8 research outputs found

    Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling

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    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level

    Secondary structures in 5’UTR correlate with translational repression of target genes.

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    <p>(<b>A</b>) A hairpin structure in the <i>CD69</i> 5’UTR. (<b>B</b>) Ribosome accumulation in <i>CD69</i> 5’UTR correlated with miR-17~92 family miRNA expression levels. Note that the hairpin structure co-localizes with the ribosome footprint peak in the <i>CD69</i> 5’UTR. <i>Actb</i> was used as control. (<b>C</b>) Deletion of the miR-17~92 family miRNAs shifted <i>CD69</i> mRNA from light to heavy polysomes. (<b>D</b>) Increased <i>CD69</i> expression in TKO B cells was mainly due to translation de-repression. Experiments in <b>B-D</b> were performed with 25.5h activated B cells.</p

    Ribosome accumulation in 5’UTR correlates with translational repression of target genes.

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    <p>(<b>A-C</b>) Ribosome accumulation in 5’UTRs of ribo-upregulated TKO targets in WT B cells (<b>A</b>), ribo-downregulated TG targets in TG B cells (<b>B</b>), but not in 5’UTRs of other targets (<b>C</b>). Ribosome occupancy in 5’UTR was normalized to the overall ribosome footprint abundance of the same gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.ref096" target="_blank">96</a>]. The first nucleotide of start codon is set as position 0 (grey dashed line). (<b>D</b>) Inverse correlation between ribosome occupancy in 5’UTR and the overall ribosome density on target mRNA in WT B cells. (<b>E</b>) High GC content in 5’UTRs of ribo-upregulated TKO targets.</p

    Target genes exhibit different sensitivity to miR-17~92 expression level changes.

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    <p><b>(A)</b> Minimal overlap between ribo-upregulated TKO targets and ribo-downregulated TG targets. <b>(B-D)</b> The responses of ribosome density of ribo-upregulated TKO targets <b>(B)</b> and ribo-downregulated TG targets <b>(C)</b> to three miR-17~92 expression levels. Translated genes lacking miR-17~92 binding sites were used as control <b>(D)</b>. Colored bars indicate median values and error bars represent interquartile ranges. Each dot represents relative ribosome density of a unique gene. Numbers indicate p-values. <b>(E)</b> Different sensitivity of individual target genes to miR-17~92 expression level changes. Protein levels were determined by immunoblot and normalized to β-Actin (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s011" target="_blank">S11</a> Figs</b>). Target gene protein levels in WT B cells were arbitrarily set as 1.0 (n≥4). Vertical lines indicate error bars. <b>(F)</b> Relative mRNA levels of individual target genes in TKO, WT and TG B cells as determined by microarray (n = 3). <b>(G)</b> A hypothetical curve depicting target gene protein level change as a function of miRNA concentration. For a miRNA-target mRNA interaction in a given cellular context, there are a threshold level and a saturation level of miRNA concentration. miRNA suppresses target gene expression in a dose-dependent manner when miRNA concentration is between the threshold and saturation levels. Suppression does not occur when miRNA concentration is below the threshold level, while suppression reaches a maximal when miRNA concentration is above the saturation level. <b>(H)</b> The hypothetical response curves of group1, group2 and group3 target genes to miR-17~92 expression level changes. Note that the difference in amplitude for individual target genes is not depicted in this graph.</p

    Quantification of miR-17~92 miRNAs and binding sites in primary B cells.

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    <p>(<b>A,B</b>) Quantitative Northern blot to determine miR-17~92 miRNA copy numbers. Indicated amounts of synthetic miR-17, miR-18a, miR-19b and miR-92 were added to naïve and activated TKO B cells before RNA extraction. The copy numbers of each miRNA subfamily were determined by Northern blot comparing WT B cells and TKO B cells with graded amounts of spike-in synthetic miRNAs, using a mixture of probes corresponding to all members of a miRNA subfamily (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s029" target="_blank">S7 Table</a></b>). Naïve B cells were activated with LPS and IL-4 for indicated amounts of time (h, hour). (<b>C-E</b>) Summary of miR-17~92 family miRNA copy numbers (<b>C</b>), conserved miR-17~92 family miRNA binding sites (<b>D</b>) (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s030" target="_blank">S8 Table</a></b>), and ratios of conserved miR-17~92 family miRNA binding sites to miRNAs (<b>E</b>) in naïve and activated B cells.</p

    Regulation of target gene sensitivity to miRNA suppression by 5’UTR.

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    <p>(<b>A</b>) An engineered psiCheck2 vector (psiCheck-2-pd) for investigating the effect of 5’UTR and 3’UTR on reporter gene expression. TSS, transcription start site. (<b>B</b>) Experimental scheme of reporter assays in primary B cells. FACS plots show electroporation efficiency using a GFP-expressing plasmid. (<b>C,D</b>) Dual luciferase reporter assay to determine the effect of 5’UTR and 3’UTR on the reporter gene protein (luciferase activity) (<b>C</b>) and mRNA (qRT-PCR) levels (<b>D</b>). Closed and open circles indicate reporters with wild-type (wt) and mutated (mut) <i>CD69</i> 3’UTR, respectively. A comparison of renilla luciferase activity normalized to firefly luciferase activity (hRluc/Fluc) between psiCheck-2-pd containing mut and wt <i>CD69</i> 3’UTR reveals the sensitivity of the renilla luciferase mRNA (hRluc) to miR-17~92-mediated suppression. Results of normalized hRlcu/Fluc (n = 10) are from three independent experiments. Each experiment contained 3–4 replicates.</p

    The impact of miR-17~92 on target gene mRNA and protein levels.

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    <p>(<b>A</b>) The distribution of mRNA abundance in naïve and activated B cells as determined by ERCC-RNA-seq analysis. Numbers in parenthesis represent the number of unique genes significantly transcribed (greater than 0.5 copy per cell). Y-axis (counts) indicates the number of genes of a given abundance (X-axis, bin size = 0.2). (<b>B,C</b>) Microarray analysis of TKO, WT, and TG B cells. Numbers in parenthesis indicate the numbers of transcribed genes and transcribed miR-17~92 targets analyzed by microarray. (<b>D</b>) The protein and mRNA levels of 13 target genes showing reduced protein levels in 25.5h activated TG B cells as determined by Immunoblot (n = 5). mRNA levels were determined by qRT-PCR and microarray (n = 3). Target gene expression levels were normalized to β-Actin, and their relative expression in WT naïve B cells was arbitrarily set as 1.0.</p

    Transgenic miR-17~92 expression shifts target mRNAs from heavy to light polysomes.

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    <p>(<b>A</b>) A representative polysome profile of activated B cells, from two biological replicates for each genotype. Numbers inside the graph indicate the number of ribosomes associated with mRNA. (<b>B</b>) Distribution of miR-21 and miR-17~92 in the sucrose gradient in WT B cells. (<b>C</b>) Distribution of miR-17~92 target mRNAs in the sucrose gradient in WT and TG B cells. β-Actin mRNA (<i>Actb</i>) is enriched in heavy polysome fractions and is used as an internal control.</p
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