Targeting Aminoacyl tRNA Synthetases for Antimalarial Drug Development.

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.

A role for tRNA modifications in genome structure and codon usage.

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.

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

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.

Archeal tRNA meets biotechnology: From vaccines to genetic code expansion.

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.

tRNAstudio: facilitating the study of human mature tRNAs from deep sequencing datasets.

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.

Human tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins.

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.

Differential expression of human tRNA genes drives the abundance of tRNA-derived fragments.

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.

tRNA deamination by ADAT requires substrate-specific recognition mechanisms and can be inhibited by tRFs.

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.

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation.

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.

The Expansion of Inosine at the Wobble Position of tRNAs, and Its Role in the Evolution of Proteomes.

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.

SunRiSE - measuring translation elongation at single-cell resolution by means of flow cytometry.

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.

Synthesis and Application of 3-Bromo-1,2,4,5-Tetrazine for Protein Labeling to Trigger Click-to-Release Biorthogonal Reactions.

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.

Genetic code and metabolism: The perpetual waltz.

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.

Detection of Inosine on Transfer RNAs without a Reverse Transcription Reaction.

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.

Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla.

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.

A new set of assays for the discovery of aminoacyl-tRNA synthetase inhibitors.

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.

Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation.

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.

Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method.

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base "mutations" when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes.

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.