tRNA Pseudouridine Synthase Research Articles

Insight into a region of chickpea (Cicer arietinum L.) Chromosome 2 revealed potential candidate genes linked to Foc4 Fusarium wilt resistance.

Two markers on Chromosome 2 of chickpea (Cicer arietinum ) are reportedly associated with resistance to race 4 Fusarium wilt, and are frequently used in breeding. However, the genes in this region that actually confer wilt resistance are unknown. We aimed to characterise them using both in silico approaches and marker trait association (MTA) analysis. Of the 225 protein-encoding genes in this region, 51 showed significant differential expression in two contrasting chickpea genotypes under wilt, with potential involvement in stress response. From a diverse set of 244 chickpea genotypes, two sets of 40 resistant and 40 susceptible genotypes were selected based on disease incidence and amplification pattern of the TA59 marker. All cultivars were further genotyped with 1238 single nucleotide polymorphisms (SNPs) specific to the 51 genes; only seven SNPs were significantly correlated with disease. SNP Ca2_24099002, specific to the LOC101498008 (Transmembrane protein 87A) gene, accounted for the highest phenotypic variance for disease incidence at 16.30%, whereas SNPs Ca2_25166118 and Ca2_27029215, specific to the LOC101494644 (β-glucosidase BoGH3B-like) and LOC101505289 (Putative tRNA pseudouridine synthase) genes, explained 10.51% and 10.50% of the variation, respectively, in the sets with contrasting disease susceptibility. Together with the TA59 and TR19 markers, these SNPs can be used in a chickpea breeding scheme to develop wilt resistance.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Correction: Macrophage-Specific TLR2 Signaling Mediates Pathogen-Induced TNF-Dependent Inflammatory Oral Bone Loss.

<h3>Abstract</h3> Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter, and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, HITS-CLIP (High throughput sequencing crosslinking immunoprecipitation) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR-8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation and function.

Genomic and transcriptomic profiling of phoenix colonies

Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing. Phoenix colonies were found to have a single nucleotide polymorphism (SNP) in the PA4673 gene, which is predicted to encode a GTP-binding protein. No SNPs were identified within VBNC-like colonies compared to the founder population. RNA sequencing did not detect change in expression of PA4673 but revealed multiple differentially expressed genes that may play a role in phoenix colony emergence. One of these differentially expressed genes, PA3626, encodes for a tRNA pseudouridine synthase which when knocked out led to a complete lack of phoenix colonies. Although not immediately clear whether the identified genes in this study may have interactions which have not yet been recognized, they may contribute to the understanding of how phoenix colonies are able to emerge and survive in the presence of antibiotic exposure.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

TRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA.

tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, m5U54, and m7G46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact the binding affinity or catalysis by tRNA elbow modification enzymes. Specifically, we focused on the Escherichia coli enzymes TruB, TrmA, and TrmB which generate Ψ55, m5U54, and m7G46, respectively. tRNAs containing a single modification were prepared, and the binding and activity preferences of purified E. coli TrmA, TruB, and TrmB were examined in vitro. TruB preferentially binds and modifies unmodified tRNA. TrmA prefers to modify unmodified tRNA, but binds most tightly to tRNA that already contains Ψ55. In contrast, binding and modification by TrmB is insensitive to the tRNA modification status. Our results suggest that TrmA and TruB are likely to act on mostly unmodified tRNA precursors during the early stages of tRNA maturation whereas TrmB presumably acts on later tRNA intermediates that are already partially modified. In conclusion, we uncover the mechanistic basis for the preferred modification order in the E. coli tRNA elbow region.

Identifying the Molecular Targets of an Anti-pathogenic Hydroalcoholic Extract of Punica granatum Peel Against Multidrug-resistant Serratia marcescens.

Antibiotic-resistant members of the family Enterobacteriaceae are among the serious threats to human health globally. This study reports the anti-pathogenic activity of Punica granatum peel extract (PGPE) against a multi-drug resistant, beta-lactamase producing member of this family i.e. Serratia marcescens. This study aimed at assessing the anti-pathogenic activity of PGPE against the gramnegative bacterial pathogen S. marcescens and identifying the molecular targets of this extract in the test bacterium. Effect of PGPE on S. marcescens growth and quorum sensing (QS)-regulated pigment production was assessed through broth dilution assay. In vivo anti-infective and prophylactic activity of PGPE was assessed employing the nematode worm Caenorhabditis elegans as a model host. Differential gene expression in PGPE-exposed S. marcescens was studied through a whole transcriptome approach. PGPE was able to modulate QS-regulated pigment production in S. marcescens without exerting any heavy growth-inhibitory effect at concentrations as low as ≥2.5 μg/mL. It could attenuate the virulence of the test bacterium towards the worm host by 22-42% (p≤0.01) at even lower concentrations (≥0.5 μg/mL). PGPE also exerted a post-extract effect on S. marcescens. This extract was found to offer prophylactic benefit too, to the host worm, as PGPE-pre-fed worms scored better (34-51%; p≤0.001) survival in face of subsequent bacterial attack. Differential gene expression analysis revealed that PGPE affected the expression of a total of 66 genes in S. marcescens by ≥1.5 fold. The anti-virulence effect of PGPE against S. marcescens is multifaceted, affecting stress-response machinery, efflux activity, iron homeostasis, and cellular energetics of this bacterium notably. Among the major molecular targets identified in this study are LPS export transporter permease (LptF), t-RNA pseudouridine synthase (TruB), etc.

Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification.

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone.

