Arginine tRNA Research Articles

Epileptic encephalopathy linked to a DALRD3 missense variant that impairs tRNA modification

Epileptic encephalopathies are severe epilepsy syndromes characterized by early onset and progressive cerebral dysfunction. A nonsense variant in the DALR Anticodon Binding Domain Containing 3 (DALRD3) gene has been implicated in epileptic encephalopathy but no other disease-associated variants in DALRD3 have been described. In human cells, the DALRD3 protein forms a complex with the METTL2 methyltransferase to generate the 3-methylcytosine (m3C) modification in specific arginine tRNAs. Here, we identify an individual with a homozygous missense variant in DALRD3 who displays developmental delay, cognitive deficiencies, and multifocal epilepsy. The missense variant substitutes an arginine residue to cysteine (R517C) within the DALR domain of the DALRD3 protein that is required for binding tRNAs. Cells derived from the individual homozygous for the DALRD3-R517C variant exhibit reduced levels of m3C modification in arginine tRNAs, indicating that the R517C variant impairs DALRD3 function. Notably, the DALRD3-R517C protein displays reduced association with METTL2 and loss of interaction with substrate tRNAs. Our results uncover another loss-of-function variant in DALRD3 linked to epileptic encephalopathy disorders. Importantly, these findings underscore DALRD3-dependent tRNA modification as a key contributor to proper brain development and function.

Genomic factors shaping codon usage across the Saccharomycotina subphylum.

Codon usage bias, or the unequal use of synonymous codons, is observed across genes, genomes, and between species. It has been implicated in many cellular functions, such as translation dynamics and transcript stability, but can also be shaped by neutral forces. We characterized codon usage across 1,154 strains from 1,051 species from the fungal subphylum Saccharomycotina to gain insight into the biases, molecular mechanisms, evolution, and genomic features contributing to codon usage patterns. We found a general preference for A/T-ending codons and correlations between codon usage bias, GC content, and tRNA-ome size. Codon usage bias is distinct between the 12 orders to such a degree that yeasts can be classified with an accuracy greater than 90% using a machine-learning algorithm. We also characterized the degree to which codon usage bias is impacted by translational selection. We found it was influenced by a combination of features, including the number of coding sequences, BUSCO count, and genome length. Our analysis also revealed an extreme bias in codon usage in the Saccharomycodales associated with a lack of predicted arginine tRNAs that decode CGN codons, leaving only the AGN codons to encode arginine. Analysis of Saccharomycodales gene expression, tRNA sequences, and codon evolution suggests that avoidance of the CGN codons is associated with a decline in arginine tRNA function. Consistent with previous findings, codon usage bias within the Saccharomycotina is shaped by genomic features and GC bias. However, we find cases of extreme codon usage preference and avoidance along yeast lineages, suggesting additional forces may be shaping the evolution of specific codons.

Copper catalyzed cycloaddition for the synthesis of non isomerisable 2′ and 3′-regioisomers of arg-tRNAarg

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2′- or 3′-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2′- or 3′-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5′-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5′-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2′ and 3′) which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

A computational screen for alternative genetic codes in over 250,000 genomes.

The genetic code has been proposed to be a 'frozen accident,' but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force that likely helped drive these codons to low frequency and enable their reassignment.

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.

Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.

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.

Interactions of Destruxin A with Silkworms' Arginine tRNA Synthetase and Lamin-C Proteins.

Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae, has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms’ arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with KD value 5.53 × 10−5 and 8.64 × 10−5 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect.

