A commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA).
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ABSTRACT:
SUBMITTER: Prifti DK
PROVIDER: S-EPMC9249204 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.
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Project description:Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have placed RIC-8 in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the Galphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of Galphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and Galphai regulate a similar process. We find that mammalian Ric-8A dissociates Galphai-GDP/LGN/NuMA complexes catalytically, releasing activated Galphai-GTP in vitro. Ric-8A-stimulated activation of Galphai caused concomitant liberation of NuMA from LGN. We conclude that Ric-8A efficiently utilizes GoLoco/Galphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division.
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Project description:The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.
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Project description:Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.
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Project description:The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.
2023-05-29 | GSE227893 | GEO
Project description:BackgroundNuclear mitotic apparatus protein 1 (NuMA1) had been reported to produce three groups of isoforms categorized as long, middle, and short groups, of which short NuMA displayed distinct localization patterns compared to long and middle isoforms. However, the function of short NuMA was not clear in the progress of cancer formation. This study aimed to unveil the role of short NuMA in cancer pathogenesis.MethodsThe expression levels of short isoforms were explored in paired gastric carcinoma (GC) samples and different cell lines. Furthermore, the short isoform behaved as a putative tumor suppressor based on cell proliferation and cell colony formation assays. Pull-down assay and whole-genome gene expression analysis were carried out to search candidate interaction partners of short NuMA.ResultsThe expression of short NuMA was highly expressed in S and G2 phases of the cell cycle; compared with nontumor tissues, short NuMA downregulated in nine GCs (GC1 [0.131, P = 5 × 10-4]; GC2 [0.316, P = 3 × 10-5]; GC3 [0.111, P = 6 × 10-4]; GC4 [0.456, P = 0.011]; GC5 [0.474, P = 0.001]; GC6 [0.311, P = 0.004]; GC7 [0.28, P = 3 × 10-5]; GC8 [0.298, P = 0.007]; and GC9 [0.344, P = 0.002]). Besides, high expression of short NuMA significantly inhibits cell growth (2.43 × 105 vs. 2.97 × 105, P = 0.0029) and cell clone information in vitro (70 vs. 2, P = 1.67 × 10-45). Short NuMA could bind with alpha-actinin-4 (ACTN4), a putative tumor promoting gene. Overexpression of short NuMA could tremendously decrease the expression of MYB proto-oncogene like 2 (MYBL2) of about 92-fold, which played an important role in the cell cycles.ConclusionsShort isoform of NuMA might be functioned as a putative role of tumor suppressor. Further studies should be made to illuminate the relationship between ACTN4, MYBL2, and tumor progression.
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| S-EPMC3349707 | biostudies-literature
Project description:The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.
2023-05-29 | GSE227892 | GEO
Project description:The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.
2023-05-29 | GSE227890 | GEO