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      An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

      research-article
      1 , 2 , 3 , 4 , 5 , 3 , 4 , 1 , 3 , 4 , 6 , 7 , 8 , 9 , 10 , 10 , 1 , 1 , 1 , 3 , 4 , 11 , 11 , 12 , 3 , 4 , 13 , 14 , 14 , 14 , 14 , 14 , 14 , 15 , 16 , 17 , 18 , 19 , 1 , 20 , 20 , 21 , 21 , 22 , 23 , 24 , 25 , 26 , 26 , 26 , 26 , 6 , 6 , 17 , 6 , 6 , 27 , 28 , 29 , 1 , 18 , 17 , 30 , 1 , UK10K Consortium 31 , University of Washington Center for Mendelian Genomics 32 , 33 , 34 , 16 , 35 , 36 , 21 , 37 , 6 , 6 , 38 , 15 , 35 , 36 , 11 , 39 , 9 , 13 , 10 , 2 , 8 , + , 7 , 40 , , + , 2 , , + , 3 , 4 , , + , 1 ,
      Nature cell biology
      cilia, ciliopathies, reverse genetics, whole-genome siRNA screen, Jeune syndrome, Joubert syndrome

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          Abstract

          Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

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          Most cited references46

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          Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.

          DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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            Is Open Access

            Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists

            Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
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              • Article: not found

              Proteomic characterization of the human centrosome by protein correlation profiling.

              The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nat. Cell Biol.
                Nature cell biology
                1465-7392
                1476-4679
                11 August 2015
                13 July 2015
                August 2015
                01 February 2016
                : 17
                : 8
                : 1074-1087
                Affiliations
                [1 ]Section of Ophthalmology and Neuroscience, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds, LS9 7TF, UK
                [2 ]Genetics and Genomic Medicine and Birth Defects Research Centre, Institute of Child Health, University College London, London, WC1N 1EH, UK
                [3 ]Department of Human Genetics, Radboud University Medical Center, Nijmegen, 6525 GA, The Netherlands
                [4 ]Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, 6525 GA, The Netherlands
                [5 ]Pediatric Genetics Section, Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg 79112, Germany
                [6 ]Department of Medical Genetics and Alberta Children’s Hospital Research Institute for Child and Maternal Health, Calgary, T3B 6A8, AB, Canada
                [7 ]Department of Pediatrics, University of Washington, Seattle, WA 98195, USA
                [8 ]Structural and Computational Biology, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
                [9 ]School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland
                [10 ]Department of Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg University of Mainz, 55122 Mainz, Germany
                [11 ]Division of Experimental Ophthalmology and Medical Proteome Center, Center of Ophthalmology, University of Tübingen, 72074 Tübingen, Germany
                [12 ]Section of Genetics, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds, LS9 7TF, UK
                [13 ]Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave, Box 268, New York, NY 10065, USA
                [14 ]BioScreening Technology Group, Biomedical Health Research Centre, St. James’s University Hospital, Leeds, LS9 7TF, UK
                [15 ]Department of Nephrology and Hypertension, University Medical Centre Utrecht, Utrecht, 3584 CX, The Netherlands
                [16 ]Centre for Molecular and Biomolecular Informatics, Radboud University Medical Center, Nijmegen, 6525 GA, The Netherlands
                [17 ]Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, K1H 8L1, ON, Canada
                [18 ]Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
                [19 ]Animal Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
                [20 ]Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
                [21 ]Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia
                [22 ]Department of Ophthalmology, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia
                [23 ]Department of Ophthalmology, College of Medicine, Alfaisal University, Riyadh, 11533, Saudi Arabia
                [24 ]Robarts Research Institute, University of Western Ontario, London, N6G 2V4, ON, Canada
                [25 ]Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA
                [26 ]Department of Pediatrics and Child Health & Department of Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, R3E 3P5, MB, Canada
                [27 ]Department of Pediatrics, University of California San Francisco, San Francisco, CA 92093, USA
                [28 ]Department of Radiology, University of California San Francisco, San Francisco, CA 92093, USA
                [29 ]Department of Pathology, Radboud University Medical Center, Nijmegen, 6525 GA, The Netherlands
                [30 ]Department of Ophthalmology, Leeds Teaching Hospitals NHS Trust, St. James’s University Hospital, Leeds, LS9 7TF, UK
                [31 ]UK10K Consortium, Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK
                [32 ]University of Washington Center for Mendelian Genomics, University of Washington, Seattle, WA 98195, USA
                [33 ]Department of Medical Genetics, Belfast City Hospital and Queens University, Belfast, BT12 6BA, UK
                [34 ]Department of Pediatrics and Adolescent Medicine, University Hospital Muenster, 48149 Muenster, Germany
                [35 ]Moorfields Eye Hospital NHS Foundation Trust and NIHR Ophthalmology Biomedical Research Centre, London, EC1V 2PD, UK
                [36 ]UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
                [37 ]Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia
                [38 ]Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, Faculty of Health & Life Sciences, University of Liverpool, Liverpool, L69 3BX, UK
                [39 ]Research Unit of Protein Science, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt, 85764 Neuherberg, Germany
                [40 ]Divisions of Developmental Medicine and Genetic Medicine, Seattle Children’s Research Institute, University of Washington, Seattle, WA 98105, USA
                Author notes
                [+]

                corresponding authors

                Article
                EMS63737
                10.1038/ncb3201
                4536769
                26167768
                55bb93a2-1c13-44a7-9da7-9548e77dc14c

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                Cell biology
                cilia,ciliopathies,reverse genetics,whole-genome sirna screen,jeune syndrome,joubert syndrome

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