027-32, 2015 May 12.
Article in English | MEDLINE | ID: mdl-25918376

ABSTRACT

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNA(Pro) and Cys-tRNA(Pro), respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed "ProXp-ST1" and "ProXp-ST2," respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.


Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Protein Biosynthesis , RNA Editing , RNA, Transfer/chemistry , Serine/chemistry , Threonine/chemistry , Amino Acids/chemistry , Bacillus/metabolism , Catalysis , Cell Proliferation , Computational Biology , Escherichia coli/metabolism , Hydrolysis , Protein Structure, Tertiary , Reproducibility of Results , Substrate Specificity , Temperature
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