Translational control by the eIF4 factors

描述(申请人提供)：在翻译水平上控制基因表达使细胞能够快速而准确地对内部和外部变化做出反应，这一过程中的缺陷可能会产生可怕的后果。癌症、心脏病和其他发育和神经疾病与磷酸化级联反应的缺陷有关，磷酸化级联反应负责修饰真核细胞翻译起始因子(EIF)，并导致翻译效率和特定mRNAs的翻译发生全球变化，但在大多数情况下，这些磷酸化事件的分子结果尚不清楚。我们工作的长期目标是在核糖体翻译预起始复合体(PIC)的mRNA招募水平上了解翻译控制的分子基础。通过进一步研究EIF的磷酸化如何改变翻译效率，我们将深入了解癌症和其他疾病病因的基本分子机制。在翻译启动过程中，EIF和其他因素在PIC和mRNAs的元件内相互作用，以确定翻译什么mRNAs。我们从纯化的酵母成分中重组了体外翻译起始，并就eIF4B和eIF4F在促进mRNA招募步骤中的作用取得了重要发现。我们证明了eIF4B直接与核糖体结合，并促进eIF4F与启动机制的功能相互作用，以促进mRNA募集到PIC，挑战了以前的观点，即eIF4B作为RNA结合蛋白激活mRNAs进行翻译。我们也有证据表明eIF4G以一种不依赖于mRNA的方式与PIC相互作用。这导致我们假设，与其像所建议的那样独立于核糖体激活mRNPs，eIF4因子与PIC结合形成直接招募和解开mRNA的holoPIC。为了研究这种可能性，并了解招募mRNAs的基本系统，我们将表征eIF4因子与PIC的相互作用。然后，我们将询问这些相互作用发生了什么，并评估当eIF4因子被磷酸化时，翻译和表型的结果变化。在未来，这将使我能够提出更多重要的问题，涉及响应单个磷酸化事件而翻译的mRNAs，以及一般的翻译启动机制如何被修改以允许对特定mRNAs的翻译控制，这使我有别于我的导师Jon Lorsch。在追求这些问题的过程中，我将获得新的技能，并建立一个令人兴奋的项目，使我成为教师职位的更具竞争力的候选人。该奖项将提供资源，帮助我实现成为一名独立研究员的目标，并使我能够更快地为成功的R01申请获得初步数据。

DESCRIPTION (provided by applicant): Controlling gene expression at the level of translation allows cells to respond rapidly and precisely to internal and external changes, and defects in this process can have dire consequences. Cancer, heart disease, and other developmental and neurological disorders have been linked to defects in phosphorylation cascades that are responsible for modifying eukaryotic translation initiation factors (eIFs) and actuating global changes in translation efficiency and in the translation of specific mRNAs, yet the molecular outcome of these phosphorylation events is not understood in most cases. The long-term goal of our work is to understand the molecular basis for translational control at the level of mRNA recruitment to the ribosomal translation preinitiation complex (PIC). By further investigating how phosphorylation of eIFs changes the efficiency of translation, we will make insights into the basic molecular mechanisms underlying the etiology of cancer and other diseases. During translation initiation, eIFs and other factors interact within the PIC and elements of mRNAs to determine what mRNAs are translated. We have reconstituted translation initiation in vitro from purified yeast components and made important findings regarding the roles of eIF4B and eIF4F in promoting the mRNA recruitment step. We demonstrated that eIF4B binds directly to the ribosome, and promotes functional interaction of eIF4F with the initiation machinery to promote mRNA recruitment to the PIC, challenging the previously held view that eIF4B acts as an RNA-binding protein to activate mRNAs for translation. We also have evidence that eIF4G interacts with the PIC in an mRNA-independent manner. This leads us to hypothesize that rather than activating mRNPs independently of the ribosome as has been suggested, the eIF4 factors bind to the PIC to form a holoPIC that directly recruits and unwinds mRNA. To investigate this possibility, and to understand the basic system in place for recruiting mRNAs, we will characterize the interactions of the eIF4 factors with the PIC. We will then ask what happens to these interactions and assess the resulting changes in translation and phenotype when the eIF4 factors are phosphorylated. In the future this will allow me to ask additional important questions about the mRNAs that are translated in response to single phosphorylation events and how the general translation initiation machinery is modified to allow translational control of specific mRNAs, distinguishing me from my mentor, Jon Lorsch. In the course of pursuing these questions, I will gain new skills and set up an exciting project that will make me a more competitive candidate for faculty positions. The award would provide the resources to help me achieve my goal of becoming an independent researcher, and allow me to acquire preliminary data for a successful R01 application more quickly.