Small translated ORFs in the 3'UTR enhance translation in vertebrates

项目摘要 在高等生物中信使RNA(MRNAs)编码单一蛋白质的流行学说 最近几年经历了戏剧性的修改。核糖体和蛋白质组图谱显示了大量 小的已翻译开放阅读框架(ORF)在先前描述的“非翻译区”(UTRs)和 长的非编码RNA。事实上，从小的开放阅读框中提取的一些多肽与各种 基本流程(例如，开发)。5‘非编码区中小ORF的翻译，称为上游ORF (UORFs)，已被证明对基因调控具有深刻的调控作用，不依赖于编码的 多肽。此外，uORF的翻译在诸如癌症等病理条件下是不同的，并且突变影响 UORF与各种人类疾病有关。我们和其他人也指出了 在3‘非编码区中翻译的小ORF，在人类细胞中称为下游开放阅读框架(DORF)， 斑马鱼的胚胎。然而，与uORF相反，没有对DORF函数进行系统研究，并且 它们与人类健康和疾病的关系仍未得到检验。此外，鉴于它们在3‘UTR中的位置， DORF参与翻译机制的分子机制仍然完全不清楚。 我们的长期目标是了解转录后调控(信使核糖核酸半衰期和翻译)如何形成 脊椎动物的基因表达及其对人类疾病的影响。这个应用程序的中心假设是 DORF的翻译调控着基因的表达。我们的初步数据强烈表明，与 UORF，DORF强烈促进了规范ORF的翻译，并作为一种未刻画的 以及有效的脊椎动物调控机制。目标是：1)确定涉及的因素 加强主要开放源码的翻译。2)剖析了驱动DORF翻译的法规信息；3) 描述DORF介导的调节的生物学影响。建议进行这项研究的理由是 了解DORF介导的调节机制，以便评估可能的生物学 在压力或疾病条件下DORF失调的重要性。这项建议在概念上是创新的。 因为它是基于对一种新颖、广泛而有效的翻译监管机制的探索 在脊椎动物中保存。从技术上讲，这一提议将结合基因组图谱(RNA-seq、核糖体 生物化学工具：RNA下拉，然后是蛋白质组学、CRISPR-Cas-9和- 12a(编辑)和我们的新型Cas13d工具(在胚胎中击倒)；结合了人类细胞和斑马鱼胚胎。 这个项目的结果将有助于理解DORF是如何被翻译的，如何塑造基因表达和 产生表型。核糖体的这一新功能增加了最近出现的 关于基因表达的翻译(例如，uORF，密码子最优化)。了解DORF生物学将提供一个入门机会 点，甚至可能是一种将突变与人类疾病联系起来的诊断工具。鉴定分子 参与这一途径的机制可能为治疗干预提供靶点。

Project Summary The prevailing doctrine that messenger RNAs (mRNAs) in higher organisms encode for a single protein has undergone a dramatic revision in recent years. 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. Indeed, some of the peptides derived from small ORFs have been implicated in various fundamental processes (e.g., development). Translation of small ORFs in the 5’UTR, known as upstream-ORFs (uORFs), has been shown to have a profound regulatory effect on gene regulation, independent of the encoded peptide. Further, translation of uORFs vary under pathologic conditions such as cancer, and mutations affecting uORFs are associated with various human diseases. We and others have also indicated the existence of translated small ORFs in the 3’UTR known as downstream open reading frames (dORFs) in human cells and zebrafish embryos. However, contrary to uORFs, there has been no systematic study of dORF functions, and their relationship to human health and disease remains untested. Further, given their location in the 3’UTR, the molecular mechanism by which dORFs engage the translational machinery remain completely unknown. Our long-term goal is to understand how post-transcriptional regulation (mRNA half-life and translation) shapes gene expression in vertebrates, and its impact on human disease. The central hypothesis of this application is that translation of dORFs regulates gene expression. Our preliminary data strongly indicate that, contrary to uORFs, dORFs strongly enhance translation of the canonical ORF and emerge as an uncharacterized and potent regulatory mechanism across vertebrates. The objectives are to: 1) Identify factors involved in enhancing translation of the main ORF. 2) Dissect the regulatory information driving dORF translation, and 3) Characterize the biological impacts of dORF-mediated regulation. The rationale for the proposed research is to gain a mechanistic understanding of dORF-mediated regulation in order to assess the possible biological importance of dORF dysregulation under stress or disease conditions. This proposal is conceptually innovative as it is based on the exploration of a novel, yet widespread and potent translation regulatory mechanism conserved across vertebrates. Technically, this proposal will combine genomic profiles (RNA-seq, Ribosome profiling); reporter (cytometry); biochemistry tools: RNA pulldowns follow by proteomics, CRISPR-Cas-9 and - 12a (to edit) and our novel Cas13d tool (knock-down in embryos); combining human cell and zebrafish embryos. The outcomes from this project will help understand how dORFs are translated, shape gene expression and generate phenotypes. This novel function of the ribosome adds to the recently emerging regulatory effects of translation on gene expression (e.g. uORF, codon optimality). Understanding dORF biology will provide an entry point and perhaps even a diagnostic tool to associate mutations with human diseases. Identifying the molecular machinery involved in this pathway might provide targets for therapeutic interventions.