BIOCHEMICAL ANALYSES OF MUSCLEBLIND COMPLEXES IN MYOTONIC DYSTROPHY
Comai Lucio
University Of Southern Californiacity: Los Angeles country: United States (us)
Grant 5R01NS060839-05 from National Institute Of Neurological Disorders And Stroke
Abstract: The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of altered RNA splicing that are controlled by the formation of pathological MBNL1 mega-complexes in myotonic dystrophy 1 (DM1) patient cells. The genetic defect in DM1 results in the production of mutant RNAs encoding expanded CUG tracts. Abnormally expanded CUG tracts have been shown to form aberrant mega-complexes that contain the alternative splice factor, MBNL1, within the nucleus. Several lines of evidence implicate the formation of these high molecular weight complexes in altered splicing of a subset of physiologically important RNAs and in the subsequent development of DM1 pathology in vivo. To determine the mechanism whereby formation of the MBNL1 mega-complexes alters the splice code in DM1 we propose to purify both normal MBNL1 complexes and the aberrant MBNL1 mega-complexes that develop in DM1 myoblasts. In complementary experiments the role of these complexes in dictating RNA splice site choice will be defined. The Aims of this application are 1. Purification and functional characterization of normal MBNL1 complexes in spliceosome assembly and RNA catalysis. 2. Purification of MBNL1 mega-complexes from DM1 myoblasts and definition of the mechanics of mega-complex formation in vivo. 3. Elucidation of the mechanisms by which formation of MBNL1 mega-complexes alters the splice code in DM1 myoblasts
Keywords: 19q; 3` Untranslated Regions; Adult; Affinity Chromatography; Alternative Splicing; Antibodies; Antibody Affinity; Behavior; Binding (Molecular Function); Biochemical; Catalysis; Cell Nucleus; Cells; Chromosomes; Co-Immunoprecipitations; Code; Complex; Data; Defect; Development; Exons; Fractionation; Gel Chromatography; Genes; Human; In Vitro; in vivo; Individual; Inherited; Introns; Laboratories; Maintenance; Mass Spectrum Analysis; Mechanics; Mediating; Molecular; Molecular Weight; Mus; Muscular Dystrophies; mutant; Mutation; Myoblasts; Myotonic Dystrophy; Names; Non-Insulin-Dependent Diabetes Mellitus; Normal Cell; Nuclear; Nuclear Extract; Pancreatic ribonuclease; Pathogenesis; Pathology; Patients; Phenotype; Play; Principal Investigator; Production; programs; Protein Kinase; Proteins; Protocols documentation; Research; research study; Resistance; RNA; RNA Binding; RNA Sequences; RNA Splice Sites; RNA Splicing; Role; Scheme; Silver Staining; Site; Skeletal muscle structure; Spliceosome Assembly Pathway; Testing
Project start date: 2007-02-01
Project end date: 2012-11-30
Budget start date: 1-DEC-2011
Budget end date: 30-NOV-2012
5R01NS060839-05 (2012): $457324
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Comai Lucio
THE WERNER SYNDROME PROTEIN IN CPT-INDUCED DNA DAMAGE
Comai Lucio
University Of Southern Californiacity: Los Angeles country: United States (us)
Grant 5R01AG023873-05 from National Institute On Aging
Abstract: Werner syndrome (WS) is an autosomal recessive disorder leading to premature onset aging and aging-related diseases including cancer and atherosclerosis. WS results from the loss of function of the WRN gene. The WRN gene encodes a RecQ helicase protein with a unique exonuclease activity (WRN) whose cellular function is poorly understood. Cells from WS patients demonstrate premature senescence and sensitivity to DNA damaging agents such as camptothecin (CPT). Importantly, we have shown that WRN binds to Ku70/80, a heterodimeric complex known to play a critical role in the repair of DNA damage. This observation strongly supports the idea that WRN functions in a DNA damage response pathway. We therefore hypothesize that WRN is required for S-phase checkpoint activation or is directly involved in the repair of DNA lesions following exposure of cells to CPT. Specifically, in Aim I we will test whether loss of WRN leads to defective S-phase checkpoint controls in CPT-treated cells. Experiments proposed in Aim 2 will study the dynamics of the recruitment of WRN and its associated factors to chromatin in response to CPT-induced DNA damage. In Aim 3, we will test the hypothesis that loss of WRN results in genetic instability at the rDNA locus leading to aberrant ribosomal RNAs biosynthesis upon DNA damage. In Aim 4, biochemical insights into these processes will be obtained by studying the response to CPT-induced DNA damage of cells expressing mutant WRN proteins deficient in exonuclease or helicase activity, or lacking conserved structural domains. Taken together, these experiments should provide important mechanistic insights into the process of human aging
