Alternative Splicing in Regulation of Cholesterol Synthesis and Uptake

Summary

Principal Investigator: Marisa Wong Medina
Abstract: DESCRIPTION (provided by applicant): LDL cholesterol levels are of fundamental importance in determining risk for cardiovascular disease. Recently, alternative splicing of the two most critical regulators of intracellular cholesterol, 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol biosynthesis, and the LDL receptor (LDLR), responsible for uptake of LDL, have been associated with variation in plasma LDL as well as with the magnitude of LDL reduction by simvastatin. Recent evidence has indicated that alternative splicing of four genes involved in cholesterol metabolism (HMGCR, LDLR, HMG-CoA synthase and mevalonate kinase) is coordinately regulated by sterols such that sterol loading increases alternative splicing while sterol depletion suppresses alternative splicing. In addition, genome-wide transcription analysis of simvastatin incubated human lymphocyte cell lines demonstrated that 95 of ~300 known components of supraspliceosomes were responsive to statin (FDR<0.0001). Among these, several splicing factors were implicated in mediating sterol regulation of alternative splicing on the basis of additional lines of evidence including: (1) correlations of variation in gene expression with both cell surface LDLR and plasma LDL concentrations;(2) DNA polymorphisms associated with plasma LDL levels;(3) siRNA knock-down resulting in changes in pre-mRNA splicing;and (4) in silico prediction of known binding motifs. These findings lead to the hypotheses that intracellular cholesterol levels regulate splicing factor(s) to generate coordinated changes in alternative splicing of multiple genes involved in cholesterol synthesis and uptake, and that variation in this process is a determinant of cellular and plasma cholesterol metabolism. Thus, the overall objectives of this proposal are: (1) to demonstrate that alternative splicing is a novel mechanism involved in regulating cellular cholesterol synthesis and uptake as well as plasma LDL levels;and (2) to identify non-genetic and genetic modifiers of this process. To determine if sterol regulated alternative splicing occurs in a larger number of genes in the cholesterol biosynthesis pathway;changes in alternative splicing will be quantified in HepG2 cells, primary human hepatocytes, and immortalized human lymphocyte cell lines treated with specific inhibitors and products of this pathway (Aim 1). The splicing factors responsible for orchestrating these coordinated changes will be identified and validated using siRNA, overexpression constructs and mini-gene constructs (Aim 2). Lastly, the physiological relevance of these observations will be assessed by testing for associations of genetically regulated alternative splicing with both in vivo plasma LDL levels and in vitro cholesterol-related phenotypes. SNP functionality will be confirmed by site directed mutagenesis of mini-gene constructs (Aim 3). Demonstration of the role of alternative splicing in the regulation of cholesterol metabolism and identification of genetic determinants of this process will aid in delineating molecular pathways contributing to inter-individual variation in plasma LDL and thus improve our understanding of cardiovascular disease development and risk.
Funding Period: 2010-08-01 - 2015-05-31
more information: NIH RePORT

Top Publications

  1. pmc Coordinately regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake
    Marisa Wong Medina
    Department of Atherosclerosis Research, Children s Hospital Oakland Research Institute, Oakland, California, United States of America
    PLoS ONE 6:e19420. 2011
  2. pmc RHOA is a modulator of the cholesterol-lowering effects of statin
    Marisa W Medina
    Children s Hospital Oakland Research Institute, Oakland, California, United States of America
    PLoS Genet 8:e1003058. 2012
  3. pmc Multivariate methods and software for association mapping in dose-response genome-wide association studies
    Chad C Brown
    Department of Statistics, North Carolina State University, Raleigh, NC, USA
    BioData Min 5:21. 2012
  4. pmc A common polymorphism in the LDL receptor gene has multiple effects on LDL receptor function
    Feng Gao
    Children s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA
    Hum Mol Genet 22:1424-31. 2013
  5. pmc Alternative splicing in the regulation of cholesterol homeostasis
    Marisa W Medina
    Children s Hospital Oakland Research Institute, Oakland, CA 94609, USA
    Curr Opin Lipidol 24:147-52. 2013
  6. pmc HNRNPA1 regulates HMGCR alternative splicing and modulates cellular cholesterol metabolism
    Chi Yi Yu
    Children s Hospital Oakland Research Institute, Oakland, CA 94609, USA
    Hum Mol Genet 23:319-32. 2014
  7. pmc Statin-induced changes in gene expression in EBV-transformed and native B-cells
    Eugene Bolotin
    Children s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA
    Hum Mol Genet 23:1202-10. 2014

