Specific tRNA Species Research Articles

In situ Nanopore sequencing reveals metabolic characteristics of the Qilian glacier meltwater microbiome.

Nanopore metagenomic sequencing enables rapid annotating microbiological ecosystems, and the previous glacier-related sequencing applications (e.g., targeted ice sheets, ice lake, and cryoconite holes) inspire us to explore high-altitude glacier meltwater at Qilian Mountain, China (3000 to 4000 m above sea level, MASL). Our findings suggest that (1) despite only several hundred meters apart, the microbial communities and functionalities are quite different among vertical alpine distributions; (2) the high-altitude Qilian meltwater microbiome serve several main metabolic functions, including sulfur oxidation, selenite decomposing, photosynthesis, energy production, enzymic, and UV tolerant activities. Meanwhile, our Nanopore metagenomic results indicate that the microbial classifications and functionalities (e.g., chaperones, cold-shock, specific tRNA species, oxidative stress, and resistance to toxic compounds) of Qilian meltwater are highly consistent with the other glacial microbiome, emphasizing that only certain microbial species can survive in the cold environment and the molecular adaptions and lifestyles remain stable all over the world. Besides, we have shown Nanopore metagenomic sequencing can provide reliable prokaryotic classifications within or among studies, which therefore can encourage more applications in the field given faster turnaround time. However, we recommend accumulating at least 400 ng nucleic acids (after extraction) and maximizing Nanopore library preparation efficiency before on-site sequencing to obtain better resolutions.

Production and purification of homogenous recombinant human selenoproteins reveals a unique codon skipping event in E. coli and GPX4-specific affinity to bromosulfophthalein

Selenoproteins are translated via animal domain-specific elongation machineries that redefine dedicated UGA opal codons from termination of translation to selenocysteine (Sec) insertion, utilizing specific tRNA species and Sec-specific elongation factors. This has made recombinant production of mammalian selenoproteins in E. coli technically challenging but recently we developed a methodology that enables such production, using recoding of UAG for Sec in an RF1-deficient host strain. Here we used that approach for production of the human glutathione peroxidases 1, 2 and 4 (GPX1, GPX2 and GPX4), with all these three enzymes being important antioxidant selenoproteins. Among these, GPX4 is the sole embryonically essential enzyme, and is also known to be essential for spermatogenesis as well as protection from cell death through ferroptosis. Enzyme kinetics, ICP-MS and mass spectrometry analyses of the purified recombinant proteins were used to characterize selenoprotein characteristics and their Sec contents. This revealed a unique phenomenon of one-codon skipping, resulting in a lack of a single amino acid at the position corresponding to the selenocysteine (Sec) residue, in about 30% of the recombinant GPX isoenzyme products. We furthermore confirmed the previously described UAG suppression with Lys or Gln as well as a minor suppression with Tyr, together resulting in about 20% Sec contents in the full-length proteins. No additional frameshifts or translational errors were detected. We subsequently found that Sec-containing GPX4 could be further purified over a bromosulfophthalein-column, yielding purified recombinant GPX4 with close to complete Sec contents. This production method for homogenously purified GPX4 should help to further advance the studies of this important selenoprotein.

Bacterial tRNase-Based Gene Therapy with Poly(β-Amino Ester) Nanoparticles for Suppressing Melanoma Tumor Growth and Relapse.

Here, a novel anticancer gene therapy with a bacterial tRNase gene, colicin D or virulence associated protein C (VapC), is suggested using biodegradable polymeric nanoparticles, such as poly(β-amino esters) (PBAEs) as carriers. These genes are meticulously selected, aiming at inhibiting translation in the recipients by hydrolyzing specific tRNA species. In terms of nanoparticles, out of 9 PBAE formulations, a leading polymer, (polyethylene oxide)4 -bis-amine end-capped poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (B4S5E5), is identified that displays higher gene delivery efficacy to cancer cells compared with the leading commercial reagent Lipofectamine 2000. Interestingly, the B4S5E5 PBAE nanoparticles complexed with colicin D or VapC plasmid DNA induce significant toxicity highly specific to cancer cells by triggering apoptosis. In contrast, the PBAE nanoparticles do not induce these cytotoxic effects in noncancerous cells. In a mouse melanoma model of grafted murine B16-F10 cells, it is demonstrated that treatment with PBAE nanoparticles complexed with these tRNase genes significantly reduces tumor growth rate and delays tumor relapse. Moreover, increased stability of PBAE by PEGylation further enhances the therapeutic effect of tRNase gene treatment and improves survival of animals. This study highlights a nonviral gene therapy that is highly promising for the treatment of cancer.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels.

