Change of the mRNA level upon Puromycin treatment
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE85794 | GEO | 2025/07/21
REPOSITORIES: GEO
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Project description:Puromycin and its derivative O-propargyl puromycin (OPP) enable detection of nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here we have used puromycin N-acetyltransferase (PAT), for cell-specific metabolic labelling with puromycin or OPP. This approach, which we named Puromycin Inactivation for Cell-Selective proteome Labelling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing PAT and detection of translation in other cell types. We performed two mass spectrometry experiments using cell-specific expression of PAT, metabolic labeling of cells with OPP and streptavidin pulldown of biotinylated de novo synthesized proteins. In the first proof-of-principle experiment (Part I) we mixed human (HEK293) and mouse (Neuro2a) cells and labeled them with OPP, comparing this with another sample where HEK293 cells expressed PAT. We show specific reduction of human-specific peptides in de novo synthesized proteome in HEK-PAT cells. In the second experiment (Part II), we used a primary co-culture of rat cortical neurons (expressing PAT in some samples) and glia, performed OPP labeling and pulldown of de novo synthesized proteins.
2023-10-24 | PXD043498 | Pride
Project description:Puromycin and its derivative O-propargyl puromycin (OPP) enable detection of nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here we have used puromycin N-acetyltransferase (PAT), for cell-specific metabolic labelling with puromycin or OPP. This approach, which we named Puromycin Inactivation for Cell-Selective proteome Labelling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing PAT and detection of translation in other cell types. We performed two mass spectrometry experiments using cell-specific expression of PAT, metabolic labeling of cells with OPP and streptavidin pulldown of biotinylated de novo synthesized proteins. In the first proof-of-principle experiment (part I) we mixed human (HEK293) and mouse (Neuro2a) cells and labeled them with OPP, comparing this with another sample where HEK293 cells expressed PAT. We show specific reduction of human-specific peptides in de novo synthesized proteome in HEK-PAT cells. In the second experiment (part II), we used a primary co-culture of rat cortical neurons (expressing PAT in some samples) and glia, performed OPP labeling and pulldown of de novo synthesized proteins.
2023-10-24 | PXD043494 | Pride
Project description:Evaluation and comparison of biological responses of rats and gene expression responses of the kidney in rats exposed to different doses of Puromycin [CAS:53-79-2;CHEBI:17939]
2004-09-01 | E-TOXM-12 | biostudies-arrayexpress
Project description:Mast cell leukemia is a rare and aggressive disease, predominantly with KIT D816V mutation. With poor response to conventional poly-chemotherapy, mast cell leukemia responded to the midostaurin treatment with a 50% overall response rate (ORR), but complete remission rate is approximately 0%. Therefore, the potential mechanisms of midostaurin resistance and the exact impacts of midostaurin on both gene expression profile and mast cell leukemia microenvironment in vivo are essential for design tailored combination therapy targeting both the tumor cells and the tumor microenvironment. Here we report a 59-year-old male mast cell leukemia patient with KIT F522C mutation treated with midostaurin. Single-cell sequencing of peripheral blood and whole exome sequencing (WES) of bone marrow were performed before and 10 months after midostaurin treatment. In accordance with the clinical response, compared to the pretreatment aberration, the decline of mast cells and increase of T-, NK, B-cells in peripheral blood, and the decrease of the KIT F522C mutation burden in bone marrow were observed. Meanwhile, the emergence of RUNX1 mutation, upregulations of genes expression (RPS27A, RPS6, UBA52, RACK1) on tumor cells, and increased frequencies of T and NK cells with TIGIT, CTLA4, and LAG3 expression were observed after midostaurin treatment, predicting the disease progression of this patient. As far as we know, this is the first case reporting the clinical, immunological, and molecular changes in mast cell leukemia patients before and after midostaurin treatment, illustrating the in vivo mechanisms of midostaurin resistance in mast cell leukemia, providing important clues to develop a sequential option to circumvent tumor progression after targeting oncogene addiction and prolong patients’ survival.
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Project description:Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. We combine quantitative proteomics, pulse labeling with puromycin, and stable isotope-labeled amino acids to analyze thousands of NPCs. Our approach enables global analyses of translational responses and co-translational modifications, providing a framework for dissecting co-translational regulations proteome-wide. HeLa cells were pre-incubated with either DMSO or a CK2 inhibitor CX4945 20 for 10 min, and subsequently, cells were pulse-labeled with puromycin and SILAC amino acids for 2 hr in the presence of DMSO or CX4945, and immunoprecipitation of puromycylated proteins was performed in triplicates.
2022-02-09 | PXD024080 | JPOST Repository
Project description:Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.
2019-02-18 | PXD012727 | Pride
Project description:We use explicit-solvent, molecular dynamics simulations to study the change in polar properties of a solvent-accessible surface for proteins undergoing phosphorylation. We analyze eight different pairs of proteins representing different structural classes in native and phosphorylated states and estimate the polarity of their surface using the molecular hydrophobicity potential approach. Whereas the phosphorylation-induced hydrophobicity change in the vicinity of phosphosites does not vary strongly among the studied proteins, the equivalent change for complete proteins covers a surprisingly wide range of effects including even an increase in the overall hydrophobicity in some cases. Importantly, the observed changes are strongly related to electrostatic properties of proteins, such as the net charge per residue, the distribution of charged side-chain contacts, and the isoelectric point. These features predefine the level of surface hydrophobicity change upon phosphorylation and may thus contribute to the phosphorylation-induced alteration of the interactions between a protein and its environment.
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Project description:Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.
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Project description:We demonstrate that Myodonta-clade-specific 4.5S RNAH (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of multiple genes containing the antisense insertion of SINE B1 The depletion of endogenous 4.5SH with antisense oligonucleotides changes the subcellular distribution of these target transcripts containing the antisense SINE B1 insertions.
2015-09-30 | GSE57264 | GEO
Project description:We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA-miRNA-Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.
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