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YAMAT-seq of mature tRNA pools in the bacterium Pseudomonas fluorescens SBW25 and derivatives

ABSTRACT: Mature tRNA pools were measured using an adaptation of Y-shaped Adapter-ligated MAture tRNA sequencing (YAMAT-seq) (Shigematsu et al., 2017) in nine strains derived from the model bacterium, Pseudomonas fluorescens SBW25. The aim of the experiment was to determine the effect on the mature tRNA pool of (i) removing the single-copy serCGA gene by genetic engineering, and (ii) duplicating the serTGA gene during a subsequent, compensatory evolution experiment. We found that (i) results in the loss of tRNA-Ser(CGA) from the mature tRNA pool, and (ii) results in a 2-fold higher expression of tRNA-Ser(UGA). tRNA-Ser(UGA) presumably substitutes for tRNA-Ser(CGA) by wobble base pairing to translate codon 5'-UCG-3'.

ORGANISM(S): Pseudomonas fluorescens

PROVIDER: GSE144791 | GEO | 2020/11/02

REPOSITORIES: GEO

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YAMAT-seq of mature tRNA pools in the bacterium Pseudomonas fluorescens SBW25 and derivatives.

Project description:Mature tRNA pools were measured using an adaptation of YAMAT-seq (Shigematsu et al., 2017; doi:10.1093/nar/gkx005 ) and further described in (Ayan et al., 2020; doi:10.7554/eLife.57947) in eight strains dervied from the model bacterium, Pseudomonas fluorescens SBW25. The aim of the experiment was to determine the effect on the mature tRNA pool of (i) removing multi-copy tRNA genes by genetic engineering, and (ii) duplicating tRNA genes during a subsequent, compensatory evolution experiment. We found that (i) results in a reduction in some tRNA isotypes in the mature tRNA pool, and (ii) results in an increase in a compensatory increase in their expression.

2022-12-02 | GSE196705 | GEO

tRNAs marked with CCACCA are targeted for degradation

Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included

2011-11-17 | E-GEOD-33756 | biostudies-arrayexpress

tRNAs marked with CCACCA are targeted for degradation

2011-11-17 | GSE33756 | GEO

YAMAT-seq of mature tRNA pools in the bacterium Pseudomonas fluorescens SBW25 and derivatives

Project description:YAMAT-seq of mature tRNA pools in the bacterium Pseudomonas fluorescens SBW25 and derivatives

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YAMAT-seq of mature tRNA pools in the bacterium Escherichia coli str. K-12 substr. MG1655 and derivatives

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Effects of tRNA[Ser]Sec anticodon stem loop modifications on selenoprotein expression

Project description:Expression of selenoproteins requires the co-translational incorporation of selenocysteine (Sec) in response to an in-frame UGA codon. The machinery of UGA/Sec re-coding is complex and many factors affect the hierarchy of expression among selenoproteins, including modification of tRNA[Ser]Sec. Its hyper-modification in the anticodon stem loop is influenced by selenium bioavailability, and a mutation in adenosine 37 (A37) that abrogates isopentenylation, has a profound effect on selenoprotein expression in mice. Patients with mutations in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder and hence we wondered whether mutations in TRIT1 negatively affected the expression of selenoproteins. Fibroblasts from a patient carrying a pathogenic R323Q mutation in TRIT1 in homozygosity did not show decreased selenoprotein expression, although recombinant TRIT1R323Q had significantly reduced activity in vitro towards anticodon stem-loop substrates. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Selenoprotein expression as assessed by western blotting, 75Se metabolic labeling, and ribosomal profiling was not decreased despite the general reduction of N6-isopentenyl-adenosine in tRNAs. We show that 5-methylcarboxymethylation and 2’O-methylation of U34 occur independently of isopentenylation of A37 in tRNA[Ser]Sec. Reanalyzing previously published ribosomal profiling datasets, we demonstrate that (i) failure of 5-carboxymethylation at U34 is associated with reduced expression of GPX1, but not GPX4, and that (ii) FTSJ1 is not the elusive U34-2’O-methyltransferase involved in the methylation of tRNA[Ser]Sec.

