Specialized regulation of non-AUG translation

项目摘要/摘要 尽管长期以来人们普遍认为8月的起始密码子标志着开放阅读的开始 框架中，核糖体足迹图谱的最新进展表明，核糖体在非 Aug密码子(例如，CUG或GUG)以惊人的频率出现。规范的Aug起始密码子由 启动子tRNAi在扫描预引发复合体内，但在很大程度上仍不清楚非AUG是如何开始的 相遇 密码子被识别或管理。考虑到已经显示了非Aug翻译的异常形式 引起多种神经退行性疾病并促成癌症恶性，更清楚地认识 如何在分子水平上控制非AUG翻译是非常需要的。使用的是表达 相同的报告蛋白，但来自不同的起始密码子，我惊讶地发现，一名来自 Cug或Gug起始密码子不被特征良好的翻译抑制剂抑制，事实上，它增加了 随着时间的推移。这与我在记者启动时观察到的预期翻译受阻形成了鲜明对比 来自一个典型的八月。这些数据有力地表明，核糖体翻译来自非8月的蛋白质 起始密码子与起始于8月密码子的核糖体有根本的不同。在接受指导的过程中 阶段，我将获得生物化学和生物信息学方面的新培训，以解决如何在不同的起点进行翻译 密码子是由核糖体和mRNAs中的序列元件共同调控的。在《目标1》中，我将 亲和力Purify翻译从Aug或Gug起始密码子开始并确定 结合核糖体的组成，以及鉴定可能赋予抗药性的其他相关蛋白 翻译抑制药。这些实验将揭示核糖体如何翻译不同的 开放阅读框架可以是不同的，并由不同的调控途径控制。在目标2中，我将使用 候选基因方法和全基因组核糖体图谱相结合的方法来识别调控非 内源基因的AUG翻译。通过识别和表征关键的调控序列，这 AIM应该允许我们预测和验证哪些基因是由不同的核糖体类别翻译的 细胞。在目标3中，我将充分利用这次培训，并在我自己的实验室中确定非8月 当蛋白质合成在细胞应激过程中自然减弱时，翻译受到调节。特别是，我们将 使用核糖体图谱来识别在细胞应激和 描述这些事件如何影响应激反应和细胞存活。在短期内，建议的 由我的专家导师和合作者提供的新培训(目标1和2)，以及补充的正式培训 课程作业将提供一个基础，我可以在此基础上继续在我的独立实验室中揭示 AUG翻译受到人类生物学的调控(目标3)。学校良好的训练环境 Wilusz&Dreyfuss实验室和宾夕法尼亚大学不仅将极大地促进有指导的研究，而且 请赋予我必要的专业技能，使我能够过渡到独立的教职岗位。

PROJECT SUMMARY/ABSTRACT Although it has long been thought that an AUG start codon universally marks the beginning of an open reading frame, recent advances in ribosome footprint mapping have revealed that ribosomes initiate translation at non- AUG codons (e.g., CUG or GUG) at an astonishing frequency. Canonical AUG start codons are recognized by the initiator tRNAi within the scanning pre-initiation complex, but it is still largely unclear how non-AUG start Met codons are recognized or regulated. Considering that aberrant forms of non-AUG translation have been shown to cause multiple neurodegenerative diseases and contribute to cancer malignancy, a clearer understanding of how non-AUG translation is controlled on the molecular level is greatly needed. Using plasmids that express the same reporter protein but from different start codons, I surprisingly found that a reporter expressed from a CUG or GUG start codon was not inhibited by well-characterized translation inhibitors and, in fact, increased over time. This is in stark contrast to the expected inhibition of translation I observed when the reporter initiated from a canonical AUG. These data strongly indicate that ribosomes translating proteins from non-AUG start codons are fundamentally different from ribosomes that start at AUG codons. During the mentored phase, I will gain new training in biochemistry and bioinformatics to address how translation at different start codons is uniquely regulated by both the ribosome and sequence elements within mRNAs. In Aim 1, I will affinity purify translating reporter mRNAs that initiate from an AUG or GUG start codon and determine the composition of the bound ribosomes, as well as identify other associated proteins that may confer resistance to translation inhibitors. These experiments will reveal important insights into how ribosomes translating distinct open reading frames can be different and be controlled by separate regulatory pathways. In Aim 2, I will use a candidate gene approach along with genome-wide ribosome profiling to identify sequences that regulate non- AUG translation from endogenous genes. By identifying and characterizing the key regulatory sequences, this aim should allow us to predict and validate which genes are translated by separate ribosome classes within cells. In Aim 3, I will take full advantage of this training and determine in my own laboratory how non-AUG translation is regulated when protein synthesis is naturally attenuated during cell stress. In particular, we will use ribosome profiling to identify non-AUG open reading frames that are regulated during cell stress and characterize how these events impact the stress response and cell survival. In the short term, the proposed new training (Aims 1 & 2) by my expert mentors and collaborators, along with complementary formal coursework, will provide a foundation on which I can continue to reveal in my independent laboratory how non- AUG translation is regulated and controls human biology (Aim 3). The excellent training environment in the Wilusz & Dreyfuss laboratories and at Penn will greatly facilitate not only the mentored research, but also endow me with the necessary professional skills to transition to an independent faculty position.