Translation of small open reading frames in 3'UTR enhances translation of canonical open reading frames

项目摘要 核糖体和蛋白质组图谱显示了大量翻译的小开放阅读框架(ORF) 在先前描述的“非翻译区”(UTRs)和长的非编码RNA中。虽然有些是小的 ORF依赖于编码的多肽在各种基本过程中发挥作用(例如，发育)。 5‘非编码区中小ORF的翻译，称为上游ORF(UORF)，通常抑制基因表达， 与编码的多肽无关。在被称为下游ORF的3‘非编码区中也有小的ORF的报道 (DORF)。然而，DORF的功能及其与人类健康和疾病的关系仍不清楚。 我使用核糖体图谱数据描述了人类和斑马鱼的DORF。我的初步数据显示， 与uORF相反，DORF(3‘UTR中的小ORF)的翻译强烈地增强了 这是一种典型的开放阅读框架，在脊椎动物中仍是一种未知的调节机制。这个 目标是：1)在单分子水平上剖析DORF如何促进规范CDS的翻译。 以及2)确定癌症中的交替多聚腺苷酸化是否影响DORF调节而致癌。 建议进行这项研究的基本原理是从机制上理解DORF介导的调控。 并评估DORF失调在疾病条件下(如癌症)可能的生物学意义。 这一建议在概念上是创新的，因为它基于对一部小说的探索，但却得到了广泛的翻译 调节机制在脊椎动物中是保守的。从技术上讲，这一提议将结合单分子 不同人类细胞系的成像、基因组图谱(rna-seq、核糖体图谱)和报告方法 (包括癌细胞)和公布的患者数据。 该项目的成果将强调核糖体作为主要基因表达调节因子的作用，以及 盾牌照亮了小兽人的重要性。这项关于DORF的翻译动力学工作将提供关键的 对这一未知调控途径的分子机制的洞察。探索DORF失调 在由于APA引起的癌症中，即使在DNA没有任何突变的情况下，也会突出显示mRNA本身是疾病的驱动因素，而且它 这也预示了DORF在检测甚至治愈癌症方面可能的临床影响。 我的长期兴趣是研究癌症中的基因表达失调。这个培训奖将增加我的 具有癌症生物学、分子生物学和生物信息学的知识背景。它还将促进技术上的 培训单分子成像、核糖体分析、癌症细胞生物学检测。总体而言，这项提议将 帮助我未来独立的癌症分子/基因组事业。

Project Summary Ribosome and proteomic profiling have revealed a large number of small translated open reading frames (ORF) within previously described “untranslated regions” (UTRs) and long non-coding RNAs. While some of the small ORFs depend on the encoded peptide to function in various fundamental processes (e.g., development). Translation of small ORFs in the 5’UTR, known as upstream-ORFs (uORFs), usually represses gene expression, independent of the encoded peptide. Small ORFs have also been reported in 3'UTR, termed downstream ORF (dORF). However, the dORF function and their relationship to human health and disease remain unknown. I characterized dORFs from human and zebrafish using ribosome profiling data. My preliminary data indicates, contrary to uORFs, translation of dORFs (small ORF in the 3’UTR) strongly enhances translation of the canonical ORFs and remains an uncharacterized regulatory mechanism across vertebrates. The objectives are: 1) Dissect at the single molecular level how dORF enhances translation of the canonical CDS. And 2) Determine whether alternative polyadenylation in cancer influences dORF regulation to cause cancer. The rationale for the proposed research is to gain a mechanistic understanding of dORF-mediated regulation and to assess the possible biological importance of dORF dysregulation under disease conditions (e.g. Cancer). This proposal is conceptually innovative as it is based on the exploration of a novel, yet widespread translation regulatory mechanism conserved across vertebrates. Technically, this proposal will combine single molecular imaging, genomic profiles (RNA-seq, Ribosome profiling), and reporter approaches in different human cell lines (including cancer cells) and published patient data. The outcomes from this project will emphasize the role of ribosome as a master gene expression regulator, and shield light on the importance of small ORFs. This translation kinetics work about dORF will provide critical insights into the molecular mechanism of this uncharacterized regulatory pathway. Exploring dORF dysregulation in cancer due to APA will highlight the mRNA itself as disease driver even without any mutation in DNA, and it also indicates possible clinical impact of dORF to detect and even cure cancer. My long-term interest is to study gene expression dysregulation in cancer. This training award will increase my knowledge background of cancer biology, molecular biology and bioinformatics. It will also promote the technical training of single molecular imaging, ribosome profiling, cell biology assays for cancer. Overall, this proposal will help me for future independent cancer molecular/genomic career.