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RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes to identify binding sites for the tetracycline analogs Col-3 and doxycycline on the human ribosome

ABSTRACT: Following the identification of the 80S ribosome as a putative target of the tetracycline analogs Col3 and doxycycline, we next sought to identify the precise binding sites for these tetracyclines within the ribonucleoprotein complex. We incorporated dual bioorthogonal handles into tetracycline-based probes, containing both a photoactive diazirine to enable direct probe crosslinking to the human ribosome and an azide handle to allow selective enrichment of crosslinked biomolecules via copper-free click chemistry. The Col-3 and doxycycline probes were each incubated with A375 cells, followed by irradiation at 365 nm to induce photolysis of the diazirine moiety and subsequent crosslinking to adjacent ribosomal components. Pulldown and RNA-Seq of the crosslinked RNAs from our experiments were used to identify enrichment of reverse transcription (RT) stops at ribosomal RNA sites caused by local crosslinking of our probes. This RNA-Seq based RT stop enrichment analysis was compared to results using an non-specific aniline control probe and untreated controls.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Jonathan Mortison

PROVIDER: E-MTAB-7147 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes, with and without competition by parent tetracyclines, to show specific binding of the tetracycline analogs Col-3 and doxycycline on rRNA substructures of the human ribosome

Project description:Upon finding that the tetracyclines COL-3 and doxycycline target unique rRNA substructures of the 80S ribosome (E-MTAB-7147), we confirmed these putative ribosomal binding sites by showing that tetracycline-based crosslinking probes are specifically competed from these rRNA structures by their parent drugs. In short, we incorporated dual bioorthogonal handles into tetracycline-based probes, containing both a photoactive diazirine to enable direct probe crosslinking to the human ribosome and an azide handle to allow selective enrichment of crosslinked biomolecules via copper-free ‘click’ chemistry. The COL-3 and doxycycline probes were each incubated with A375 cells, followed by irradiation at 365 nm to induce photolysis of the diazirine moiety and subsequent crosslinking to adjacent ribosomal components. Pulldown and RNA-Seq of the crosslinked RNAs from our experiments were used to identify enrichment of reverse transcription (RT) stops at ribosomal RNA sites caused by local crosslinking of our probes. In these experiments, UV crosslinking was preformed with the COL-3 and doxycycline probe compounds in the presence and absence of free COL-3 and doxycycline (i.e., parent tetracycline molecules lacking the bifunctional diazirine-azide moiety), which were used as competitors to specifically diminish crosslinking to the ribosomal RNA. Crosslinking to ribosomal RNA was determined using RNA-Seq based RT stop enrichment, which was was also compared to inactive tetracycline probes containing a modified bifunctional linker, a non-specific aniline probe, and untreated controls.

2018-08-31 | E-MTAB-7161 | biostudies-arrayexpress

RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes to identify binding sites for the tetracycline analogs Col-3 and doxycycline on the human ribosome

Project description:RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes to identify binding sites for the tetracycline analogs Col-3 and doxycycline on the human ribosome

| PRJEB28264 | ENA

Expression data in HTETOP cells following tetracycline or dexrazoxane treatment

2009-02-19 | GSE14886 | GEO

Project description:RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes, with and without competition by parent tetracyclines, to show specific binding of the tetracycline analogs Col-3 and doxycycline on rRNA substructures of the human ribosome

| PRJEB28296 | ENA

Expression data in HTETOP cells following tetracycline or dexrazoxane treatment

Project description:HTETOP cells, derived from the human fibrosarcoma cell line HT1080, express human topoisomearse II α (TOP2A) exclusively from a tetracycline (TET)-regulated transgene, we used HTETOP cells to differentiate between TOP2A-dependent and –independent apoptotic effects of doxorubicin and dexrazoxane. We used microarrays to detect global transcriptional changes in HTETOP cells following tetracycline doxycycline or dexrazoxane treatment. Keywords: Drug treatment comparison HTETOP cells were treated with 1 µg/ml doxycycline to inhibit TOP2A expression or vehicle for 24 hours, doxycycline-induced trascriptional changes were identified by comparison of the two groups. HTETOP cells were treated with 100 µM dexrazoxane to inhibit TOP2A activity or vehicle for 24 hours, global trascriptional changes were identified by comparison of the two groups.