A tiling array-based comparative genomic hybridization approach to predict copy number variations between Plasmodium falciparum field isolates from the Indian Sub-continent

Background: There are several techniques to analyse copy number variation in both research and clinical settings, such as whole genome amplification (sWGA), SNP arrays and one of the most commonly used techniques, array based comparative genomic hybridization (aCGH). In the latter, copy number comparison is obtained between differentially labelled target and reference DNAs by measuring ratio of fluorescence intensity of probes indicating loss or gain in the chromosomal region. Methods: Here we carry out a comparative analysis between two Plasmodium falciparum parasite isolates (Pf-isolate-2 and Pf-isolate-1) causing malaria using array CGH. The array contains approximately 418,577, 60mer custom-designed probes with an average probe spacing of 56 bp. The significant major variations (amplifications and deletions) copy number variations (CNV) in Pf-isolate-2 (Pf-2) in comparison with Pf-isolate-1 (Pf-1), are reported. Results: CNVs have been seen in all the chromosomes in Pf-2, most of the deletions have been seen mostly in sub-telomeric and telomeric regions of the chromosomes that comprises of variant surface antigen family genes. Apart from the subtelomeric regions other parts of the chromosomes have also shown CNVs. Novel variations , like continuous amplification of 28kb region (249817-278491) of chromosome-8, which covers for 3 genes two of which codes for conserved Plasmodium proteins with unknown function (MAL8P1.139, PF08_0122) and tRNA pseudouridine synthase, putative (PF08_0123). Amplifications in regions harboring genes like GTP cyclohydrolase I (GCH-1, PFL1155W) and ribosomal protein, L24, putative (PFL1150C) of chromosome 12 were seen. Conclusion: Other than known variations reported earlier, some novel variations have also been seen in the chromosomes of Pf-2. This is an experimental case study reporting major amplifications and deletions in Pf-isolate-2 in comparison with Pf-isolate-1 using a tiling array based comparative genomic hybridization approach.

RNA modification enzyme TruB is a tRNA chaperone

Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB's RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870-1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.

A novel papillation assay for the identification of genes affecting mutation rate in Pseudomonas putida and other pseudomonads

Formation of microcolonies (papillae) permits easy visual screening of mutational events occurring in single colonies of bacteria. In this study, we have established a novel papillation assay employable in a wide range of pseudomonads including Pseudomonas aeruginosa and Pseudomonas putida for monitoring mutation frequency in distinct colonies. With the aid of this assay, we conducted a genome-wide search for the factors affecting mutation frequency in P. putida. Screening ∼27,000 transposon mutants for increased mutation frequency allowed us to identify 34 repeatedly targeted genes. In addition to genes involved in DNA replication and repair, we identified genes participating in metabolism and transport of secondary metabolites, cell motility, and cell wall synthesis. The highest effect on mutant frequency was observed when truA (tRNA pseudouridine synthase), mpl (UDP-N-acetylmuramate-alanine ligase) or gacS (multi-sensor hybrid histidine kinase) were inactivated. Inactivation of truA elevated the mutant frequency only in growing cells, while the deficiency of gacS affected mainly stationary-phase mutagenesis. Thus, our results demonstrate the feasibility of the assay for isolating mutants with elevated mutagenesis in growing as well as stationary-phase bacteria.

Purification, crystallization and preliminary X-ray crystallographic study of the tRNA pseudouridine synthase TruB from Streptococcus pneumoniae.

The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine (Ψ) at residue 55 of elongator tRNAs. In order to better elucidate the functions of TruB in the formation of pseudouridine, the three-dimensional structure of full-length TruB was determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported. The crystal belonged to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 Å, β = 102.05°, and diffracted to a resolution of 1.7 Å. The crystal is most likely to contain one molecule in the asymmetric unit, with a VM value of 2.40 Å(3) Da(-1).

X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold

X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold

X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold

Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 Å resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.

Expression, purification, crystallization and preliminary diffraction studies of the tRNA pseudouridine synthase TruD from Escherichia coli.

Pseudouridine, the 5-ribosyl isomer of uridine, is the most common modification of structural RNA. The recently identified pseudouridine synthase TruD belongs to a widespread class of pseudouridine synthases without significant sequence homology to previously known families. TruD from Escherichia coli was overexpressed, purified and crystallized. The crystals diffract to a minimum Bragg spacing of 2.4 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.4, b = 108.6, c = 111.7 A.

Crystal structure of the apo forms of psi 55 tRNA pseudouridine synthase from Mycobacterium tuberculosis: a hinge at the base of the catalytic cleft.

The three-dimensional structure of the RNA-modifying enzyme, psi55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-A resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central beta-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.

The truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes.

Invasive strains of Pseudomonas aeruginosa can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of P. aeruginosa was screened to identify P. aeruginosa genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the fimV gene. A fimV mutant was unable to induce the expression of exoS, exoT and exsA genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a fimV-containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the fimV mutant was not restored by the introduction of a fimV gene alone, although it restored the twitching motility. A gene downstream of fimV, encoding a tRNA pseudouridine synthase (truA) homologue, was able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. Indeed, a truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that truA controls tRNAs that are critical for the translation of type III genes or their regulators.

Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit.

RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA. This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs. We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA. Comparison of the RNA-free and -bound forms of TruB reveals that this enzyme undergoes significant conformational changes on binding to its substrate. These conformational changes include the ordering of the "thumb loop," which binds right into the RNA hairpin loop, and a 10 degree hinge movement of the C-terminal domain. Along with the result of docking experiments performed on apo TruB, we conclude that TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA.

tRNA pseudouridine synthase I (PsiSI) catalyzes the conversion of uridine to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. PsiSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and PsiSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for PsiS.

Functional Effect of Deletion and Mutation of theEscherichia coli Ribosomal RNA and tRNA Pseudouridine Synthase RluA

The Escherichia coli gene rluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of both rluA- strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA-) either in rich or minimal medium at 24, 37, or 42 degrees C, but when both strains were grown together, a strong selection against the deletion strain was observed.