(from the application): Aging affects expression of a number of genes including those that control cell growth and differentiation. Regulation of gene expression occurs at levels of transcription, KNA splicing, stability and translation as well as at the level of post translational modifications. RNA binding proteins are key factors that control RNA processing. This application investigates the role of RNA binding proteins in aging. The major hypothesis of this application is that the activity of RNA binding proteins is affected by aging which, in turn, leads to increased expression of aging associated proteins. Age dependent regulation of two targets of RNA binding proteins, C/EBPB and p21, will be examined to test this hypothesis. We observed that stability of p21 mRNA is increased in senescent fibroblasts. This observation suggests that RNA binding proteins stabilize p21 mRNA during senescence. An RNA binding protein, CUGBP1, binds to the 5' region of p21 mRNA and induces production of p21 in a cell-free translation system.
Specific Aim 1 investigates the role of RNA binding proteins in the stabilization of p21 mRNA during senescence. Additional RNA binding proteins that differentially interact with p21 mRNA will be identified, isolated and used for the study of their role in p21 regulation during senescence. Regulation of p21 mRNA stability/translation by RNA binding proteins will be examined in transient experiments and in stable clones. It is well documented that aging alters the production of C/EBPB isoforms. Levels of a dominant negative isoform of C/EBPB, LIP, are induced in livers of older animals. The production of LIP in liver occurs via initiation of translation from the third initiation codon of a single C/EBPB mRNA. The RNA binding protein, CUGBP1, induces translation of LIP in a cell-free translation system and is associated with polysomes producing LIP in liver. This application will test the hypothesis that aging mediated induction of LIP occurs via alterations in activity of CUGBP1 or/and other RNA binding proteins. Because phosphorylation of CUGBP1 is increased in old animals, we suggest that aging induces the binding activity of CUGBP1 via phosphorylation. Activated CUGBP1 binds to the 5' region of C/EBPB mRNA and induces translation of LIP in older animals.
In Specific Aim II, we will define age dependent sites of phosphorylation in CUGBP1 and examine the role of CUGBP1 in LIP production. We will also examine age dependent alterations of RNA binding proteins in quiescent rat liver and in liver after partial hepatectomy.
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