1. Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome
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Joel T. Haas, Jan Albert Kuivenhoven, Justina C. Wolters, Andrzej J. Rzepiela, Mathilde Varret, Ann Verhaegen, Valérie Carreau, Szymon Stoma, Anne Philippi, Alaa Othman, Jerome Robert, N. Dalila, Belle V. van Rosmalen, An Verrijken, Arnold von Eckardstein, Bart van de Sluis, Silvija Radosavljevic, Paolo Zanoni, Simon F. Norrelykke, Roger Meier, M. Yalcinkaya, Bart Staels, Andreas Geier, Lucia Rohrer, Michael Stebler, Michele Visentin, Antoine Rimbert, Catherine Boileau, Antonio Gallo, Melinde Wijers, Nicolette C. A. Huijkman, Steve E. Humphries, Jonas Weyler, Freerk van Dijk, Michaela Keel, Srividya Velagapudi, Jean-Pierre Rabès, Marieke Smit, Anne Tybjærg-Hansen, Adriaan van der Graaf, Luisa Vonghia, Yara Abou-Khalil, Sven Francque, Grigorios Panteloglou, Marta Futema, Luc Van Gaal, University hospital of Zurich [Zurich], Universität Zürich [Zürich] = University of Zurich (UZH), Institute for Molecular Systems Biology [ETH Zurich] (IMSB), Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Récepteurs Nucléaires, Maladies Métaboliques et Cardiovasculaires - U1011 (RNMCD), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), CHU Lille, Scientific Center for Optical and Electron Microscopy (ScopeM), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), University of Groningen [Groningen], unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), St George's, University of London, Laboratoire de Recherche Vasculaire Translationnelle (LVTS (UMR_S_1148 / U1148)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Sorbonne Paris Nord, AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Saint-Joseph de Beyrouth (USJ), University of Copenhagen = Københavns Universitet (UCPH), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), AP-HP. Université Paris Saclay, Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Antwerp University Hospital [Edegem] (UZA), University of Antwerp (UA), University of Amsterdam [Amsterdam] (UvA), Columbia University [New York], University Hospital of Würzburg, University College of London [London] (UCL), 603091, ANR-10-LABX-46, 2015T068, CVON2017-2020, AOM06024, 2014/267, Pfizer: 24052829, European Molecular Biology Organization, EMBO: ALTF277-2014, Seventh Framework Programme, FP7, Sixth Framework Programme, FP6: LSHM-CT-2005-018734, Fondation Maladies Rares, FMR, International Atherosclerosis Society, IAS, British Heart Foundation, BHF, European Commission, EC, European Research Council, ERC: 694717, ANR 16-RHUS-0006, Agence Nationale de la Recherche, ANR: ANR-16-RHUS-0007, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, SNF: 310030-185109, 31003A-160126, Fonds De La Recherche Scientifique - FNRS, FNRS, Fonds Wetenschappelijk Onderzoek, FWO: 1802154 N, PG008/08, RG3008, Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO: 184.021.007, Universität Zürich, UZH: FK-20-037, We acknowledge the use of data from BIOS-consortium ( http://wiki.bbmri.nl/wiki/BIOS_bios ) which is funded by BBMRI-NL (NWO project 184.021.007). Flow cytometry was performed with equipment of the flow cytometry facility, University of Zurich., This work was conducted as part of the TransCard project of the seventh Framework Program (FP7) granted by the European Commission, to J. Albert Kuivenhoven, A. Tybjaerg-Hansen, and A. von Eckardstein (number 603091) as well as partially the FP7 RESOLVE project (to J.T. Haas, B. Staels, A. Verhaegen, S. Francque, L. Van Gaal, and A. von Eckardstein) and the European Genomic Institute for Diabetes (EGID, ANR-10-LABX-46 to B. Staels). Additional work by A. von Eckardstein’s team was funded by the Swiss National Science Foundation (31003A-160126, 310030-185109) and the Swiss Systems X program (2014/267 [Medical Research and Development (MRD)] HDL-X). P. Zanoni received funding awards from the Swiss Atherosclerosis Society (Arbeitsgruppe Lipide und Atherosklerose [AGLA] and the DACH Society for Prevention of Cardiovascular Diseases). G. Panteloglou received funding from the University of Zurich (Forschungskredit, grant no. FK-20-037). J. Albert Kuivenhoven is an Established Investigator from the Dutch Heart Foundation (2015T068). J. Albert Kuivenhoven was also supported by GeniusII (CVON2017-2020). The Laboratory for Vascular Translational Science (L.V.T.S.) team is supported by Fondation Maladies Rares, Programme Hospitalier de Recherche Clinique (PHRC) (AOM06024), and the national project CHOPIN (CHolesterol Personalized Innovation), granted by the Agence Nationale de la Recherche (ANR-16-RHUS-0007). Y. Abou Khalil is supported by a