Detail Information

Publications7

  1. pmc Coordinately regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake
    Marisa Wong Medina
    Department of Atherosclerosis Research, Children s Hospital Oakland Research Institute, Oakland, California, United States of America
    PLoS ONE 6:e19420. 2011
    ..Thus coordinated regulation of alternative splicing may contribute to cellular cholesterol homeostasis as well as plasma LDL levels...
  2. pmc RHOA is a modulator of the cholesterol-lowering effects of statin
    Marisa W Medina
    Children s Hospital Oakland Research Institute, Oakland, California, United States of America
    PLoS Genet 8:e1003058. 2012
    ....
  3. pmc Multivariate methods and software for association mapping in dose-response genome-wide association studies
    Chad C Brown
    Department of Statistics, North Carolina State University, Raleigh, NC, USA
    BioData Min 5:21. 2012
    ..However, previous studies may have over-simplified the complex differences in dose-response profiles between genotypes, resulting in a loss of power...
  4. pmc A common polymorphism in the LDL receptor gene has multiple effects on LDL receptor function
    Feng Gao
    Children s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA
    Hum Mol Genet 22:1424-31. 2013
    ....
  5. pmc Alternative splicing in the regulation of cholesterol homeostasis
    Marisa W Medina
    Children s Hospital Oakland Research Institute, Oakland, CA 94609, USA
    Curr Opin Lipidol 24:147-52. 2013
    ..In this review, we highlight recent studies examining alternative splicing as a modulator of cellular cholesterol homeostasis and as an underlying mechanism of dyslipidemia...
  6. pmc HNRNPA1 regulates HMGCR alternative splicing and modulates cellular cholesterol metabolism
    Chi Yi Yu
    Children s Hospital Oakland Research Institute, Oakland, CA 94609, USA
    Hum Mol Genet 23:319-32. 2014
    ..These results suggest that HNRNPA1 plays a role in the variation of cardiovascular disease risk and statin response...
  7. pmc Statin-induced changes in gene expression in EBV-transformed and native B-cells
    Eugene Bolotin
    Children s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA
    Hum Mol Genet 23:1202-10. 2014
    ..This dataset is now uploaded to GEO at the accession number GSE51444...

Research Grants30

  1. CELLULAR AND MOLECULAR BIOLOGY OF LIPOPROTEIN METABOLISM
    Michael C Phillips; Fiscal Year: 2013
    ..The reasons for this protective effect are not understood fully and this project seeks to uncover the molecular mechanisms underlying the beneficial properties of HDL. ..
  2. DEVELOPMENT AND CONTROL OF PULMONARY ALVEOLAR STABILITY
    Samuel Hawgood; Fiscal Year: 2013
    ..abstract_text> ..
  3. Molecular Mechanisms linking Aging, Abeta Proteotoxicity and Neurodegeneration
    Jeffery W Kelly; Fiscal Year: 2013
    ..abstract_text> ..
  4. Cholesterol Homeostasis in Malignant Glioma
    ELIZABETH ANNE KOLAR; Fiscal Year: 2013
    ..This research may lead to new therapeutic strategies for human cancers and further our understanding of the role of lipid metabolism in malignancy. ..
  5. REGULATION OF CHOLESTEROL HOMEOSTASIS BY NONCODING RNAS
    Daniel S Ory; Fiscal Year: 2013
    ..The goal of this proposal is to study how these genes modulate the distribution and level of cholesterol in cells. The proposed studies may identify new targets for drug development for lowering cholesterol in disease states. ..
  6. Control of Sterol and Lipoprotein Homeostasis by miRNA
    Angel Baldán; Fiscal Year: 2013
    ..The results of these studies might lead to novel, improved ways to manage patients with hypercholesterolemia and/or patients with cholestasis. ! ..
  7. LIPID AND LIPOPROTEIN METABOLISM IN ATHEROSCLEROSIS
    Alan M Fogelman; Fiscal Year: 2013
    ..These six Projects will be supported by four cores and together will form a highly interactive and synergistic Program Project that is focused on lipid and lipoprotein metabolism in atherosclerosis. ..
  8. Inflammatory responses of vascular cells
    Paul L Fox; Fiscal Year: 2013
    ..abstract_text> ..
  9. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
    ..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
  10. MicroRNAs as physiological and pathological regulators of cholesterol homeostasis
    Kathryn J Moore; Fiscal Year: 2013
    ..These studies may uncover novel therapeutic strategies for the treatment of cardiovascular disease. ..
  11. MOLECULAR BASIS OF CHOLESTEROL METABOLISM
    Joseph L Goldstein; Fiscal Year: 2013
    ..Such an integrated interdisciplinary approach is possible only through continued support of this PPG. ..
  12. Integrative Genomics of Vanin Gene Expression in Relation to CVD Risk
    Eric Moses; Fiscal Year: 2013
    ..The knowledge gained may lead to better methods for identifying those at greatest risk of CVD and point to new strategies for drug therapy. ..