Preparation of tRNAArg for Arginylation Assay by In Vitro Transcription.

This chapter describes the preparation of tRNAArg by in vitro transcription. tRNA produced by this method can be efficiently utilized for in vitro arginylation assays, following aminoacylation with Arg-tRNA synthetase, either directly during the arginylation reaction or separately to produce the purified preparation of Arg-tRNAArg. tRNA charging is described in other chapters of this book.

Preparation of an Enriched tRNAArg Fraction for Arginylation by Expression in E. coli.

The method described here provides a fast and efficient way to obtain an enriched preparation of tRNA of interest, which is also posttranscriptionally modified by the intracellular machinery of the host cells, E. coli. While this preparation also contains a mixture of total E. coli tRNA, the enriched tRNA of interest is obtained in high yields (milligram) and is highly efficient for biochemical assays in vitro. It is routinely used in our lab for arginylation.

Enzymatic Aminoacylation of tRNAArg Using Recombinant Arg-tRNA Synthetase.

This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g., for measuring the kinetics or determining the effect of different compounds and chemicals on the reaction. In such cases, tRNAArg can be pre-charged with Arg and purified away from the RARS enzyme prior to arginylation.

Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis.

Posttranslational protein arginylation catalyzed by arginyl transferases is a mechanism to regulate multiple physiological processes. This protein arginylation reaction uses a charged Arg-tRNAArg as the donor of arginine (Arg). The inherent instability of the ester linkage of the arginyl group to the tRNA, which is sensitive to hydrolysis at the physiological pH, makes it difficult to obtain structural information on how the arginyl transfer reaction is catalyzed. Here, we describe a methodology to synthesize stably charged Arg-tRNAArg that would facilitate structural analysis. In the stably charged Arg-tRNAArg, the ester linkage is replaced with an amide linkage, which is resistant to hydrolysis even at alkaline pH.

The CGA codon decoding through tRNAArg (ICG) supply governed by Tad2/Tad3 in Saccharomyces cerevisiae.

The CGA codon is a rare codon in Saccharomyces cerevisiae and is known to be inefficiently decoded by wobble pairing with Arg-tRNA(ICG). The tRNAArg (ICG) is post-transcriptionally edited from tRNAArg (ACG) by the anticodon first adenosine deamination enzyme Tad2/Tad3 complex. Experimental consecutive CGA codons cause ribosome stalling to result in the reduction of the encoding protein product. In this study, the additional supply of tRNAArg (ACG) genes that produce decoding Arg-tRNA(ICG) promoted the product level from the CGA12-luc reporter, revealing that the product reduction is essentially due to inefficient decoding and deficiency in the tRNA supply. The mature tRNAArg (ICG) and the precursor tRNAArg (ACG) ratios examined for cellular tRNA fraction revealed that the tRNAArg (ICG) ratio is maintained at less than 30% and is responsive to the Tad2/Tad3 expression level.

Functional Interplay between Arginyl-tRNA Synthetases and Arginyltransferase.

Protein arginylation, mediated by arginyltransferase ATE1, is a post-translational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance among ATE1, RARS and translation are unknown. Here, we addressed the question of how these two enzymes can partition Arg-tRNAArg to functionally distinct pathways using an intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We found that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or the availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1's redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence among translation, arginyl-tRNA synthesis and arginylation.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops.Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine.

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex.

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

tRNAArg-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation.

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNAArg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNAArg fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg:tRFArg compared with wild type, suggesting a functional link between tRFArg and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.

Adaptation of codon usage to tRNA I34 modification controls translation kinetics and proteome landscape.

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification.

In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific tRNA arginine species for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein to recognize particular arginine tRNAs destined for m3C modification. DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in the DALRD3 gene that impairs m3C formation in human patients exhibiting developmental delay and early-onset epileptic encephalopathy. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification and suggest a crucial biological role for DALRD3-dependent tRNA modification in proper neurological development.

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.

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.

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.

Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans.

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m3C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m3C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m3C formation in mammals as well. Here, we report the discovery and characterization of three distinct m3C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m3C in specific tRNAs and that METTL8 only contributes m3C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m3C in total tRNA, and primer extension analysis located METTL2-modified m3C at position 32 of tRNAThr isoacceptors and tRNAArg(CCU) We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m3C modification in mRNA, and the discovery of METTL8 as an mRNA m3C writer enzyme opens the door to future studies of other m3C epitranscriptomic reader and eraser functions.

Hili Inhibits HIV Replication in Activated T Cells.

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNAArg(UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4+ T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

Dimerization of Arginyl-tRNA Synthetase by Free Heme Drives Its Inactivation in Plasmodium falciparum.

Excess cellular heme is toxic, and malaria parasites regulate its levels during hemoglobin digestion. Aminoacyl-tRNA synthetases are ubiquitous enzymes, and of these, arginyl-tRNA synthetase (RRS) is unique as its enzymatic product of charged tRNA is required for protein synthesis and degradation. We show that Plasmodium falciparum arginyl-tRNA synthetase (PfRRS) is an active, cytosolic, and monomeric enzyme. Its high-resolution crystal structure highlights critical structural differences with the human enzyme. We further show that hemin binds to and inhibits the aminoacylation activity of PfRRS. Hemin induces a dimeric form of PfRRS that is thus rendered enzymatically dead as it is unable to recognize its cognate tRNA(arg). Excessive hemin in chloroquine-treated malaria parasites results in significantly reduced charged tRNA(arg) levels, thus suggesting deceleration of protein synthesis. These data together suggest that the inhibition of Plasmodium falciparum arginyl-tRNA synthetase can now be synergized with existing antimalarials for more potent drug cocktails against malaria parasites.