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

The first Ralstonia-infecting bacteriophage from soil of the United States, designated RsoM1USA, was isolated from a tomato field in Florida. Electron microscopy revealed that phage RsoM1USA is member of the genus P2virus in the family Myoviridae with an icosahedral head of about 66 nm in diameter, a contractile tail of about 152 nm in length, and a long “neck.” Phage RsoM1USA infected 12 of the 30 tested R. solanacearum species complex strains collected worldwide in each of the three Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii. The phage completed its infection cycle 180 min post infection with a burst size of about 56 particles per cell. Phage RsoM1USA has a genome of 39,309 nucleotides containing 58 open reading frames (ORFs) and is closely related to Ralstonia phage RSA1 of the species Ralstonia virus RSA1. The genomic organization of phage RsoM1USA is also similar to that of phage RSA1, but their integrases share no sequence homology. In addition, we determined that the integration of phage RsoM1USA into its susceptible R. solanacearum strain K60 is mediated by the 3′ 45-base portion of the threonine tRNA (TGT), not arginine tRNA (CCG) as reported for phage RSA1, confirming that the two phages use different mechanism for integration. Our proteomic analysis of the purified virions supported the annotation of the main structural proteins. Infection of a susceptible R. solanacearum strain RUN302 by phage RsoM1USA resulted in significantly reduced growth of the infected bacterium in vitro, but not virulence in tomato plants, as compared to its uninfected RUN302 strain. Due to its differences from phage RSA1, phage RsoM1USA should be considered the type member of a new species with a proposed species name of Ralstonia virus RsoM1USA.

Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3.

Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.

Mitochondrial complex IV mutation increases reactive oxygen species production and reduces lifespan in aged mice.

Mitochondrial DNA (mtDNA) mutations can negatively influence lifespan and organ function. More than 250 pathogenic mtDNA mutations are known, often involving neurological symptoms. Major neurodegenerative diseases share key etiopathogenetic components ie mtDNA mutations, mitochondrial dysfunction and oxidative stress. Here, we characterized a conplastic mouse strain (C57BL/6J-mtNOD) carrying an electron transport chain complex IV mutation that leads to an altered cytochrome c oxidase subunit III. Since this mouse also harbours adenine insertions in the mitochondrial tRNA for arginine, we chose the C57BL/6J-mtMRL as control strain which also carries a heteroplasmic stretch of adenine repetitions in this tRNA isoform. Using MitoSOX fluorescence, we observed an elevated mitochondrial superoxide production and a reduced gene expression of superoxide dismutase 2 in the 24-month-old mtNOD mouse as compared to control. Together with the decreased expression of the fission-relevant gene Fis1, these data confirmed that the ageing mtNOD mouse had a mitochondrial dysfunctional phenotype. On the functional level, we could not detect significant differences in synaptic long-term potentiation, but found a markedly poor physical constitution to perform the Morris water maze task at the age of 24months. Moreover, the median lifespan of mtNOD mice was significantly shorter than of control animals. Our findings demonstrate that a complex IV mutation leads to mitochondrial dysfunction that translates into survival.

Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells

Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that a selective loss of arginine tRNA charging during limitation for arginine regulates translation through ribosome pausing at two of six arginine codons. Surprisingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation leads to loss of tRNA charging and ribosome pausing. Ribosome pausing decreases protein production and triggers premature ribosome termination without reducing mRNA levels. Together, our results suggest that amino acids that are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on codon-specific ribosome pausing.

Lysogenic Conversion of the Phytopathogen Ralstonia solanacearum by the P2virus ϕRSY1.

A P2-like phage ϕRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ϕRSY1 showed high sequence similarity to the Ralstonia phage ϕRSA1 and the GMI1000 prophage ϕRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ϕRSY1 and ϕRSX use a 15-base 3′ portion of the serine tRNA(GGA) gene as attB, while ϕRSA1 uses a 45-base 3′ portion of the arginine tRNA(CCG) gene. The different orientation of int in the genomes means that the gene arrangements in the prophage states are reversed in ϕRSY1 and ϕRSA1. Several putative gene products of ϕRSY1 may affect the bacterium’s fitness. ϕRSY1 contains an open reading frame (ORF) that seems to encode a protein similar to Vgr in the type VI secretion system of various bacterial species. ϕRSY1 lysogens showed phenotypic changes including enhanced twitching motility, large colony formation, and easy aggregation of cells, suggesting involvement of this ORF in the changes. In view of these phage gene arrangements, we surveyed prophages in the genomes of various R. solanacearum strains and found that the P2-like phages of R. solanacearum (14 phages) consist of two major groups: the ϕRSY1-type and the ϕRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.

