Structure and Function of the Decapping Enzyme Complex

描述（由申请人提供）：mRNA的破坏是真核基因表达中的关键事件，在早期动物发育、细胞生长、增殖和适应应激中起关键作用。专用的mRNA稳定性途径确保消除含有提前终止密码子的异常转录物，并且严格控制关键转录物（例如编码细胞因子、白介素和原癌基因的转录物）的丰度。在这些途径中的一个关键的、受调控的步骤是通过Dcp 2去除5'端帽结构，其通过将mRNA体的5'单磷酸暴露于5'至3'外切核糖核酸酶来破坏mRNA。Dcp 2的活性由多种途径特异性共激活剂通过尚不清楚的机制刺激。以芽殖酵母为模型系统，我们将联合收割机结合生物化学、生物物理和遗传学方法来确定必需激活剂Dcp 1如何调节Dcp 2的催化活性。在具体目标1中，具有不可水解底物的Dcp 1/Dcp 2复合物的晶体学研究将指导突变体的动力学和遗传分析，以剖析开帽的化学步骤;在具体目标2中，我们将使用NMR光谱来确定Dcp 1是否通过在非活性和活性形式之间改变构象平衡来增强Dcp 2的催化活性;在具体目标3中，我们将确定Dcp 1/Dcp 2与脱帽蛋白家族成员Edc 1的增强子之间的三元复合物的晶体结构，以确定共激活剂的刺激机制。这些综合研究将揭示影响数千个人类基因丰度的几种mRNA稳定性途径中的关键步骤。公共卫生相关性：数千个人类基因的表达受到信使RNA（mRNA）协同破坏的调节。在人类疾病中观察到控制这一过程的蛋白质因子的突变，包括几种癌症和遗传性疾病。我们试图了解控制mRNA去帽的分子机制：将mRNA破坏的倒数第二个不可逆步骤。这些研究提供的生物化学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.