Abstract

Normal levels and developmental control of globin synthesis in fetal and adult erythrocytes is critically dependent on the unusual stability of the encoding mRNAs. Our prior studies establish a link between stabilization of human (h)a-globin mRNA and formation of a sequence specific RNA-protein (RNP) complex ('a-Complex') at its 3' untranslated region. We hypothesize that this a-Complex, composed of a defined polypyrimidine tract bound by a sequence-specific RNA binding protein, aCP, stabilizes a-globin mRNA by controlling one or more rate-limiting steps in mRNA decay. These observations will be extended in this proposal by focusing on three Specific Aims.
In Aim I we focus on characterization of structural determinants and mechanism(s) of ha-globin mRNA stabilization. Using a set of Tet-transactivator cell lines we will characterize and compare a-globin mRNA decay pathways in erythroid and nonerythroid environments, identify and characterize influences of 5'UTR and coding sequences on a-Complex function, and determine whether aCP is fully sufficient to mediate ha-globin mRNA stabilization.
In Aim II we will characterize protein-protein interactions involved in a-Complex action. Interactions of aCP with candidate partner proteins will be assessed and their functional importance tested using an in vitro mRNA decay assay.
In Aim m we will characterize the corresponding roles of nuclear and cytoplasmic aCP in assembly and function of the a-Complex. We will identify elements in aCP that dictate its subcellular localization, determine whether aCP associates with ha-globin mRNA in the nucleus, determine the site of cytoplasmic aCP action on ha-globin mRNA, and design dominant-negative mutations of aCP to probe its distinct nuclear and cytoplasmic functions. The characterization of the determinants and mechanisms involved in stabilization of ha-globin mRNA outlined in this proposal is central to a understanding of globin gene expression in health and disease and to the design of therapeutic approaches to a broad spectrum of hereditary anemias.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065449-04
Application #
6645365
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Evans, Gregory
Project Start
2000-09-05
Project End
2004-08-31
Budget Start
2003-09-01
Budget End
2004-08-31
Support Year
4
Fiscal Year
2003
Total Cost
$277,375
Indirect Cost

  Institution

Name
University of Pennsylvania
Department
Genetics
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104

  Publications

Grevet, Jeremy D; Lan, Xianjiang; Hamagami, Nicole et al. (2018) Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361:285-290
Ghanem, Louis R; Kromer, Andrew; Silverman, Ian M et al. (2018) Poly(C)-Binding Protein Pcbp2 Enables Differentiation of Definitive Erythropoiesis by Directing Functional Splicing of the Runx1 Transcript. Mol Cell Biol 38:
Ji, Xinjun; Humenik, Jesse; Yang, Daphne et al. (2018) PolyC-binding proteins enhance expression of the CDK2 cell cycle regulatory protein via alternative splicing. Nucleic Acids Res 46:2030-2044
Ghanem, Louis R; Kromer, Andrew; Silverman, Ian M et al. (2016) The Poly(C) Binding Protein Pcbp2 and Its Retrotransposed Derivative Pcbp1 Are Independently Essential to Mouse Development. Mol Cell Biol 36:304-19
Kini, Hemant K; Silverman, Ian M; Ji, Xinjun et al. (2016) Cytoplasmic poly(A) binding protein-1 binds to genomically encoded sequences within mammalian mRNAs. RNA 22:61-74
Ji, Xinjun; Park, Juw Won; Bahrami-Samani, Emad et al. (2016) ?CP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome. Nucleic Acids Res 44:2283-97
Sriswasdi, Sira; Harper, Sandra L; Tang, Hsin-Yao et al. (2014) Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry. Proc Natl Acad Sci U S A 111:1801-6
Kini, Hemant K; Kong, Jian; Liebhaber, Stephen A (2014) Cytoplasmic poly(A) binding protein C4 serves a critical role in erythroid differentiation. Mol Cell Biol 34:1300-9
Ghanem, Louis R; Chatterji, Priya; Liebhaber, Stephen A (2014) Specific enrichment of the RNA-binding proteins PCBP1 and PCBP2 in chief cells of the murine gastric mucosa. Gene Expr Patterns 14:78-87
Harper, Sandra L; Sriswasdi, Sira; Tang, Hsin-Yao et al. (2013) The common hereditary elliptocytosis-associated *-spectrin L260P mutation perturbs erythrocyte membranes by stabilizing spectrin in the closed dimer conformation. Blood 122:3045-53

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