The broad objective of this proposal is to understand posttranscriptional control of early animal development. One mechanism that regulates maternal mRNA expression is cytoplasmic polyadenylation. CPEB, a sequence-specific RNA binding protein, is at the heart of this process;it nucleates a number of factors on specific mRNAs and controls translation by modulating poly(A) tail length. Polyadenylation does not happen en masse, but instead occurs in sequential waves that are most evident during oocyte maturation in Xenopus. The first wave happens at meiosis I (MI) while the second occurs at meiosis II (MII). One determinant for first or second wave polyadenylation is the amount of CPEB in the cell. During the MI to MII transition, some of the CPEB is destroyed, which is important for second wave polyadenylation. CPEB destruction requires multiple cdk1 phosphorylations, subsequent ubiquitination, and Pin1 (peptidylprolyl cis/trans isomerase) activity. Pin1 activity increases during meiosis and binds and controls CPEB destruction. Moreover, Pin1 mediates CPEB destruction in mammalian cells as well. Pin1 also binds phospho-maskin, the CPEB- and eIF4E- binding factor that coincides with maskin phosphorylation and dissociation from eIF4E, allowing initiation complex assembly. The goals of the first specific aim are to determine how Pin1 mediates CPEB destruction and MI to MII transitioning, and whether it regulates translation by disrupting the maskin-eIF4E interaction. The second specific aim intends construct an integrated network map of polyadenylation during maturation. To do so, CLIP (crosslink IP) will be employed;it will identify not only the mRNAs to which CPEB binds, but where on the mRNA CPEB binds. This analysis will define, on a genome wide scale, what constitutes a CPEB binding element (CPE). These experiments will be complemented by the identification of all the mRNAs that undergo cytoplasmic polyadenylation, and which of 2 non-canonical poly(A) polymerases are used. Translational control by CPEB regulates not only early development, but neuronal synaptic cellular senescence as well. Thus, these experiments have important implications of human infertility, neurodegeneration, and cancer etiology.
8. Project Narrative The regulation of gene expression by the protein CPEB controls early animal development, higher brain functions, and anti-cancer mechanisms. Thus, detailed biochemical analysis of CPEB is essential for these critical biological processes, which clearly impacts human health.
|Ivshina, Maria; Lasko, Paul; Richter, Joel D (2014) Cytoplasmic polyadenylation element binding proteins in development, health, and disease. Annu Rev Cell Dev Biol 30:393-415|
|Nechama, Morris; Lin, Chien-Ling; Richter, Joel D (2013) An unusual two-step control of CPEB destruction by Pin1. Mol Cell Biol 33:48-58|
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|Cao, Quiping; Padmanabhan, Kiran; Richter, Joel D (2010) Pumilio 2 controls translation by competing with eIF4E for 7-methyl guanosine cap recognition. RNA 16:221-7|
|Lin, Chien-Ling; Evans, Veronica; Shen, Shihao et al. (2010) The nuclear experience of CPEB: implications for RNA processing and translational control. RNA 16:338-48|
|Kan, Ming-Chung; Oruganty-Das, Aparna; Cooper-Morgan, Amalene et al. (2010) CPEB4 is a cell survival protein retained in the nucleus upon ischemia or endoplasmic reticulum calcium depletion. Mol Cell Biol 30:5658-71|
|Kim, Jong Heon; Richter, Joel D (2008) Measuring CPEB-mediated cytoplasmic polyadenylation-deadenylation in Xenopus laevis oocytes and egg extracts. Methods Enzymol 448:119-38|
|Kim, Jong Heon; Richter, Joel D (2007) RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB. Genes Dev 21:2571-9|
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