Posttranscriptional regulation (PTR) has come into vogue since the discovery of microRNAs and their ability to regulate developmental processes. However, RNA-binding proteins (RBPs) are well-established regulators of mRNAs and also play major roles in modulating PTR. This proposal will examine the cooperation and/or competition between microRNAs and the Hu RBPs in regulating mRNAs encoding members of the Wnt/B-catenin Pathway. The Wnt pathway plays important roles in cell-cell communication during development and in tumors. Recently, we have identified 61 Wnt pathway members whose mRNA associate with HuR in Jurkat T cells. Given that the Wnt pathway has been heavily studied in neuronal systems, this proposal will examine the regulation of these mRNAs by Hu proteins and predicted microRNAs during neuronal differentiation of P19 embryonic carcinoma cells treated with retinoic acid. I hypothesize that Hu proteins collaborate with microRNAs during neuronal differentiation to regulate the production of Wnt pathway components, and that this results in coordination of expression per the posttranscriptional RNA operon (PTRO) model derived previously in the Keene laboratory.
The specific aims of the project are: 1. Identify the mRNA targets associated with HuR during neuronal development. We will use ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-Chip) to identify subsets of messages associated with HuR and investigate their dynamics during P19 neuronal development. 2. Investigate the interaction between HuR and miRNAs in neurons. RIP-Chip analysis, along with real time PCR and Northern analysis will be used to identify the subset of miRNAs associated with the HuR mRNP in neurons. 3. Investigate HuR's posttranscriptional role during neuronal development.
This aim will study the impact of HuR on the expression and regulation of the Wnt pathway during neuronal development. By overexpressing and/or knocking down HuR in P19 cells and looking at the effects on Wnt family member RNA and protein expression and transcriptional activity we can begin to understand the regulatory role of HuR. Relevance: This project will investigate the posttranscriptional regulation of the Wnt pathway during neuronal development. By better understanding this oft-overlooked form of regulation, we will identify potential targets for therapeutic intervention in diseases involving the Wnt pathway. ? ?

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F03A-M (20))
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Riddle, Robert D
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Duke University
Schools of Medicine
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Mansfield, Kyle D; Keene, Jack D (2012) Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation. Nucleic Acids Res 40:2734-46
Mansfield, Kyle D; Keene, Jack D (2009) The ribonome: a dominant force in co-ordinating gene expression. Biol Cell 101:169-81