2009-02-19 | E-GEOD-14886 | biostudies-arrayexpress

The study of EF4 mediated gene expression under tetracycline stress in two Escherichia coli strains

Project description:Purpose: We use the gene expression data to estimate the effects of tetracycline on gene expression and average ribosome density. Methods: The mRNAs were extracted with TRIzol reagent. The mRNAs were fragmented into 280 bp and the sequencing process was conducted on HiSeq 2500 platform. We use cutadapt, bowtie2, Plastid and DEseq2 software to analyze the expression levels of genes in two Escherichia coli strains. Results: The gene expression in EF4 knockout Escherichia coli strain was similar with BW25113 strain under normal condition. Under tetracycline treatment, many genes' expression were differentially regulated. Interestingly, we found that the gene expression of ribosomal proteins was up-regulated in WT strain comparing with EF4 knockout E. coli strain. Conclusions: Our results suggest that EF4 affects the average ribosome density and global gene expression in two Escherichia coli strain under tetracycline treatment.

2021-01-09 | GSE109025 | GEO

Tetracycline-treated Litomosoides sigmodontis vs. untreated control Litomosoides sigmodontis

Project description:The filarial nematodes Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause elephantiasis, dermatitis and blindness, resulting in severe morbidity in developing countries. 1.3 billion people are at risk of infection. Targeting the essential Wolbachia endobacteria of filarial nematodes with doxycycline has proven to be an effective therapy, resulting in a block in embryogenesis and worm development, and macrofilaricidal effects. However, doxycycline is contraindicated for a large portion of the at-risk population. To identify new targets for anti-wolbachial therapy, understanding the molecular basis of the Wolbachia-filaria symbiosis is required. We performed cross-species hybridization by using the Brugia malayi microarray to identify differentially expressed genes in the rodent filaria Litomosoides sigmodontis after depletion of Wolbachia which therefore might have a role in symbiosis. Female adult Litomosoides sigmodontis from patent infections were treated with tetracycline to deplete endosymbiotic Wolbachia bacteria. RNA from tetracycline-treated Litomosoides sigmodontis was compared to untreated age-matched control worms. This experiment was performed for three different timepoints: day 6, 15 and 36 of tetracycline treatment. One biological replicate was performed each with two technical replicates (dye-flip replicates).

2010-04-07 | E-GEOD-20976 | biostudies-arrayexpress

Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes

Project description:Purpose: We use the ribosome profiling protocol to understand EF4 mediated translation events. Methods: We used ribosome profiling data to analyze by plastid software. The ribosome were purified by sucrose gradient separation. Results: Using sequencing data, we found that EF4 mediates the 1-nucleotide conformational change of ribosomes. We also found that many genes involved in translation after tetracycline treatment. Finally, we found that EF4 stalls the elongating ribosomes globally. Conclusions: Our results suggest that EF4 mediates both 30S biogenesis and translation elongation process, in particular, EF4 stalls the elongating ribosomes globally.

2018-05-14 | GSE106448 | GEO

Acute expression of cyclin D1 in mouse mammary gland using the tetracycline system

Project description:Analysis of mammary glands from tet-inducible (rtTA) transgenic mice expressing cyclin D1 (Ccnd1). MMTV-rtTA transgenic mice (MMTV-Mouse Mammary Tumor Virus promoter) were cross-mated to cyclin D1 transgenic mice under the control of the tet operon. 8-week-old tetracycline-inducible cyclin D1/rtTA bi-transgenic pregnant female mice (12 days postcoitus) were treated with doxycycline through drinking water supplementation at a final concentration of 2 mg/ml. Control mice were rtTA transgenics alone and were treated in the same manner. After 7 days of doxycycline treatment, the mice were sacrificed and mammary glands taken for RNA isolation. Results provide insight into the in vivo gene expression pattern regulated by cyclin D1 through acute induction.

2012-02-10 | GSE35076 | GEO

WblC- and tetracycline-dependent shifts in the composition of Streptomyces coelicolor ribosomal proteome

Project description:WblC, also known as WhiB7, is a widely conserved WhiB-like transcription factor in actinomycetes that activates transcription of many targets upon antibiotic challenge to bring about intrinsic resistance to a wide range of translation-targeting antibiotics. As we found that WblC controls many genes involved in translation and that WblC promotes translation rate upon antibiotic stress in the model actinomycetes Streptomyces coelicolor, we speculated that WblC might alter the protein composition of ribosome during antibiotic stress. To test this, we prepared 70S ribosome fraction from wild-type S. coelicolor cells untreated or treated with tetracycline and ΔwblC mutant treated with tetracycline, and then compared the protein compositions of each 70S samples by mass spectrometric quantification.

2020-05-27 | PXD015538 | Pride

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