Keywords: (S)-4-ethyl-4-hydroxy-1H-pyrano-[3`, 4`[{..}]6, 7]indolozino[1, 2-b]quinoline-3, 14(4H, 12H)-dione; 1H-Pyrano(3`, 4`[{..}]6, 7)indolizino(1, 2-b)quinoline-3, 14(4H, 12H)-dione, 4-ethyl-4-hydroxy-, (S)-; 1H-Pyrano[3`, 3`.6, 7]indolizino[1, 2-b]quinoline-3, 14(4H, 12H)-dione, 4-ethyl-4hydroxy-(S)-(9CI); 20-(S)-camptothecine; 21, 22-secocamptothecin-21-oic acid lactone; Activation Analysis; Age; age dependent; Age of Onset; age related; Allergy; Analyses, Activation; Assay; atheromatosis; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; atherosclerotic vascular disease; base; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biologic Assays; Biological Assay; Camptothecin; Cancers; Carcinogen-DNA Adducts; Cell Cycle Checkpoint; Cell Function; Cell physiology; Cell Process; Cells; Cellular Function; Cellular Physiology; Cellular Process; Chromatin; Chromosomal Instability; Chromosome Instability; Complex; Defect; Deoxyribonucleic Acid; Detection; Disease; disease/disorder; Disorder; DNA; DNA Adducts; DNA Damage; DNA Damage Repair; DNA Double Strand Break; DNA Helicases; DNA Injury; DNA lesion; DNA Nicking-Closing Protein; DNA Relaxing Enzyme; DNA Relaxing Protein; DNA Repair; DNA Repair Pathway; DNA Topoisomerase I; DNA Topoisomerases, Type I; DNA Type 1 Topoisomerase; DNA Untwisting Enzyme; DNA Untwisting Protein; DNA unwinding enzyme; DNA Unwinding Proteins; DNA, Ribosomal; DNA-PKcs; Exonuclease; experiment; experimental research; experimental study; failure; Failure (biologic function); fatal attempt; fatal suicide; FLR; G22P1; G22P1 gene; gene product; Generalized Growth; Genetic; Genome; Genome Stability; genotoxic agent; Genotoxins; Goals; Growth; helicase; Human; human TOP1 protein; human WRN protein; Human, General; Hypersensitivity; Individual; insight; intent to die; Ku70; loss of function; malignancy; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Mediating; Molecular; Molecular Interaction; Monitor; Mutagens; mutant; neoplasm/cancer; nicking closing enzyme; Normal Cell; ontogeny; ori Region; pathway; Pathway interactions; Patients; Phase; Play; Predisposition; Principal Investigator; Process; Progeria, Adult; programs; Programs (PT); Programs [Publication Type]; Proteins; rDNA; RECQ3; RECQ3 protein, human; RECQL2 protein, human; recruit; Recruitment Activity; relaxing enzyme; repair; repaired; Replication Origin; Research; research study; response; Ribosomal DNA; Ribosomal RNA; RNA biosynthesis; Role; rRNA; senescence; Site; social role; Stability, Genomic; Subcellular Process; suicidality; Suicide; Susceptibility; swivelase; Testing; Tissue Growth; TLAA; TOP1; TOP1 protein, human; Topo I; Topoisomerase; Topoisomerase I; Topoisomerase, DNA, I; Topoisomerase-I Inhibitor; Toxic effect; Toxicities; Type I DNA Topoisomerase; Type I DNA Topoisomerases; Unscheduled DNA Synthesis; untwisting enzyme; Werner Syndrome; Werner syndrome helicase, human; Werner Syndrome Protein; Werner`s syndrome; WRN; WRN gene; WRN Protein; WRN protein, human
Project start date: 2004-06-01
Project end date: 2011-05-31
Budget start date: 1-JUN-2008
Budget end date: 31-MAY-2011
5R01AG023873-05 (2008): $296867
ROLE OF THE WERNER SYNDROME PROTEIN COMPLEX IN THE METABOLISM OF CHROMOSOME ENDS
Comai Lucio
University Of Southern Californiacity: Los Angeles country: United States (us)
Grant 5R01AG034156-02 from National Institute On Aging
Abstract: The goal of this study is to gain mechanistic insights on the function of the Werner syndrome protein (WRN) complex in the processes that maintain telomere integrity and prevent the formation of extrachromosomal telomeric circles. WRN is a nuclear protein with helicase and exonuclease activities, whose loss-of-function mutations are associated with the premature aging and cancer prone disease Werner syndrome (WS). Genetic and biochemical evidence implicate WRN in telomere metabolism and suggest that abnormal telomere length homeostasis contributes to the pathology of WS. Our biochemical studies have shown that WRN operates as a functional unit with the Ku70/80 heterodimer and have further demonstrated that loss of WRN function causes the production of telomeric circles in fibroblasts expressing telomerase. To determine the mechanism whereby the WRN complex regulates telomere homeostasis, we propose to mechanistically characterize the functional interplay between WRN, Ku and telomere-specific factors found at chromosome ends and