Comparative tRNA-seq for rapid profiling of tRNA modifications in a non-model organism
Ontology highlight
ABSTRACT:
ORGANISM(S): Vibrio cholerae Escherichia coli
PROVIDER: GSE147614 | GEO | 2020/05/15
REPOSITORIES: GEO
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Project description:RNA modifications have a substantial impact on tRNA function. While modifications in the anticodon loop play an important role in translational fidelity, modifications in the tRNA core influence tRNA structural stability. In bacteria, tRNA modifications play important roles in the stress response and expression of virulence factors. While tRNA modifications are well characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa is lacking. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which we used to identify tRNA modifications in P. aeruginosa. We determined conservation for many modifications between the two organisms. We also identified potential sites of tRNA modification in P. aeruginosa tRNA that are not present in E. coli. One of these sites is found at the same positions as acacp3U, a modification previously identified in Vibrio cholerae. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which may play roles in determining virulence and pathogenicity.
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Project description:Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from mature tRNA with a simple-to-use computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in multiple eukaryotes and is applicable to any organism with a known genome.
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Project description:In marine Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome via homologous recombination. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in two Vibrio campbellii strains, DS40M4 and NBRC 15631, enables high frequencies of natural transformation. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4. This result is in contrast to Vibrio cholerae that requires the quorum-sensing regulator HapR to activate the competence regulator QstR. However, similar to V. cholerae, QstR is necessary for transformation in DS40M4. To investigate the difference in transformation frequencies between BB120 and DS40M4, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. BB120 encodes homologs of all known competence genes, but most of these genes were not induced by ectopic expression of TfoX, which likely accounts for the non-functional natural transformation in this strain. Comparison of transformation frequencies among Vibrio species indicates a wide disparity among even closely related strains, with Vibrio vulnificus having the lowest functional transformation frequency. We show that ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus.
2019-11-14 | GSE136941 | GEO
Project description:Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq). Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species.
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Project description:N1-methyl adenosine (m1A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red‑m1A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-ATT and 5.8S rRNA.
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Project description:Identification of post translational modifications on Vibrio cholerae protein VesB (from purified VesB and culture supernatant) using in-gel digestion with trypsin, LC-MS/MS, database searching.
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Project description:Investigation of whole genome gene expression level changes in a Vibrio cholerae O395N1 delta-nqrA-F mutant, compared to the wild-type strain. Total RNA recovered from wild-type cultures of VIbrio cholerae O395N1 and its nqrA-F mutant strain. Each chip measures the expression level of 3,835 genes from Vibrio cholerae O1 biovar eltor str. N16961 with twenty average probes/gene, with five-fold technical redundancy.
2014-04-01 | E-GEOD-56387 | biostudies-arrayexpress
Project description:Abstract: tRNAs are highly modified in the elbow region and harbor 5-methyluridine at position 54 and pseudouridine at position 55 in the T arm, which are generated by the enzymes TrmA and TruB, respectively. Although all elongator tRNAs contain these modifications across all domains of life, the cellular relevance of these modifications and their corresponding modifying enzymes remains elusive. In addition to modifying every tRNA, Escherichia coli TrmA and TruB have both been shown to fold tRNA independently of its modification activity acting as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wildtype. To identify how TrmA and TruB contribute to cellular fitness, we have systematically assessed the effects of deleting trmA and/or trmB in E. coli. Since tRNA folding is a pre-requisite for tRNA aminoacylation, we determined cellular aminoacylation levels revealing a global decrease in aminoacylation for all tRNAs in ΔtrmA and ΔtruB. Moreover, the absence of 5-methyluridine 54 or pseudouridine 55 alters tRNA modification at other positions: whereas acp3U47 is decreased, thiouridine levels are increased. Understanding the importance of TrmA and TruB for tRNA aminoacylation and modification, we then analyzed how these global tRNA changes in ΔtrmA and ΔtruB strains affect translation. Whereas global protein synthesis is not significantly changed in ΔtrmA and ΔtruB, the abundances of many specific proteins are altered, and transcriptomics experiments suggest that the dysregulation of many proteins is controlled at the translational level. In conclusion, we demonstrate that universally conserved modifications of the tRNA elbow are critical for global tRNA function by enhancing other tRNA modifications, tRNA folding, tRNA aminoacylation and translation of specific genes thereby contributing to cellular fitness.
2023-05-31 | GSE233667 | GEO
Project description:Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs (in even simple systems) is obliquely known. Limited analysis suggests tRNA levels change during proliferation, differentiation, cancer, and neurodegeneration; possibly mediating changes in translation efficiency and mRNA stability. A major limitation for the field has been the ability to subject tRNA pools to high-throughput analysis as they are highly structured, modified, and of high sequence similarity. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), an easily implemented high-throughput technique to monitor tRNA abundance and sequence variants (possibly due to RNA modifications). With QuantM-tRNA seq we provide a comprehensive analysis of the tRNA transcriptome from distinct mammalian tissues. We observe dramatic distinctions in isodecoder expression and likely RNA modifications between unique tissues with a particularly strong signature within the central nervous system. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar. These findings suggest that anticodon pools are buffered via an unknown mechanism to achieve uniform decoding throughout the body.
2020-08-04 | GSE141436 | GEO
Project description:We exposed wild-type Vibrio cholerae E7496, multiple Vibrio cholerae virulence factor deleted genes with intact hemolysin A gene [CVD109] and without hemolysin A gene [CVD110] in E7946, and E.coli OP50 to wild-type C.elegans N2 for 18 hours. We used microarrays to detail the global gene expression and identified distinct classes of up-regulated and down-regulated genes during this process. C. elegans were exposed to Vibrio cholerae and E.coli then hybridization on Affymetrix microarray chips.
2011-12-02 | E-GEOD-34026 | biostudies-arrayexpress