The long-term goal of this grant is to establish a comprehensive understanding of post-transcriptional controls critical to mammalian erythroid differentiation. Erythroid differentiation is marked by a set of dramatic morphologic and functional changes, much of this process occurring in a transcriptionally-silent environment. For this reason erythroid differentiation is heavily dependent on post-transcriptional controls. RNA binding proteins (RBPs), the major regulators of post-transcriptional controls, impact on transcript processing in the nucleus as well as the stability and function of the mRNA in the cytoplasm. RBPs vary widely in their structures, binding specificities, and cellular compartmentalization and can modulate RNA functions by direct actions and/or via the recruitment of effector complexes. Identification of RBPs, their mRNA targets, and corresponding post-transcriptional controls have advanced our understanding of numerous developmental processes and have revealed unanticipated pathophysiologic pathways. By defining post-transcriptional controls in erythroid differentiation, we will expand our understanding of inherited and acquired disorders of erythropoiesis and establish a template for similar investigations in other systems. To achieve this goal, we will carry out a novel and state-of-the-art transcriptome-wide analyses to reveal the full range of mRNA-protein (mRNP) interactions that accompany the dynamic process of erythroid terminal differentiation. This approach combines biochemical methodologies with innovative informatic pipelines specifically designed for comprehensive descriptions of complex RNA-protein interactions. This unbiased analysis will be combined with a series of targeted in-depth mechanistic studies that focus on two sets of mRNA/protein interactions that we have identified to play central roles in erythroid differentiation. The combined output of these complementing approaches will establish the unique network of post-transcriptional controls in this developmentally robust and clinically relevant model system. This proposal encompasses three Specific Aims.
Aim 1. Characterize the roles of the polyC-binding proteins, ?1 and ?2, as post- transcriptional integrators of erythroid differentiation.
Aim 2. Identify the critical role(s) of the polyA binding protein, PABPC, in defining and sustaining the erythroid transcriptome.
Aim 3. Map the global structure of mRNA/protein interactions in erythroid cells and define the dynamic nature of these interactions in the differentiation process. Combining transcriptome-wide analyses with targeted mechanistic studies will generate a powerful access to the complex array of RNA/protein interactions that constitute critical determinants of erythroid differentiation. Success in these studies will fundamentally alter our understanding of this intensively studied pathway; serve as a prototype for investigations of post- transcriptional controls in other systems, and present novel targets fo future diagnostic and therapeutic innovations.

Public Health Relevance

The vast majority of disease processes in the human population reflect defects in gene structure and/or regulation of gene expression. The expression of a gene is dependent on multiple biochemical steps that copy a gene from DNA to RNA and then process the RNA so that it can be effectively translated into proteins. The controls over RNA processing and expression are complex and variable. By delineating these biochemical steps we will establish a knowledge base from which we can better understand abnormalities of gene regulation that underlie many inherited and acquired disease processes and will identify targets for novel diagnostic and therapeutic approaches.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065449-16
Application #
9011539
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Qasba, Pankaj
Project Start
2000-09-05
Project End
2019-11-30
Budget Start
2015-12-01
Budget End
2016-11-30
Support Year
16
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Genetics
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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
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. (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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