define the process activated by loss of WRN function responsible for the formation of telomeric circles. To accomplish these objectives we propose the following three aims. In Aim 1, we will characterize the role of WRN and its interacting partner Ku70/80 in the regulation of telomeric termini in human cells. In Aim 2, we will dissect the molecular mechanisms leading to the formation of extrachromosomal t- circles resulting from loss of WRN function. In Aim 3, we will characterize the biochemical properties of WRN in the context of model telomeric substrates in vitro. The studies proposed in this application will elucidate the role of WRN at telomeres and provide valuable information for understanding how loss of WRN function promotes genome instability, premature aging and the early onset of diseases such as cancer and cardiovascular disease. It is recognized that the incidence of these diseases increases progressively with age. Therefore, the changes at the molecular, cellular and physiologic levels that occur during aging profoundly influence the development and progression of these diseases. Functional analysis of WRN will provide a clearer understanding of these changes and will help in the development of therapeutic agents aimed at preventing the early onset of age-associated diseases. Werner syndrome (WS) is a genetic disease caused by loss of the Werner syndrome protein and predisposes to premature aging and early onset of diseases such as cancer and cardiovascular diseases. The function of the Werner syndrome protein and the role of this protein in cellular homeostasis and preventing normal cells from becoming unhealthy are poorly understood. The studies proposed in this application will be important for elucidating the molecular mechanisms underlying the development of WS pathology, which in turn could lay the foundation for the development of therapeutic approaches to improve the effectiveness of treatments to prevent cancer and other age-onset diseases in the general population
Keywords: Affect; Age; Age of Onset; Aging; Apoptosis; Arabidopsis; base; Binding (Molecular Function); Biochemical; Biochemical Genetics; Biological Assay; Cardiovascular Diseases; Cell Aging; Cell physiology; Cells; chromatin immunoprecipitation; Chromosomes; Complex; Data; Development; Disease; Disease Progression; DNA; DNA Binding Domain; DNA Damage; DNA Structure; Dominant-Negative Mutation; early onset; Electrophoresis, Gel, Two-Dimensional; Employee Strikes; Enzymes; Excision; Exonuclease; extrachromosomal DNA; Family member; Fibroblasts; Foundations; G22P1 gene; General Population; Genes; Genetic; Genetic Recombination; Genome; Genomic Instability; Goals; helicase; Hereditary Disease; Homeostasis; homologous recombination; Human; human WRN protein; improved; In Vitro; in vivo; Incidence; insight; Length; Link; Longevity; loss of function mutation; Maintenance; Malignant Neoplasms; malignant phenotype; Measures; Mediating; member; Metabolism; Modeling; Molecular; Molecular Conformation; mutant; Names; Nature; Normal Cell; Nuclear Protein; Nuclear Proteins; Nucleic Acids; overexpression; Pathology; Pathway interactions; Patients; Phenotype; Physiological; Plants; Play; premature; Premature aging syndrome; prevent; Process; Production; Property; protein complex; protein protein interaction; Proteins; public health relevance; reconstitution; Regulation; repaired; research study; Residual state; response; Role; senescence; Signal Transduction; Southern Blotting; Telomerase; telomere; Telomere Maintenance; Telomere Shortening; Testing; Therapeutic Agents; therapeutic development; Treatment Effectiveness; Werner Syndrome; Work; XRCC3 gene
Relevance: Public Health Relevance Statement Werner syndrome (WS) is a genetic disease caused by loss of the Werner syndrome protein and predisposes to premature aging and early onset of diseases such as cancer and cardiovascular diseases. The function of the Werner syndrome protein and the role of this protein in cellular homeostasis and preventing normal cells from becoming unhealthy are poorly understood. The studies proposed in this application will be important for elucidating the molecular mechanisms underlying the development of WS pathology, which in turn could lay the foundation for the development of therapeutic approaches to improve the effectiveness of treatments to prevent cancer and other age-onset diseases in the general population
Project start date: 2010-04-01
Project end date: 2015-03-31
Budget start date: 1-APR-2011
Budget end date: 31-MAR-2012
PFA/PA: PA-07-070
5R01AG034156-02 (2011): $349051