grant from Ministère de l’Education Nationale et de la Technologie (France). J.T. Haas was supported by an EMBO Long Term Fellowship (ALTF277-2014). B. Staels is a recipient of an ERC Advanced Grant (no. 694717). Both are also supported by PreciNASH (ANR 16-RHUS-0006). Research at the Antwerp University Hospital was supported by the European Union: FP6 (HEPADIP Contract LSHM-CT-2005-018734). S. Francque has a senior clinical research fellowship from the Fund for Scientific Research (FWO) Flanders (1802154 N). S.E. Humphries received grants RG3008 and PG008/08 from the British Heart Foundation, and the support of the UCLH NIHR BRC. S.E. Humphries directs the UK Children’s FH Register which has been supported by a grant from Pfizer (24052829) given by the International Atherosclerosis Society., This work was conducted as part of the TransCard project of the seventh Framework Program (FP7) granted by the European Commission, to J. Albert Kuivenhoven, A. Tybjaerg-Hansen, and A. von Eckardstein (number 603091) as well as partially the FP7 RESOLVE project (to J.T. Haas, B. Staels, A. Verhaegen, S. Francque, L. Van Gaal, and A. von Eckardstein) and the European Genomic Institute for Diabetes (EGID, ANR-10-LABX-46 to B. Staels). Additional work by A. von Eckardstein's team was funded by the Swiss National Science Foundation (31003A-160126, 310030-185109) and the Swiss Systems X program (2014/267 [Medical Research and Development (MRD)] HDL-X). P. Zanoni received funding awards from the Swiss Atherosclerosis Society (Arbeitsgruppe Lipide und Atherosklerose [AGLA] and the DACH Society for Prevention of Cardiovascular Diseases). G. Panteloglou received funding from the University of Zurich (Forschungskredit, grant no. FK-20-037). J. Albert Kuivenhoven is an Established Investigator from the Dutch Heart Foundation (2015T068). J. Albert Kuivenhoven was also supported by GeniusII (CVON2017-2020). The Laboratory for Vascular Translational Science (L.V.T.S.) team is supported by Fondation Maladies Rares, Programme Hospitalier de Recherche Clinique (PHRC) (AOM06024), and the national project CHOPIN (CHolesterol Personalized Innovation), granted by the Agence Nationale de la Recherche (ANR-16-RHUS-0007). Y. Abou Khalil is supported by a grant from Minist?re de l'Education Nationale et de la Technologie (France). J.T. Haas was supported by an EMBO Long Term Fellowship (ALTF277-2014). B. Staels is a recipient of an ERC Advanced Grant (no. 694717). Both are also supported by PreciNASH (ANR 16-RHUS-0006). Research at the Antwerp University Hospital was supported by the European Union: FP6 (HEPADIP Contract LSHMCT- 2005-018734). S. Francque has a senior clinical research fellowship from the Fund for Scientific Research (FWO) Flanders (1802154 N). S.E. Humphries received grants RG3008 and PG008/08 from the British Heart Foundation, and the support of the UCLH NIHR BRC. S.E. Humphries directs the UK Children's FH Register which has been supported by a grant from Pfizer (24052829) given by the International Atherosclerosis Society., ANR-16-RHUS-0006,PreciNASH,PreciNASH(2016), ANR-16-RHUS-0007,CHOPIN,CHOPIN(2016), Center for Liver, Digestive and Metabolic Diseases (CLDM), Restoring Organ Function by Means of Regenerative Medicine (REGENERATE), HAL UVSQ, Équipe, Récepteurs Nucléaires, Maladies Métaboliques et Cardiovasculaires (RNMCD - U1011), Service d'Endocrinologie, Métabolisme et Prévention des Maladies Cardio-vasculaires [CHU Pitié-Salpêtrière], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
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Spliceosome ,Physiology ,RNA Splicing ,Population ,Hypercholesterolemia ,Familial hypercholesterolemia ,030204 cardiovascular system & hematology ,Biology ,03 medical and health sciences ,0302 clinical medicine ,[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,Gene expression ,medicine ,Humans ,education ,Gene ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,education.field_of_study ,Intron ,Nuclear Proteins ,Hep G2 Cells ,medicine.disease ,Molecular biology ,Endocytosis ,[SDV.MHEP.CSC] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,Lipoproteins, LDL ,Cholesterol ,HEK293 Cells ,Cardiovascular diseases ,Liver ,Receptors, LDL ,Mutation ,LDL receptor ,Hepatocytes ,Spliceosomes ,lipids (amino acids, peptides, and proteins) ,Human medicine ,Cardiology and Cardiovascular Medicine - Abstract
Background: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR . The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25 , are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25 , overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.
- Published
- 2022