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.

Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism.

BackgroundPrimordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity.ResultsTwo presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant. WDR4 is the human ortholog of the yeast Trm82, an essential component of the Trm8/Trm82 holoenzyme that effects a highly conserved and specific (m7G46) methylation of tRNA. The human mutation and the corresponding yeast mutation result in a significant reduction of m7G46 methylation of specific tRNA species, which provides a potential mechanism for primordial dwarfism associated with this lesion, since reduced m7G46 modification causes a growth deficiency phenotype in yeast.ConclusionOur study expands the number of biological pathways underlying primordial dwarfism and adds to a growing list of human diseases linked to abnormal tRNA modification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0779-x) contains supplementary material, which is available to authorized users.

The biased nucleotide composition of the HIV genome: a constant factor in a highly variable virus

Viruses often deviate from their hosts in the nucleotide composition of their genomes. The RNA genome of the lentivirus family of retroviruses, including human immunodeficiency virus (HIV), contains e.g. an above average percentage of adenine (A) nucleotides, while being extremely poor in cytosine (C). Such a deviant base composition has implications for the amino acids that are encoded by the open reading frames (ORFs), both in the requirement of specific tRNA species and in the preference for amino acids encoded by e.g. A-rich codons. Nucleotide composition does obviously affect the secondary and tertiary structure of the RNA genome and its biological functions, but it does also influence phylogenetic analysis of viral genome sequences, and possibly the activity of the integrated DNA provirus. Over time, the nucleotide composition of the HIV-1 genome is exceptionally conserved, varying by less than 1% per base position per isolate within either group M, N, or O during 1983–2009. This extreme stability of the nucleotide composition may possibly be achieved by negative selection, perhaps conserving semi-stable RNA secondary structure as reverse transcription would be significantly affected for a less A-rich genome where secondary structures are expected to be more stable and thus more difficult to unfold.This review will discuss all aspects of the lentiviral genome composition, both of the RNA and of its derived double-stranded DNA genome, with a focus on HIV-1, the nucleotide composition over time, the effects of artificially humanized codons as well as contributions of immune system pressure on HIV nucleotide bias.

NEFA/nucleobindin-2 is a target autoantigen of the anti-Wa antibody and is associated with transfer RNA

Objectives The anti-Wa antibody found in systemic sclerosis patients reacts with a transfer RNA (tRNA)-associated 48-kDa protein and immunoprecipitates several tRNAs. We investigated the Wa antigen and its binding to tRNA species.Methods We performed molecular cloning of the Wa antigen and made its recombinant protein. To investigate Wa antigen distribution in the cell, we performed an indirect immunofluorescence study. To determine the Wa-bound tRNA species, we performed a reverse transcription (RT)-polymerase chain reaction (PCR) using the RNAs immunoprecipitated by anti-Wa antibody as templates, and synthetic primers of mammalian tRNA sequences. To clarify the tissue expression of Wa antigen, we performed quantitative and semi-quantitative PCR of the cDNA.Results We demonstrated that the Wa antigen was identical to NEFA (DNA binding/EF-hand/acidic amino acid rich region), otherwise known as nucleobindin-2. A full-length and an alternative splice variant cDNA lacking exon 11 were isolated by cloning NEFA cDNA. Anti-Wa-positive sera stained both the nucleus and cytoplasm of HEp-2 cells. RT-PCR suggested that Wa binds at least six tRNA species. In human tissues, NEFA is expressed predominantly in exocrine glands.Conclusions We have demonstrated that the Wa antigen is NEFA or nucleobindin-2, which binds specific tRNA species, and is distributed in specific human tissues.

