Defining the regulatory roles of alternative ribosome initiation and novel peptides

项目摘要/摘要 人类基因组计划完成15年多后，我们对《注解》的理解 基因组仍然不完整。不属于“注释基因组”的替代核糖体起始点 编码新的开放阅读框架(ORF)，它是注释蛋白质的变体，上游是不同的ORF 或位于注释蛋白的下游，甚至位于长的非编码RNA上的ORF。这些新奇的ORF正在涌现 作为一种快速对特定基因和蛋白质合成网络重新编程的翻译控制机制， 特别是在压力和细胞状态转变期间，如肿瘤形成。此外，这些新的ORF 编码仍未确定特征的推定多肽。因此，理解翻译控制 机制，以及编码多肽的调节功能，可以揭示基本的生物学和 治疗学的靶点。现有对替代起始或替代ORF编码肽的研究已经采取了 由于缺乏在全基因组范围内描述它们的工具，这是一种特别的方法。我们的目标是发展和 应用深度测序和基于CRISPR的高通量方法来绘制这些基因的功能角色 全基因组非常规翻译，以肿瘤发生学为模型系统。功能基因组学 解决我们对功能性替代ORF理解的不足的方法是独一无二的。在……里面 在这项提案中，我们的目标是描述非常规起始点在 在不同时间点使用核糖体分析和RNA-SEQ进行肿瘤形成(目标1)。这将定义新的ORF 以及如何调节这些ORF的翻译以促进表达 致癌基因和多肽。然后，我们将使用CRISPR筛查来确定肿瘤生长所需的ORF， 并通过表征定位、物理相互作用和遗传来定义新型多肽的功能 互动(目标2)。最后，我们将机械地询问替代Start站点的使用情况，以调查如何 翻译在单元状态改变期间进行调优(目标3)。总体而言，该提案的结果将解决长期- 关于翻译控制的问题，并揭示了新蛋白质的调节作用。组合在一起 K99阶段获得的指导支持、技能和数据将为陈博士提供实现以下目标的跳板 在R00阶段及以后作为调查员的独立性。我们的研究结果将为我们提供新的 对翻译控制的基本方面的洞察，并将定义与生物学和 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.