2021-11-17 | GSE183923 | GEO

Analysis of the Hfq regulon in Pseudomonas fluorescens SBW25.

Project description:Hfq is a transcriptional and translational pleiotropic regulator in several bacteria. RNA-Seq, Ribo-Seq and Proteomic analyses were carried out in the wild-type and a hfq deletion strain of Pseudomonas fluorescens SBW25 with the intention to separate the influence of Hfq on the transcript stability and translation. This submission relates to the RNA-Seq data only. RNA was extracted from two replicate cultures each of SBW25-WT and SBW25-Δhfq strains and, after removal of ribosomal RNA, subjected to RNA-Seq in an Illumina NextSeq500 machine. The resulting sequence data was analysed by mapping to the reference sequence of Pseudomonas fluorescens SBW25 as available in the Genbank accession NC_012660.

2019-02-07 | E-MTAB-5396 | biostudies-arrayexpress

METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

Project description:A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear-encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.

2022-05-19 | PXD030418 | Pride

Ribosome profiling of Pseudomonas fluorescens SBW25 and its hfq deletion mutant.

Project description:Hfq is a transcriptional and translational pleiotropic regulator in several bacteria. RNA-Seq, Ribo-Seq and Proteomic analyses were carried out in the wild-type and a hfq deletion strain of Pseudomonas fluorescens SBW25 with the intention to separate the influence of Hfq on the transcript stability and translation. This submission relates to the Ribosome profiling data only. Ribosomes were purified from MNase treated cell lysates by density gradient ultracentrifugation from two replicate cultures each of SBW25-WT and SBW25-hfq strains. RNA was extracted from the purified ribosomes and subjected to RNA-Seq in an Illumina HiSeq2000 machine. The resulting sequence data was analysed by mapping to the reference sequence of Pseudomonas fluorescens SBW25 as available in the Genbank accession NC_012660.

2017-08-24 | E-MTAB-5408 | biostudies-arrayexpress

Expression analysis of the gacS mutant of Pseudomonas fluorescens SBW25

Project description:Pseudomonas species are ubiquitous in plant-associated environments and produce an array of volatiles, enzymes and antimicrobials. The biosynthesis of many metabolites is regulated by the GacS/GacA two-component regulatory system. Transcriptome analysis of Pseudomonas fluorescens SBW25 revealed that 702 genes were differentially regulated (fold change>4, P<0.0001) in a gacS::Tn5 mutant, with 300 and 402 genes up- and down-regulated, respectively. Genes that were significantly down-regulated are involved in viscosin biosynthesis (viscABC), protease production (aprA), motility, biofilm formation, and secretory systems. Genes that were significantly up-regulated are involved in siderophore biosynthesis and oxidative stress. In contrast to previous studies with gac-mutants of other Pseudomonas species/strains, the gacS mutant of SBW25 inhibited growth of oomycete, fungal and bacterial pathogens significantly more than parental strain SBW25. A potential candidate for this enhanced antimicrobial activity was a large nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode for an 8-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis of an NRPS gene in this cluster, however, did not lead to a reduction in the antimicrobial activity of the gacS mutant. Collectively these results indicate that a mutation in the GacS/GacA regulatory system causes major transcriptional changes in P. fluorescens SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms. This expression study used total RNA recovered from four separate wild-type cultures of Pseudomonas fluorescens SBW25 and four separate cultures of the gacS mutant. Expression design was based on the updated genome sequence of Pseudomonas fluorescens SBW25, NC_012660.1 and associated plasmid pQBR0476 with nineteen 60-mer probe per gene. Each probe is replicated 3 times. The design includes random GC and other control probes.

2013-06-17 | E-GEOD-43443 | biostudies-arrayexpress

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