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.

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.

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.

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.

Effect of consecutive rare codons on the recombinant production of human proteins in Escherichia coli.

In Escherichia coli, the expression of heterologous genes for the production of recombinant proteins can be challenging due to the codon bias of different organisms. The rare codons AGG and AGA are among the rarest in E. coli. In this work, by using the human gene RioK2 as case study, we found that the presence of consecutive AGG-AGA led to a premature stop, which may be caused by an event of -1 frameshift. We found that translational problems caused by consecutive AGG-AGA are sequence dependent, in particular, in sequences that contain multiple rare AGG or AGA codons elsewhere. Translational problems can be alleviated by different strategies, including codon harmonization, codon optimization, or by substituting the consecutive AGG-AGA codons by more frequent arginine codons. Overall, our results furthered our understanding about the relationship between consecutive rare codons and translational problems. Such information will aid the design of DNA sequence for the production of recombinant proteins.

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.

Generation of an arginine-tRNA-adapted Saccharomyces cerevisiae strain for effective heterologous protein expression.

The tRNA population reflects the codon bias of the organism and affects the translation of heterologous target mRNA molecules. In this study, Saccharomyces cerevisiae strains with modified levels of rare tRNA were engineered, that allowed efficient generation of recombinant proteins with unfavorable codon usage. We established a novel synthetic tRNA expression cassette and verified functional nonsense suppressor tRNAGlnSCUA generation in a stop codon read-through assay with a modified ß-galactosidase reporter gene. Correlation between altered tRNA and protein level was shown by survival of copper sensitive S. cerevisiae cells in the presence of copper ions by an increased transcription of tRNAArgCCG molecules, recognizing rare codons in a modified CUP1 gene. Genome integration of tRNA expression cassette led to the generation of arginine-tRNA-adapted S. cerevisiae strains, which showed elevated tRNA levels (tRNAArgCCG, tRNAArgGCG and tRNAArgUCG) pairing to rare codons. The modified strain MNY3 revealed a considerably improved monitoring of protein-protein interaction from Aspergillus fumigatus bait and prey sequences in yeast two-hybrid experiments. In future, this principle to overcome limited recombinant protein expression by tRNA adaption of expression strains instead of codon adaption might provide new designer yeast cells for an efficient protein production and for improved genome-wide protein-protein interaction analyses.

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.

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.

Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons.

The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non-proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non-proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA(Arg) in a cell-free protein synthesis system by using a tRNA(Arg) -specific tRNase, colicin D. Then by supplementing the system with tRNACCU , the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids.

Where have all the inosines gone? Conflicting evidence for A-to-I editing of the anticodon of higher eukaryotic tRNAACGArg questions the dogma of a universal wobble-mediated decoding of CGN codons.

Codon-anticodon recognition between triplets of an mRNA and a specific tRNA is the key element in the translation of the genetic code. In general, the precision of this process is dominated by a strict Watson-Crick base-pairing scheme. However, the degeneracy of the genetic code led Crick to propose the Wobble Hypothesis, permitting a less restraining interaction with the third base of the codon and involving the participation of inosine for decoding C-ending codons. The concept that the anticodon base A34 of tRNAACGArg in all eukaryotes, eubacteria, and plant chloroplasts is converted to I34 is firmly anchored in the literature despite conflicting evidence for its existence in higher eukaryote cytoplasmic tRNAACGArg. Here, we provide additional data and summarize the arguments favoring and contradicting post-transcriptional deamination of this position. A hypothesis that resolves the apparent conflict is proposed. © 2016 IUBMB Life, 68(6):419-422, 2016.

Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Identity elements for the aminoacylation of metazoan mitochondrial tRNA(Arg) have been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment.

Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.

The tRNAarg gene and engA are essential genes on the 1.7-Mb pSymB megaplasmid of Sinorhizobium meliloti and were translocated together from the chromosome in an ancestral strain.

Bacterial genomes with two (or more) chromosome-like replicons are known, and these appear to be particularly frequent in alphaproteobacteria. The genome of the N(2)-fixing alfalfa symbiont Sinorhizobium meliloti 1021 contains a 3.7-Mb chromosome and 1.4-Mb (pSymA) and 1.7-Mb (pSymB) megaplasmids. In this study, the tRNA(arg) and engA genes, located on the pSymB megaplasmid, are shown to be essential for growth. These genes could be deleted from pSymB when copies were previously integrated into the chromosome. However, in the closely related strain Sinorhizobium fredii NGR234, the tRNA(arg) and engA genes are located on the chromosome, in a 69-kb region designated the engA-tRNA(arg)-rmlC region. This region includes bacA, a gene that is important for intracellular survival during host-bacterium interactions for S. meliloti and the related alphaproteobacterium Brucella abortus. The engA-tRNA(arg)-rmlC region lies between the kdgK and dppF2 (NGR_c24410) genes on the S. fredii chromosome. Synteny analysis showed that kdgK and dppF2 orthologues are adjacent to each other on the chromosomes of 15 sequenced strains of S. meliloti and Sinorhizobium medicae, whereas the 69-kb engA-tRNA(arg)-rmlC region is present on the pSymB-equivalent megaplasmids. This and other evidence strongly suggests that the engA-tRNA(arg)-rmlC region translocated from the chromosome to the progenitor of pSymB in an ancestor common to S. meliloti and S. medicae. To our knowledge, this work represents one of the first experimental demonstrations that essential genes are present on a megaplasmid.

A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice.

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J x CAST/Ei) x A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ x CAST/Ei) x NOD/LtJ and (SKH2/J x CAST/Ei) x SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.

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.

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.

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.

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae.

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 x 10(-5) to a high of 6.9 x 10(-4) per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.