Defining the regulatory roles of alternative ribosome initiation and novel peptides

定义替代核糖体起始和新型肽的调节作用

基本信息

批准号：
10013272
负责人：
Jin Chen
金额：
$ 5万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-09 至 2020-10-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10013272
关键词：
Address Biochemical Biological Biological Models Biology Biophysics CRISPR screen CRISPR/Cas technology Cell Line Cell Survival Cell physiology Cells Cellular Stress Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Complement Data Development Disease Event FRAP1 gene Gene Expression Regulation Genes Genetic Genome Genomic approach Goals Health Transition Human Human Biology Human Genome Project Initiator Codon Knock-in Knock-out Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Mentors Microscopy Modeling Monitor Noise Normal Cell Oncogenes Oncogenic Open Reading Frames Organoids Pathway interactions Peptides Phase Phenotype Protein Biosynthesis Proteins Proteome RNA RNA-Binding Proteins Regulation Regulatory Pathway Reporter Research Personnel Ribosomal RNA Ribosomes Role Signal Pathway Site Stress System Techniques Technology Testing Therapeutic Time Training Trans-Activators Translating Translations Untranslated RNA Variant base cell transformation deep sequencing design experience experimental study follow-up functional genomics gene synthesis genome annotation genome-wide insight knock-down neoplastic cell novel programs protein expression response ribosome profiling single molecule skills targeted treatment tool transcriptome transcriptome sequencing translation factor tumor growth tumorigenesis

项目摘要

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.

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”的一部分。编码新颖的开放阅读框 (ORF)，这些开放阅读框要么是带注释的蛋白质的变体，要么是上游不同的 ORF 或注释蛋白的下游，甚至是长非编码 RNA 上的 ORF，这些新型 ORF 正在出现。作为快速重新编程特定基因和蛋白质合成网络的翻译控制机制，尤其是在应激和细胞状态转变（例如肿瘤发生）期间。此外，这些新的 ORF 也具有重要作用。编码尚未表征的假定肽，从而了解翻译控制。机制以及编码肽的调节功能可以揭示基础生物学和现有的替代起始或替代 ORF 编码肽的研究已经采取。由于缺乏在全基因组范围内分析它们的工具，我们的目标是开发和开发它们。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使用肿瘤发生作为功能基因组模型系统进行非常规翻译转录组。方法独特地解决了我们对功能替代 ORF 的理解不足。该提案的目的是描述全球对非常规引发地点的利用情况在不同时间点使用核糖体分析和 RNA-seq 进行肿瘤发生（目标 1）。哪些 ORF 被主动翻译，以及如何调节这些 ORF 的翻译以促进这些 ORF 的表达然后，我们将使用 CRISPR 筛选来识别肿瘤生长所需的 ORF，并通过表征定位、物理相互作用和遗传来定义新型肽的功能最后，我们将机械地询问替代起始站点的使用情况以调查如何进行。翻译在细胞状态变化期间进行调整（目标 3）。总体而言，该提案的结果将解决长期问题。有关翻译控制的悬而未决的问题，并揭示了新蛋白质的组合的调节作用。在 K99 阶段获得的指导支持、技能和数据将为陈博士提供实现目标的跳板作为 R00 阶段及以后的研究者的独立性，我们的研究结果将提供新的内容。对翻译控制的基本方面的见解，并将定义与生物学和相关的新范式疾病。