P21 and a family of transcription factors, CCAAT/Enhancer Binding Proteins (C/EBP), play a crucial role in the regulation of cell growth and differentiation. C/EBPalpha and p21 are strong inhibitors of cell proliferation, since both are capable of binding to cdk2 and cdk4 and inhibiting their activities. Although the expression of C/EBP proteins and p21 are primarily regulated at the level of transcription, recent observations showed that the inhibition of translation of C/EBPalpha by RNA binding proteins is a key event in development of tumors such as leukemias. We have recently found that a specific group of cytoplasmic RNA binding proteins interact with GC-rich regions within C/EBPalpha, C/EBPbeta and p21 mRNAs and are able to regulate translation of these mRNAs. Two of these RNA binding proteins were isolated and characterized in detail. Calreticulin (CRT) interacts with stem-loop structures within C/EBPalpha, C/EBPbeta and p21 mRNAs and inhibits their translation. We also found that an RNA binding protein, CUGBP1, binds to 5' regions of C/EBPbeta and p21 mRNAs and that this interaction increases translation of C/EBPbeta and p21. The major hypothesis of this application is that RNA binding proteins regulate cell growth and differentiation by controlling translation of C/EBP proteins and p21.
Three Specific Aims are designed to use a variety of biological models including a cell-free system, cultured cells and animal models address this hypothesis.
Specific Aim I examines the role of CRT-dependent inhibition of C/EBPalpha in the regulation of cell growth.
Specific Aim II investigates the role of CRT-mediated regulation of p21 in cellular senescence. Specifically, we will test the hypothesis that release of CRT-dependent inhibition of p21 translation is required for the elevation of p21 in senescent fibroblasts.
In Specific Aim III, we will test the hypothesis that competition of RNA binding proteins for regulation of p21 and C/EBPbeta translation determines cell fate. This hypothesis is based on our observations showing that CRT and CUGBP1 bind to the same sequences within p21 and C/EBPbeta mRNAs, but the results of these interactions are different. Together, these studies will provide important insight into how particular RNA binding proteins function through newly discovered pathways to control cell division.
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