Specialized regulation of non-AUG translation

项目总结/摘要 尽管一直认为AUG起始密码子普遍标志着开放阅读的开始 框架，核糖体足迹作图的最新进展表明，核糖体在非转录水平启动翻译， AUG密码子（例如，或GUG）以惊人的频率。典型的AUG起始密码子被 在扫描前起始复合物内的起始剂tRNAi，但在很大程度上仍然不清楚非AUG如何启动 遇到 密码子被识别或调节。考虑到非AUG翻译的异常形式已经被证明是 导致多种神经退行性疾病并导致癌症恶性，更清楚地了解 如何在分子水平上控制非AUG翻译是非常需要的。利用质粒表达 相同的报告蛋白，但来自不同的起始密码子，我惊讶地发现， CUG或GUG起始密码子不被充分表征的翻译抑制剂抑制，事实上， 随着时间这与我观察到的预期的翻译抑制形成鲜明对比， 这些数据有力地表明，核糖体翻译蛋白质从非AUG 起始密码子与起始于AUG密码子的核糖体根本不同。在指导期间， 阶段，我将获得生物化学和生物信息学的新培训，以解决如何在不同的开始翻译 密码子是由核糖体和mRNA内的序列元件独特调节的。在目标1中，我将 亲和纯化起始于AUG或GUG起始密码子的翻译报告基因mRNA，并确定 结合的核糖体的组成，以及确定其他相关的蛋白质，可能赋予抗性， 翻译抑制剂这些实验将揭示核糖体如何翻译不同的 开放阅读框可以是不同的，并由单独的调节途径控制。在目标2中，我将使用 候选基因方法沿着全基因组核糖体分析以鉴定调节非- 来自内源基因的AUG翻译。通过识别和表征关键调控序列， 目的应该使我们能够预测和验证哪些基因是由内部不同的核糖体类别翻译的。 细胞在目标3中，我将充分利用这一培训，并在我自己的实验室中确定非AUG 当蛋白质合成在细胞应激期间自然减弱时，翻译受到调节。特别是要 使用核糖体分析来鉴定在细胞应激期间受调节的非AUG开放阅读框， 描述这些事件如何影响应激反应和细胞存活。在短期内，拟议的 新的培训（目标1和2）由我的专家导师和合作者，沿着补充正式 课程，将提供一个基础上，我可以继续揭示在我的独立实验室如何非- AUG翻译受到调节并控制人类生物学（目的3）。优秀的培训环境， Wilusz & Dreyfuss实验室和宾夕法尼亚大学将极大地促进不仅指导研究， 赋予我必要的专业技能，过渡到一个独立的教师职位。

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.