Regulation of Cell Growth by RNA Binding Proteins

描述(申请人提供)：p21和一个转录因子家族，CCAAT/增强子结合蛋白(C/EBP)，在调节细胞生长和分化中发挥关键作用。C/EBPalpha和p21都能与CDK2和CDK4结合并抑制其活性，是细胞增殖的强有力的抑制因子。虽然C/EBP蛋白和p21的表达主要是在转录水平上调节的，但最近的观察表明，RNA结合蛋白抑制C/EBPalpha的翻译是白血病等肿瘤发生的关键事件。我们最近发现，一组特定的细胞质RNA结合蛋白与C/EBPalpha、C/EBPbeta和p21mRNAs中的GC富集区相互作用，并能够调节这些mRNAs的翻译。其中两个RNA结合蛋白被分离出来并进行了详细的鉴定。钙网蛋白(CRT)与C/EBPalpha、C/EBPbeta和p21mRNAs中的茎环结构相互作用并抑制其翻译。我们还发现，RNA结合蛋白CUGBP1与C/EBPbeta和p21mRNAs的5‘区结合，这种相互作用增加了C/EBPbeta和p21的翻译。这一应用的主要假设是，RNA结合蛋白通过控制C/EBP蛋白和p21的翻译来调节细胞的生长和分化。设计了三个特定的目标来使用各种生物模型，包括无细胞系统、培养细胞和动物模型来解决这一假设。特定目的I研究依赖CRT的C/EBPalpha抑制在细胞生长调节中的作用。特殊目的II研究CRT介导的p21调控在细胞衰老中的作用。具体地说，我们将测试这一假设，即在衰老的成纤维细胞中，需要释放依赖CRT的p21翻译抑制来提高p21的水平。在特定的目标III中，我们将检验这样的假设，即RNA结合蛋白对p21和C/EBPbeta翻译的调节竞争决定了细胞的命运。这一假设是基于我们的观察结果，即CRT和CUGBP1与p21和C/EBPbeta mRNAs中的相同序列结合，但这些相互作用的结果不同。总之，这些研究将为特定的RNA结合蛋白如何通过新发现的控制细胞分裂的途径发挥作用提供重要的见解。

DESCRIPTION (provided by applicant): 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.