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Systematic discovery of endogenous ribonucleoprotein complexes - RNA DIF-FRAC Human HEK293T Cells Replicate2

ABSTRACT: The associated files are mass spec data from size exclusion chromatographic separations of Human HEK293T with and without RNAse A treatment.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Kevin Drew

LAB HEAD: Edward Marcotte

PROVIDER: PXD014820 | Pride | 2019-10-25

REPOSITORIES: Pride

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Systematic Discovery of Endogenous Human Ribonucleoprotein Complexes.

Mallam Anna L AL Sae-Lee Wisath W Schaub Jeffrey M JM Tu Fan F Battenhouse Anna A Jang Yu Jin YJ Kim Jonghwan J Wallingford John B JB Finkelstein Ilya J IJ Marcotte Edward M EM Drew Kevin K

Cell reports 20191001 5

RNA-binding proteins (RBPs) play essential roles in biology and are frequently associated with human disease. Although recent studies have systematically identified individual RNA-binding proteins, their higher-order assembly into ribonucleoprotein (RNP) complexes has not been systematically investigated. Here, we describe a proteomics method for systematic identification of RNP complexes in human cells. We identify 1,428 protein complexes that associate with RNA, indicating that more than 20% o ...[more]

PMID: 31665645

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Systematic discovery of endogenous ribonucleoprotein complexes - RNA DIF-FRAC Human HEK293T Cells Replicate1

Project description:The associated files are mass spec data from size exclusion chromatographic separations of Human HEK293T with and without RNAse A treatment.

2019-10-18 | PXD015406 | Pride

Systematic Identification of Protein Phosphorylation-Mediated Interactions

Project description:Protein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with less than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphates. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

2020-09-22 | PXD021422 | Pride

RNAseq analyses in HEK293T-ACE2 cells infected with SARS-CoV-2 (VOC Delta, 24hours) vs untreated cells

Project description:In this study a gene expression (i.e., RNAseq) analysis was performed in HEK293T-ACE2 cellular model upon infection with viral particle belonging to VOC Delta (MOI: 0.026) for 24 hours in order to have a global picture of the transcriptome landscape in response to early phase of infection of SARS-CoV-2 ( VOC Delta infection and to evaluate the role of Ca2+ in HEK293-ACE2 cellular model and transfer to homeostasis in SARS-COV-2 patients (by Pasqualino de Antonellis1-2* and Veronica Ferrucci 1-2* (first authors) et al. and Massimo Zollo1-2# (corresponding author). Manuscript in preparation 2022 July 15th 2022. Short title "ATP2B1 (PMCA1), regulated by FOXO3, influences susceptibility to severe COVID19".

2022-12-01 | E-MTAB-11973 | biostudies-arrayexpress

TCF/LEF dependent and independent regulation of Wnt/beta-catenin transcription

Project description:During canonical Wnt signalling the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones simultaneously carrying loss-of-function alleles of all four TCF/LEF genes. Exploiting unbiased whole transcriptome sequencing studies, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.

2018-11-22 | E-MTAB-7028 | biostudies-arrayexpress

Interaction and global proteomics reveal the role of the pluripotency factor L1TD1 in embryonal tumor cell proliferation and mobility

Project description:Pluripotency factors are known to be abnormally expressed cancer cells, which often correlate with poor prognosis. The molecular mechanisms by which pluripotency factors contribute to tumor aggressiveness are yet to be elucidated. The pluripotency marker L1TD1 interacts with other pluripotency factors and is a marker for human embryonic stem cells. In cancer, L1TD1 expression has been detected in solid tumors, including Embryonal tumors of the Central Nervous System. Previously, we reported that, in medulloblastoma, L1TD1 expression correlates with metastasis and shorter overall patient survival. Here, we use Affinity purification-mass spectrometry and global proteomics to map the L1TD1 protein interaction network and assess proteins regulated by L1TD1 expression, respectively. Interactome and global proteome analysis led to the enrichment of overlapping Biological Processes attributed to L1TD1 including Cell proliferation, Cell death and cell migration. We also used L1TD1 overexpressing tumor cell populations for in vitro validation of the omic data. L1TD1 overexpressing cells presented higher cell mobility, enhanced filopodial formation and distinct cell morphology. These cells were overall more proliferative and resistant to cisplatin treatment in vitro.

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Early response of human monocyte-derived macrophages (MDM) to HIV-1

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2016-06-10 | E-GEOD-81327 | biostudies-arrayexpress

HEK 293T cells RNA-seq data treated with 5-aza-2-deoxycytidine

Project description:DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic M-bM-^@M-^\imprintM-bM-^@M-^] of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug, and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome, and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation. mRNA profile of 5 different samples of HEK 293T cells treated with 5-aza-CdR

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Exon array analysis of two human cell lines (HEK293T Ramos B cell)

Project description:In order to compare previously generated transcriptome data by second generation sequencing (Sultan et al. Science 2008), we interrogated the human Affymetrix exon chips 1.0ST using the same source of material. Two biological replicates from HEK293T and B cell lines were hybridized on the human Affymetrix exon chips 1.0ST

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single cell RNAseq on isolated spinal cord cells before and 3 day post tail amputation in Xenopus tropicalis tadpoles at stage NF50

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2021-05-21 | E-MTAB-8839 | biostudies-arrayexpress

Inactivation of the exosome ribonuclease DIS3 triggers the pluripotency factor LIN28B, repressing let-7 miRNAs and unleashing MYC and RAS.

Project description:Somatic mutations affecting DIS3, the catalytic component of the RNA exosome, have been found in up to 18% of patients affected by the hematological cancer multiple myeloma. Here we show that DIS3 targets and degrades the pluripotency factor LIN28B. In cancer cells, DIS3 inactivation leads to enhanced LIN28B expression, thus disrupting the let-7 miRNAs tumor suppressor network and ultimately increasing protein levels of crucial oncogenes such as MYC and RAS. DIS3 represents the catalytic component of the exosome. The exosome is required for cell viability and targets several RNA species, including pre-mRNAs, pre-tRNAs, pre-rRNAs, snRNAs and snoRNAs. To gain insight on the macular wiring underlying DIS3 activity in mammalian cells, we comprehensively evaluated expression profiles, including miRNAs, in various cell lines, upon DIS3 knockdown. This series of microarray experiments contains the miRNA expression profiles of independent replicates of RPMI-8226, KMS12-BM multiple myeloma cell lines and HEK-293T cells, knocked-down with a scrambled or hDIS3 sh4 and collected 72 hours after infection. 500 nanograms of total RNA were processed using the FlashTag labeling kit, which uses a tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample. The labelled RNA was then hybridized to Affymetrix GeneChip® microRNA arrays v1.0, following the Affymetrix manufacturer's instructions.

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