NEFA/nucleobindin-2 is a target autoantigen of the anti-Wa antibody and is associated with transfer RNA

The anti-Wa antibody found in systemic sclerosis patients reacts with a transfer RNA (tRNA)-associated 48-kDa protein and immunoprecipitates several tRNAs. We investigated the Wa antigen and its binding to tRNA species. We performed molecular cloning of the Wa antigen and made its recombinant protein. To investigate Wa antigen distribution in the cell, we performed an indirect immunofluorescence study. To determine the Wa-bound tRNA species, we performed a reverse transcription (RT)-polymerase chain reaction (PCR) using the RNAs immunoprecipitated by anti-Wa antibody as templates, and synthetic primers of mammalian tRNA sequences. To clarify the tissue expression of Wa antigen, we performed quantitative and semi-quantitative PCR of the cDNA. We demonstrated that the Wa antigen was identical to NEFA (DNA binding/EF-hand/acidic amino acid rich region), otherwise known as nucleobindin-2. A full-length and an alternative splice variant cDNA lacking exon 11 were isolated by cloning NEFA cDNA. Anti-Wa-positive sera stained both the nucleus and cytoplasm of HEp-2 cells. RT-PCR suggested that Wa binds at least six tRNA species. In human tissues, NEFA is expressed predominantly in exocrine glands. We have demonstrated that the Wa antigen is NEFA or nucleobindin-2, which binds specific tRNA species, and is distributed in specific human tissues.

Synonymous codon usage analysis of the mycobacteriophage Bxz1 and its plating bacteria M. smegmatis: identification of highly and lowly expressed genes of Bxz1 and the possible function of its tRNA species.

The extent of codon usage in the protein coding genes of the mycobacteriophage, Bxz1, and its plating bacteria, M. smegmatis, were determined, and it was observed that the codons ending with either G and / or C were predominant in both the organisms. Multivariate statistical analysis showed that in both organisms, the genes were separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. The second major explanatory axis differentiates the genes according to their genome type. A comparison of the relative synonymous codon usage between 20 highly- and 20 lowly expressed genes from Bxz1 identified 21 codons, which are statistically over represented in the former group of genes. Further analysis found that the Bxz1- specific tRNA species could recognize 13 out of the 21 over represented synonymous codons, which incorporated 13 amino acid residues preferentially into the highly expressed proteins of Bxz1. In contrast, seven amino acid residues were preferentially incorporated into the lowly expressed proteins by 10 other tRNA species of Bxz1. This analysis predicts for the first time that the Bxz1-specific tRNA species modulates the optimal expression of its proteins during development.

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein.

High-level expression of non-functional model proteins, derived from elongation factor EF-Tu by the deletion of an essential domain, greatly inhibits the growth of Escherichia coli partly deficient in peptidyl-tRNA hydrolase. High-level expression in wild-type cells has little effect on growth. The inhibitory effect is therefore presumably due to the sequestration of essential tRNA species, partly in the form of free peptidyl-tRNA. The growth inhibitory effect can be modulated by changing the last sense codon in the genes encoding the model proteins. Thus, replacement of Ser by Lys or His at this position increases growth inhibition. The effects of 11 changes studied are related to the rates of accumulation previously observed of the corresponding families of peptidyl-tRNA. Two non-exclusive hypotheses are proposed to account for these observations: first, the last sense codon of mRNA is a preferred site of peptidyl-tRNA drop-off in cells, due to the slow rate of translation termination compared with sense codon translation; secondly, the relatively long pause of the ribosome at the stop codon (of the order of 1 s), results in significant temporary sequestration on the ribosome of the tRNA cognate to the last sense codon.

Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR).

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [32P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNALysand the corresponding tRNALyscarrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNALeu(UUR)and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m2G content. Complementary sequencing of tRNALeu(UUR)has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA Asp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.

