Structure and Function of the Decapping Enzyme Complex

描述(申请人提供)：mRNA的破坏是真核基因表达中的一个关键事件，在动物早期发育、细胞生长、增殖和适应应激反应中发挥关键作用。专门的mRNA稳定途径确保了含有提前终止密码子的异常转录本被消除，并严格控制了关键转录本的丰度，如编码细胞因子、白介素和原癌基因的转录本。在这些途径中，一个关键的调控步骤是Dcp2去除5‘末端帽结构，Dcp2通过将mRNA主体的5’一磷酸暴露于5‘到3’外切核糖核酸酶来判定mRNA的破坏。Dcp2的活性受到多种途径特异性共激活剂的刺激，其机制尚不清楚。以发芽酵母为模型系统，我们将结合生化、生物物理和遗传学方法来确定必需激活剂Dcp1是如何调节Dcp2的催化活性的。在特定目标1中，对Dcp1/Dcp2与非水解性底物的复合体的结晶学研究将指导突变体的动力学和遗传学分析，以剖析解帽的化学步骤；在特定目标2中，我们将利用核磁共振光谱来确定Dcp1是否通过改变非活性和活性形式之间的构象平衡来增强Dcp2的催化活性；在特定目标3中，我们将确定Dcp1/Dcp2与解链蛋白家族成员增强剂Edc1之间的三元复合体的晶体结构，以确定共激活剂的刺激机制。这些综合研究将阐明影响数千个人类基因丰度的几个信使核糖核酸稳定途径的关键一步。与公共卫生相关：数千个人类基因的表达受信使RNA(信使RNA)的协调破坏调控。控制这一过程的蛋白质因素的突变在人类疾病中被观察到，包括几种癌症和遗传性疾病。我们试图了解控制信使核糖核酸解离的分子机制：使信使其被破坏的倒数第二个、不可逆转的步骤。这些研究提供的生化和结构细节可能为治疗遗传性遗传疾病和癌症铺平道路。

DESCRIPTION (provided by applicant): The destruction of mRNA is a key event in eukaryotic gene expression, playing a crucial role in early animal development, cellular growth , proliferation and adaptation to stress. Dedicated mRNA stability pathways ensure that aberrant transcripts containing premature stop codons are eliminated and the abundance of key transcripts such as those coding for cytokines, interleukins and proto-oncogenes are tightly controlled. A critical, regulated step in these pathways is the removal of the 5' terminal cap structure by Dcp2, which sentences an mRNA for destruction by exposing the 5' monophosphate of the mRNA body to 5' to 3' exoribonucleases. The activity of Dcp2 is stimulated by a variety of pathway specific co-activators through mechanisms that are not well understood. Using budding yeast as a model system, we will combine biochemical , biophysical and genetic methods to determine how the essential activator Dcp1 regulates the catalytic activity of Dcp2. In specific aim 1, crystallographic studies of the Dcp1/Dcp2 complex with non- hydrolyzable substrate will guide kinetic and genetic analyses of mutants to dissect the chemical step of decapping ; in specific aim 2, we will use NMR spectroscopy to determine if Dcp1 enhances the catalytic activity of Dcp2 by shifting a conformational equilibrium between inactive and active forms; in specific aim 3, we will determine the crystal structure of the ternary complex between Dcp1/Dcp2 with the Enhancer of decapping protein family member , Edc1, to define the mechanism of stimulation by co-activators. These integrated studies will shed light on a critical step in several mRNA stability pathways that affects the abundance of thousands of human genes. PUBLIC HEALTH RELEVANCE: The expression of thousands of human genes is regulated by the coordinated destruction of messenger RNA (mRNA). Mutations in protein factors that control this process are observed in human diseases, including several cancers and genetic disorders. We seek to understand molecular mechanisms controlling mRNA decapping: the penultimate, irreversible step committing an mRNA to destruction. The biochemical and structural details provided by these studies may pave the way to treat inherited genetic disorders and cancer.