Defining the regulatory roles of alternative ribosome initiation and novel peptides

项目概要/摘要 人类基因组计划完成15年多后，我们对“注释”的理解 基因组仍然不完整。不属于“注释基因组”的替代核糖体起始位点 编码新的开放阅读框（ORF），其是注释蛋白的变体，上游不同的ORF 或注释蛋白质的下游，甚至长的非编码RNA上的ORF。这些新的ORF正在出现 作为快速重编程特定基因和蛋白质合成网络的翻译控制机制， 特别是在应激和细胞状态转化如肿瘤发生期间。此外，这些新的ORF 编码尚未鉴定的推定肽。因此，理解翻译控制 机制，以及编码肽的调节功能，可以揭示基础生物学和 治疗的目标。现有的替代起始或替代ORF编码肽的研究已经采取了 由于缺乏在全基因组范围内对其进行分析的工具，采取了临时性办法。我们的目标是发展和 应用深度测序和高通量，基于CRISPR的方法来绘制这些基因的功能作用， 非常规翻译转录组范围内，使用肿瘤发生作为模型系统。功能基因组 这些方法是唯一准备解决我们对功能性替代ORF的理解不足的方法。在 本提案的目的是描述非常规起始位点在 使用核糖体分析和RNA-seq在不同时间点观察肿瘤发生（目的1）。这将定义新的ORF 以及这些ORF的翻译是如何被调节以促进 致癌基因和肽。然后，我们将使用CRISPR筛选来识别肿瘤生长所必需的ORF， 并通过表征定位、物理相互作用和遗传修饰来定义新肽的功能。 交互作用（目标2）。最后，我们将机械地询问替代起始站点的使用情况，以调查如何 在细胞状态改变期间，翻译被调谐（Aim 3）。总的来说，该提案的结果将解决长期- 翻译控制的问题，并揭示新蛋白质的调节作用。相结合 在K99阶段获得的指导支持、技能和数据将为陈博士提供一个跳板， 在R 00阶段及以后作为研究者的独立性。我们的研究结果将提供新的 深入了解翻译控制的基本方面，并将定义与生物学相关的新范式， 疾病

PROJECT SUMMARY/ABSTRACT More than 15 years after the completion of the Human Genome Project, our understanding of the “annotated” genome is still incomplete. Alternative ribosome initiation sites that are not part of the “annotated genome” encode novel open reading frames (ORFs) that are either variants of annotated proteins, distinct ORFs upstream or downstream of annotated proteins, or even ORFs on long non-coding RNAs. These novel ORFs are emerging as a translational control mechanism to rapidly reprogram specific genes and protein synthesis networks, especially during stress and cell-state transformations such as tumorigenesis. Furthermore, these new ORFs encode putative peptides that remain uncharacterized. Thus, understanding the translational control mechanisms, as well as the regulatory functions of the encoded peptides, could reveal fundamental biology and targets for therapeutics. Existing studies of alternative initiation or alternative ORF-encoded peptides have taken an ad hoc approach, owing to a lack of tools to profile them at genome-wide scale. Our goal is to develop and apply deep sequencing and high-throughput, CRISPR-based methods to map the functional roles of these nonconventional translation transcriptome-wide, using tumorigenesis as a model system. Functional genomic approaches are uniquely poised to address the deficit in our understanding of functional alternative ORFs. In this proposal we will aim to characterize the global utilization of nonconventional initiation sites during tumorigenesis using ribosome profiling and RNA-seq at various time points (Aim 1). This will define novel ORFs that are actively translated, and how the translation of these ORFs are regulated to promote expression of oncogenic genes and peptides. Then, we will use CRISPR screens to identify ORFs necessary for tumor growth, and define the functions of the novel peptides by characterizing localization, physical interactions, and genetic interactions (Aim 2). Finally, we will mechanistically interrogate alternative start site usage to investigate how translation is tuned during cell-state changes (Aim 3). Overall, the results from the proposal will address long- standing questions about translational control, and reveal the regulatory roles of novel proteins. The combination of mentored support, skills, and data obtained in the K99 phase will provide Dr. Chen a springboard to achieving independence as an investigator in the R00 phase and beyond. The results of our studies will provide new insights into fundamental aspects of translational control, and will define new paradigms relevant to biology and disease.