Separation of Rat Liver Cytoplasmic Trnas by High Performance Liquid Chromatography

Abstract Experiments aimed at developing a chromatographic map of cytoplasmic tRNA population from rat liver are described. HPLC tRNA fractionation by a linear gradient of acetonitrile/ammonium acetate, results in a chromatogram with more than 60 significant peaks, having area peak percentages ranging from 0.001 to 5.0. This paper presents evidence that each peak corresponds to a specific tRNA species. Specificity of tRNA-peak assignment was established by charging total tRNA with a mixture of 19 unlabeled aminoacids plus a radioactive aminoacid, and isolating the radioactive aminoacylated iso-tRNAs by gel electrophoresis, followed by HPLC acetonitrile gradient. The radioactive aminoacyl-tRNA species were eluted as well-behaved peaks. Here we describe the analysis of tRNATrp, which gave 2 well separated radioactive peaks, most likely corresponding to tRNATrp isoacceptor species postulated by the wobble hypothesis. Taken together, the present data validate this procedure for monitoring quantitatively and q...

Human immunodeficiency virus uses tRNA Lys,3 as primer for reverse transcription in HeLa-CD4 + cells

Significant amounts of different tRNA molecules are present in retroviral particles, but one specific tRNA species functions as primer in reverse transcription. It is generally believed that the HIV-1 virus uses the tRNA Lys,3 molecule as primer. This is based on sequence complementarity between the 3' end of tRNA Lys,3 and the primer-binding site (PBS) on HIV-1 genomic RNA. Recent biochemical analyses indicated that tRNA Lys,3 is indeed incorporated into viral particles. Interestingly, tRNA Lys,3 could not be detected in virions produced by HeLa-CD4 + cells [(1992) Biochem. Biophys. Res. Commun. 185, 1105-1115]. In order to test whether alternative tRNA molecules can function as primer in HIV replication, we performed a series of experiments based on the observation that tRNA primer sequences are inherited by the viral progeny. We cultured HIV-1 for prolonged periods of time in HeLa-CD4 + cells, but did not detect sequence changes in the PBS region. Furthermore, we found PBS-mutants to be replication incompetent, again suggesting that HIV-1 solely uses tRNA Lys,3 as primer. Most importantly, we obtained revertants of one such PBS-mutant, which had restored a wild-type PBS sequence. This tRNA Lys,3-mediated repair demonstrates a general requirement for this primer in HIV-1 reverse transcription.

Transcription of the fimbrial subunit gene and an associated transfer RNA gene of Pseudomonas aeruginosa

Gene fimA encoding the structural subunit of the fimbriae of Pseudomonas aeruginosa PAK is located in the centre of a 1.2-kb HindIII genomic DNA fragment [see also Sastry et al., J. Bacteriol. 164 (1985) 571–577], which in turn is located within a 6.2-kb EcoRI fragment. Immediately downstream from fimA is a putative threonine tRNA gene [Dalrymple and Mattick, Biochem. Int. 13 (1986) 547–553]. Northern blotting experiments showed that fimA is transcribed to an mRNA of approx. 650 nucleotides, which also includes the threonine tRNA sequence but no other protein-coding region. There was no indication that this mRNA is processed to release the tRNA sequence. However, the tRNA did appear to be expressed independently from its own promoter in the region 3' to fimA. When these sequences were introduced into Pseudomonas putida, we found that the level of expression of fimA from the cloned 6.2-kb EcoRI fragment was approx. 30-fold greater than that from the smaller HindIII fragment, whereas that of the specific tRNA species was unaltered. The size of the fimA transcript was also unaltered. These results provide evidence that the fimA gene is subject to specific transcriptional activation in vivo and that this activation involves sequences flanking the 1.2-kb HindIII fragment.

Blocks in Elongation and Initiation of Protein Synthesis Induced by Interferon Treatment in Mouse L Cells

Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell-free extracts from interferon-treated mouse L cells, due to a "deficiency" in some specific tRNA species. A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met-tRNA(FMET) binding is inhibited. The block in initiation appears to be secondary to the block in elongation.