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

项目摘要 核糖体和蛋白质组分析揭示了大量的小翻译开放阅读框架（ORF） 在先前描述的“非翻译区”（UTR）和长非编码RNA内。虽然一些小的 ORF依赖于编码的肽在各种基本过程中发挥作用（例如，发展）。 在5 'UTR中的小ORF的翻译，称为上游ORF（uORF），通常抑制基因表达， 独立于所编码的肽。在3 'UTR中也报道了小ORF，称为下游ORF （dORF）。然而，dORF的功能及其与人类健康和疾病的关系仍然未知。 我使用核糖体分析数据表征了来自人类和斑马鱼的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 典型的ORF，仍然是一个未表征的脊椎动物调控机制。的 目的是：1）在单分子水平上剖析dORF如何增强典型CDS的翻译。 和2）确定癌症中的选择性多聚腺苷酸化是否影响dORF调节以引起癌症。 该研究的基本原理是获得对dORF介导的调节的机制性理解 并评估疾病条件（例如癌症）下dORF失调的可能生物学重要性。 这一建议在概念上是创新的，因为它是基于对一种新颖而广泛的翻译的探索 在脊椎动物中保守的调节机制。从技术上讲，这个提议将联合收割机 不同人类细胞系中的成像、基因组图谱（RNA-seq、核糖体图谱）和报告方法 （包括癌细胞）和已发表的患者数据。 该项目的成果将强调核糖体作为主基因表达调节因子的作用， 屏蔽小ORF重要性的光。关于dORF的翻译动力学工作将提供关键的 深入了解这种未表征的调控途径的分子机制。探索dORF失调 在癌症中，由于阿帕将突出mRNA本身作为疾病驱动因素，即使没有任何DNA突变， 也表明dORF检测甚至治愈癌症的可能临床影响。 我的长期兴趣是研究癌症中的基因表达失调。这次培训奖励将提高我的 具有肿瘤生物学、分子生物学和生物信息学知识背景。它还将促进技术 单分子成像、核糖体分析、癌症细胞生物学分析的培训。总的来说，这项建议将 帮助我将来独立从事癌症分子/基因组学职业。

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.