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Enhanced interactome capture (eRIC) of steady-state and arsenite stressed HuH7 cell cytoplasmic and nuclear fractions

ABSTRACT: HuH7 cells were grown under steady-state conditions or exposed 100 ÂµM Na-arsenite for 1h. Subsequently, the cells were irradiated or not with 253 nm UV light (150 mJ/cm^2), processed to nuclear and cytoplasmic fractions, and subjected to eRIC for determining the nuclear and cytoplasmic RNA-binding proteins from the different conditions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

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

SUBMITTER: Frank Stein

LAB HEAD: Andreas E. Kulozik

PROVIDER: PXD016735 | Pride | 2020-04-06

REPOSITORIES: Pride

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			Action	DRS
	Cyto_input_merged_results_20190314_1144_peptides.txt	Txt
	Cyto_input_merged_results_20190314_1144_proteins.txt	Txt
	Cyto_input_merged_results_20190314_1144_summary.txt	Txt
	Cyto_interactome_merged_results_20190313_1019_peptides.txt	Txt
	Cyto_interactome_merged_results_20190313_1019_proteins.txt	Txt

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Publications

Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins.

Backlund Michael M Stein Frank F Rettel Mandy M Schwarzl Thomas T Perez-Perri Joel I JI Brosig Annika A Zhou Yang Y Neu-Yilik Gabriele G Hentze Matthias W MW Kulozik Andreas E AE

Nucleic acids research 20200501 9

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation fol ...[more]

PMID: 32313943

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Enhanced RNA Interactome capture (eRIC) and RNA Interactome capture (RIC) of Jurkat cells (DSMZ, ACC-282).

Project description:1.0 â€“ 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.

2018-09-03 | PXD010941 | Pride

Enhanced RNA Interactome capture (eRIC) and RNA Interactome capture (RIC) of Jurkat cells (DSMZ, ACC-282) exposed to 0.5mM DMOG or vehicle (DMSO) for 6 hours.

Project description:1.0 â€“ 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were incubated with 0.5mM DMOG (Cayman Chemical Company, 71210) or an equivalent volume of Dimethyl Sulfoxide (DMSO) (vehicle, Merck 1.02950.0500). The DMSO concentration in the medium was 0.023% v/v. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.

2018-09-03 | PXD010942 | Pride

Enhanced RNA interactome capture (eRIC) of human malignant bone tumor cells and normal bone/mesenchymal cells

Project description:Patient-derived bone tumor (osteosarcoma and giant cell tumor of bone) cells, and the normal mesenchymal stem cells and osteoblasts were cultured and subjected to UV crosslinking (UV) at 254 nm or without crosslinking (noUV) as negative controls. Subsequently, RNA-binding proteins (RBPs) were identified by eRIC.

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Enhanced RNA Interactome capture (eRIC) and total proteome of mouse brain, kidney and liver

Project description:Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Brain, kidneys and liver were immediately harvested, flash frozen in liquid nitrogen and stored at -80Ã‚Â°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 Ã‚Âµm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 Ã‚Â°C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20Ã‚Â°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed or not to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC to determine the RNA-bound proteome. eRIC eluates obtained from the respective organs of two mice were combined per independent experiment. The analyses were conducted in duplicate for each organ studied. For the no crosslink controls, organ sections from four mice were pooled to generate one unique no crosslink eRIC sample per organ studied.

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Drosophila mRNA Interactome - Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

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1 A post-transcriptional regulatory code for mRNA stability dur-ing the maternal-to-zygotic transition

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2020-05-26 | PXD009514 | Pride

Poly(A) RNA interactome capture upon HDAC inhibition by Romidepsin

Project description:Immediate early genes (IEGs) represent a unique class of genes with rapid induction kinetics and transient expression patterns, which requires IEG mRNAs to be short-lived. Here, we establish cytoplasmic polyadenylation element-binding protein 4 (CPEB4) as a major determinant of IEG mRNA instability. We identified human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), which is known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CBEP4 in HeLa cells revealed that CPEB4 preferentially binds to the 3' untranslated region (UTR) of IEG mRNAs, at U-rich sequence motifs located in close proximity to the poly(A) site. By transcriptome-wide mRNA decay measurements, we found that the strength of CPEB4 binding correlates with short mRNA half-lives, and that loss of CPEB4 expression leads to the stabilization of IEG mRNAs. Further, we demonstrate that CPEB4 mediates mRNA degradation by recruitment of the evolutionarily conserved CCR4-NOT complex, the major eukaryotic deadenylase. While CPEB4 is primarily known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as an RBP that enhances the degradation of short-lived IEG mRNAs.

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Project description:Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Kidneys were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC (PMID: 30352994). The proteins captured by eRIC were separated by gel electrophoresis, divided into 7 fractions of defined mass, and identified by MS to determine their distribution across the fractions. Crosslinking to other proteins should shift the signal to higher mass fractions. The analyses were conducted in triplicate.

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