Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

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.

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.

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.

Mitochondrial Protein Synthesis and mtDNA Levels Coordinated through an Aminoacyl-tRNA Synthetase Subunit.

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS.

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.

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.

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.

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.

Saturation of recognition elements blocks evolution of new tRNA identities.

Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure.

Inosine modifications in human tRNAs are incorporated at the precursor tRNA level.

Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

An appended domain results in an unusual architecture for malaria parasite tryptophanyl-tRNA synthetase.

Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations.

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively.

Selective inhibition of an apicoplastic aminoacyl-tRNA synthetase from Plasmodium falciparum.

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antibacterial targets and so constitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplastic ARSs remain unexplored. We have characterized the lysylation system of Plasmodium falciparum, and designed, synthesized, and tested a set of inhibitors based on the structure of the natural substrate intermediate: lysyl-adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically inhibit Plasmodium apicoplastic lysyl-tRNA synthetase and show antimalarial activities in the micromolar range.

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses.

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies.