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Cell-specific analysis of mRNA synthesis and decay

ABSTRACT: Abstract: Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Toxoplasma gondii Homo sapiens

PROVIDER: GSE4401 | GEO | 2006/03/08

SECONDARY ACCESSION(S): PRJNA94627

REPOSITORIES: GEO

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Cell-specific analysis of mRNA synthesis and decay

Project description:This SuperSeries is composed of the following subset Series: GSE2946: Synthesis vs. Abundance, Bradyzoite Development GSE2947: Thiouracil Pulse-chase, Tachyzoites and Bradyzoites mRNA stability data GSE2948: UPRT Transgenic cells, Human Arrays Abstract: Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii. Refer to individual Series

2006-03-07 | E-GEOD-4401 | biostudies-arrayexpress

TU-tagging: cell type specific RNA isolation from intact complex tissues

Project description:We show that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil (4TU) delivery can be used to label and purify cell type specific RNA from intact complex tissues. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in any organism where UPRT can be spatially expressed, including mammals and other vertebrates. To confirm that thio-labeled RNA was from UPRT-expressing cells and demonstrate the utility of TU-tagging for cell type-specific RNA isolation, we purified TU-tagged and untagged RNA and compared them using microarrays. We chose to isolate RNA from larval glia, which are present in low number, are highly dispersed, and have a complex cell morphology, making them one of the most difficult cell types to isolate by dissection or dissociation methods. We used reversed polarity (repo)-GAL4 to drive expression of UAS-UPRT specifically in glial cells of the larval brain. We purified TU-tagged and untagged RNA from 72-96h larval brains, and hybridized them to custom Agilent microarrays. We performed two biological replicate experiments.

2010-06-19 | E-GEOD-15445 | biostudies-arrayexpress

plant rabt nÂ°1-Plant RABT nÂ°1

Project description:adt12-01_uprt - plant rabt nÂ°1 - Impact of RNA labeling by Plant RABT on plant transcriptome - Determine the incidence of RNA marking by the RABT method Plantation on the plants transcriptome. In several studies Clearly et al. have described a method referred as "4TU tagging" which can be use to study mRNA synthesis and decay either in a mixed population of cells or in a specific cell type (Cleary, Meiering et al. 2005, Zeiner, Cleary et al. 2008, Miller, Robinson et al. 2009, Rabani et al., 2011). Through the specific cell type expression in drosophila and mammalian cells of theToxoplasma gondii uracil PhosphoribosylTransferase activity and the metabolization of the uracil analog 4-thiouracil (4 TU), mRNA were selectively tagged, purified and used in microarray based analysis. 6 dye-swap - treated vs untreated comparison

2014-09-10 | E-GEOD-60532 | biostudies-arrayexpress

plant rabt n°1-Plant RABT n°1

Project description:adt12-01_uprt - plant rabt n°1 - Impact of RNA labeling by Plant RABT on plant transcriptome - Determine the incidence of RNA marking by the RABT method Plantation on the plants transcriptome. In several studies Clearly et al. have described a method referred as "4TU tagging" which can be use to study mRNA synthesis and decay either in a mixed population of cells or in a specific cell type (Cleary, Meiering et al. 2005, Zeiner, Cleary et al. 2008, Miller, Robinson et al. 2009, Rabani et al., 2011). Through the specific cell type expression in drosophila and mammalian cells of theToxoplasma gondii uracil PhosphoribosylTransferase activity and the metabolization of the uracil analog 4-thiouracil (4 TU), mRNA were selectively tagged, purified and used in microarray based analysis.

2014-09-10 | GSE60532 | GEO

TU-tagging: cell type specific RNA isolation from intact complex tissues

2009-04-24 | GSE15445 | GEO

To investigate 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT). [Adult SLAM-Itseq]

Project description:To understand 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT), we exposed adult lf:UPRT zebrafish (whereby animals have hepatocyte-specific uracil phosphoribosyltransferase expression) with 4-TU for 1, 3, 6 and 9 hours. We then dissected adult livers for SLAM-ITseq to determine the optimal 4-TU labelling time in our SLAM-ITseq workflow to allow us to study hepatocyte-specific nascent transcriptional changes.

2024-01-13 | GSE230830 | GEO

A Snapshot of RNA Expression in a Single Segment of the Kidney Reveals Stimulus Specific Responses

Project description:The identification of acute kidney disease at the time of patient encounter remains a central problem in clinical medicine. A single analyte, the serum creatinine (sCr) is currently in use as a surrogate for tubular, vascular, or interstitial cellular damage. Nonetheless the sCr test is not specific to kidney injury, but rather might reflect physiological responses to a the primary disease in a distant organ. In addition, while cellular events occur over minutes or hours, sCr requires 24 hours or more to reach a worrisome clinical threshold or demonstrate further deterioration of tissue function and architecture. Here we have adapted the method of Gay et al, Genes Dev. 2013 27(1):98-115. doi: 10.1101/gad.205278.112. to allow cell specific labelling of RNA at the time of our choosing after an injury. The technique involves the cell specific expression of a uracil phosphoribosyltransferase (Uprt) and the subsequent purification of 4-thio-uracil labelled nascent RNAs. Using this technique focused on the collecting duct, we found that a model of volume depletion and a model of ischemic damage, both of which raise the sCr do not activate the same cohort of genes nor the same pathways of gene expression. Hence, a snapshot of newly synthesized RNA reveals the complexity subsumed by diagnostic classifications dependent on sCr (called 'AKI'). We suggest that the Uprt technique will allow characterization of each cell type in the nephron at multiple time points after the onset of injury. These data will replace our current diagnostic strategies with Precision Medicine.

2022-02-04 | GSE99084 | GEO

Mouse bone marrow-derived macrophages infected with T. gondii (Type I, RH strain) for 6 hr

Project description:Toxoplasma gondii is a globally distributed parasite pathogen that infects virtually all warm-blooded animals. A hallmark of immunity to acute infection is the production of IFN-γ and IL-12, followed by a protective T cell response that is critical for parasite control. Naïve T cell activation requires both TCR stimulation and the engagement of costimulatory receptors. Because of their important function in activating T cells, the expression of co-stimulatory ligands is believed to be under tight control. The molecular mechanisms governing their induction during microbial stimulation, however, are not well understood. We found that all three strains of T. gondii (Types I, II, and III) up-regulated the expression of B7-2, but not B7-1, on the surface of mouse bone marrow-derived macrophages. This induction occurred at the transcriptional level, required active parasite invasion, and was not dependent on MyD88 or TRIF. Genome-wide transcriptional analysis comparing infected and uninfected macrophages revealed the activation of MAPK signaling in infected cells. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on JNK, but not ERK or p38 signaling. We also observed that T. gondii-induced B7-2 expression on human peripheral blood monocytes is dependent on JNK signaling, indicating that a common mechanism of B7-2 regulation by T. gondii may exist in both humans and mice. We used microarrays to examine genome-wide changes in host cell gene expression during T. gondii infection at an early time point (6 hpi). BMdM were mock-treated or infected with T. gondii (Type I, RH strain) at an MOI of 2 and cultured for 6 hours. Biological triplicates were performed. Total RNA was harvested and used for cDNA synthesis, labeling, and hybridization to Affymetrix mouse 430 2.0 expression arrays.

2011-12-31 | E-GEOD-27972 | biostudies-arrayexpress

Comparative ribosome profiling uncovers a dominant role for translational control in Toxoplasma gondii

Project description:The lytic cycle of the protozoan parasite Toxoplasma gondii, which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in Toxoplasma during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of Toxoplasma gondii are lacking. We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular Toxoplasma gondii parasites to investigate translational control during the lytic cycle. Results: Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames.

2017-11-23 | GSE99395 | GEO

C. elegans L4 hypodermal SLAM-ITseq

Project description:To adapt SLAM-ITseq for Caenorhabditis elegans, we inserted a codon-optimized version of the Toxoplasma gondii UPRT gene downstream of a FRT-flanked mCh::his-58 cassette and under control of the strong and ubiquitous eft-3 promoter. We introduced a single copy of this construct into the C. elegans genome and crossed the resulting line with a dpy-7p::FLP driver. FLP-dependent expression of UPRT was confirmed by Western blotting (strain BN1190). As a proof of concept, we exposed animals to 4-TU from L3 to L4 stage and purified total RNA. RNA from animals without FLP expression was used to control for UPRT-independent labeling strain BN1158). RNA was alkylated with iodoacetamide, which leads to a T>C conversion of 4-TU-labeled positions during cDNA synthesis, and subjected to deep sequencing (see Materials and Methods). We confirmed that the presence of FLP and UPRT did not affect gene expression. Comparing the T>C conversion rate per gene revealed a significant increase in nematodes expressing UPRT in the hypodermis. Next, we performed a beta-binominal test to identify genes that were significantly labelled across all replicas, which retrieved 197 genes (p<0.003; FDR<0.1 after adjustment for multiple comparisons). Original SLAM-ITseq reference: Matsushima W, Herzog VA, Neumann T, Gapp K, Zuber J, Ameres SL, Miska EA (2018) SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development 145 (13). doi:10.1242/dev.164640

2022-12-31 | GSE192419 | GEO

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