Data on true tRNA diversity among uncultured and bacterial strains.

Complete genome sequences of two uncultured archaea (BX649197 and CR937008) and 10 uncultured bacteria (AC160099, FP245538-FP245540, FP312972, FP312974-75, FP312977, FP312985 and NZ_JPJG01000067) were used for creation of digital data of tRNA. tRNAscan-SE and ENDMEMO GC calculating tools were used for detection of tRNA, drawing their structures and calculation of GC percent. Seven archaeal and 48 bacterial tRNA were detected from above 12 sequences. Four archaeal and 30 bacterial tRNA showed cove score more than 20% are called as true tRNA. Three tRNA of uncultured bacteria (AC160099) has the presence of the variable loop. The tRNA of FP245540, FP245575, FP245577 and FP245585 has one variable loop each. The true tRNA of archaea were Alanine, Arginine and Cysteine-type tRNA, while the majority of bacteria true tRNA classified as Alanine, Glutamic acid, Isoleucine, Leucine, Methionine, Phenylalanine, Proline and Valine-type tRNA with cove score ranged from 70% to 97.15%. Archaeal and bacterial have GC content approximately 43% and 34.7–63.3% respectively. Archaeal tRNA has 60.4–64.2% GC content. Similarly, bacterial tRNA contributed 49.3–66.3% GC content to the total GC content. This generated data is useful for studies on diversity of tRNA among prokaryotes.

Translation of 'Systematization of Codons in the Genetic Code [III]' by Yu. B. Rumer (1969).

Translation of 'Systematization of Codons in the Genetic Code [III]' by Yu. B. Rumer (1969).

Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant.

It has been identified that a TEV protease (TEVp) variant, TEVp5M, displays improved solubility. Here, we constructed fifteen TEVp5M variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp5M. After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp5M on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp5M. Supply of rare tRNAs caused the decrease of activity of TEVp5M and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.

Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF-κB activation inhibited CCL20 expression in mtBALB cybrids and decreased their migratory capabilities. Thus, acquired mtDNA mutations may promote tumorigenic phenotypes through up-regulation of chemokine CCL20.

FimY of Salmonella enterica serovar Typhimurium functions as a DNA-binding protein and binds the fimZ promoter

Salmonella enterica serovar Typhimurium produces type 1 fimbriae with binding specificity to mannose residues. Elements involved in fimbrial structural biosynthesis, transport, and regulation are encoded by the fim gene cluster. FimZ, FimY, FimW, STM0551, and an arginine transfer RNA (fimU) were previously demonstrated to regulate fimbrial expression. The amino acid sequences of the C-terminal portion of FimY revealed similarity with those of LuxR-like proteins. Electrophoretic mobility shift assays indicated that FimY possessed DNA-binding capacity and bound a 605-bp DNA fragment spanning the intergenic region between fimY and fimZ, while a FimY protein harboring a double mutation in the C-terminal helix-turn-helix region containing a glycine (G) to aspartate (D) substitution at residue 189 and isoleucine (I) to lysine (K) substitution at residue 195 lost its ability to bind this DNA fragment. A lux box sequence (5′-TCTGTTATTACATAACAAATACT-3′) within the fimZ promoter was required for binding. None of the DNA fragments derived from the promoters for fimA, fimY, or fimW was shifted by FimY. Pull-down assays showed that there were physical protein/protein interactions between FimY and FimZ. We propose that in the regulatory circuit of type 1 fimbriae, FimY functions as a DNA-binding protein to activate fimZ, and a FimY-FimZ protein complex may form to regulate other fim genes. Confirming these proposals requires further study.

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ 0 cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.