Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MRNA composition of IRP2 mRNPs in mouse tissues

ABSTRACT: Affymetrix microarrays were used to determine the mRNA composition of mRNPs obtained by immunoprecipitation with IRP2 (iron regulatory protein 2). Reference Sanchez at al., 2007 Nat. Protoc. Affymetrix microarrays were used to determine the mRNA composition of mRNPs obtained by immunoprecipitation with IRPs (iron regulatory proteins). In brief, total RNA was extracted from the reported tissue of four to six C57BL6/J mice using Trizol reagent. mRNA from duodenum, liver and spleen were extracted from mice fed with an iron poor diet (<10mg/kg, Altromin Spezialfutter GmbH & Co, Lage, Germany) for 21 days starting from weaning age. mRNAs from bone marrow and brian were extracted from mice (8-10 weeks old) fed with an iron-normal diet. For each tissue, the RNA samples were pooled and the immunoprecipitations were carried out combining 50 M-BM-5g of total RNA with purified recombinant His-tagged IRP2 produced in E.coli and a mouse monoclonal anti-His tag antibody. A control reaction in which the recombinant IRP2 was omitted was performed in parallel (referred with the word M-bM-^@M-^\controlM-bM-^@M-^] in the title). All the microarray procedures were conducted at the EMBL Genomics Core Facility using standard Affymetrix protocols. In brief, approximately 120ng of immunoprecipitated RNA was used as input to a two-step amplification procedure to generate biotin-labeled RNA fragments for hybridization to the Affymetrix GeneChip Mouse Genome 430 2.0 array (Eukaryotic Sample and Array Processing manual 701024 Rev.3). Intensity values for the hybridizations were obtained either using RMA, calculations done in bioconductor (www.bioconductor.org) or MAS5, calculated using Affymetrix GCOS package. MAS5 calculated intensities were further quantile normalized using bioconductor.

ORGANISM(S): Mus musculus

SUBMITTER: Mayka Sanchez

PROVIDER: E-GEOD-17097 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins.

Sanchez Mayka M Galy Bruno B Schwanhaeusser Bjoern B Blake Jonathon J Bähr-Ivacevic Tomi T Benes Vladimir V Selbach Matthias M Muckenthaler Martina U MU Hentze Matthias W MW

Blood 20110922 22

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition wa ...[more]

PMID: 21940823

Publication: 1/2

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Project description:Aims and introduction: Cardiac fibrosis is the hallmark of cardiac disease, particularly myocardial infarction (MI), and is one of the most prevalent causes of heart failure. MI is intricately linked to inflammation, extracellular matrix (ECM) remodelling, and cardiac fibrosis, however the mechanisms underpinning these events remain poorly understood. We recently identified that ECM1 has potential to play a key role in cardiac wound healing but the cellular origins in the heart and specific mechanisms of ECM1 in fibrosis remain elusive. Therefore, we investigated the spatiotemporal, cell specific, expression profile of ECM1 in the healthy and diseased mouse and human heart and investigate ECM1 dependent human cardiac fibroblast (HuCFb) cell signalling mechanisms. Methods and results: Western blotting, immunohistochemistry (IHC) and mRNA in-situ hybridisation revealed that ECM1 protein expression was significantly upregulated in human ischaemic and dilated heart failure patients, and predominantly localised interstitially to fibrotic, inflammatory, and peri-vascular areas. ECM1 specific analysis of the Litviňuková et al. (2020) single-cell and -nuclei RNA sequencing (sc/snRNAseq) dataset of healthy human hearts, and the Farbehi et al. (2019) scRNAseq dataset of mouse hearts post-MI demonstrated that ECM1 expression originates from fibroblasts, macrophages, and pericytes/vascular mural cells in the heart. Further, we identify that post-MI ECM1 is expressed in a temporal dynamic flux from unactivated fibroblasts in sham-operated mice, to M1 macrophages (M1MΦ) at day-3, and myofibroblasts and M2MΦ at day-7. Phosphoproteomics of ECM1 treated human cardiac fibroblast cells (HuCFb) alongside ECM1 to HuCFb cell surface protein binding pull-down and mass spectrometry, revealed that ECM1 signalling goes via proteins associated with Rho protein signal transduction, cell-cell adhesion, microtubule dynamics, and cell chemotaxis pathways, potentially initiated by ECM1 to CTNND1 binding on the cell surface. Further mechanistic analyses identified that ECM1 inhibits HuCFb cell migration and stimulates inflammatory (IL-6 and IL-1β mRNA expression), fibrotic (TGF-β1, TGF-β2 and Col1a2 mRNA expression), and non-canonical Wnt signalling pathways (Wnt5a mRNA expression and JNK1/2/3 and MYPT1 phosphorylation). Conclusions: This study demonstrates that ECM1 expression originates from macrophages and fibroblasts in the mouse and human heart. ECM1 has significant effects on HuCFb migration, inflammatory, fibrotic, Rho protein, and Wnt5a/non-canonical Wnt signalling pathways. Together, ECM1 plays an important role in cardiac wound healing and may represent a novel mechanism of inflammation-fibrosis crosstalk and progression post-MI.

Dataset Information

MRNA composition of IRP2 mRNPs in mouse tissues

Publications

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins.

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