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

项目摘要 目前流行的理论认为，在高等生物中，信使RNA（mRNA）编码一种蛋白质， 近年来经历了戏剧性的修改。核糖体和蛋白质组分析揭示了大量 在先前描述的“非翻译区”（UTR）内的小翻译开放阅读框（ORF）， 长非编码RNA。事实上，一些来源于小ORF的肽已经涉及多种生物学功能。 基本过程（例如，发展）。5 'UTR中的小ORF的翻译，称为上游ORF （uORF），已被证明对基因调控具有深刻的调控作用，独立于编码的 肽。此外，uORF的翻译在病理条件下变化，例如癌症，并且突变影响 uORF与多种人类疾病相关。我们和其他人也指出， 在人细胞中，在3 'UTR中翻译的小ORF被称为下游开放阅读框（dORF）， 斑马鱼胚胎然而，与uORF相反，还没有对dORF功能的系统研究， 它们与人类健康和疾病的关系尚未得到检验。此外，考虑到它们在3 'UTR中的位置， dORF参与翻译机制的分子机制仍然完全未知。 我们的长期目标是了解转录后调控（mRNA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。本申请的中心假设是 dORF的翻译调控基因表达。我们的初步数据有力地表明，与 uORF、dORF强烈增强了典型ORF的翻译，并作为未表征的ORF出现。 和有效的调节机制。目标是：1）确定涉及的因素 增强主要ORF的翻译。2)剖析驱动dORF翻译的调控信息，以及3） 描述dORF介导调控的生物学影响。拟议研究的基本原理是 获得dORF介导的调节机制的理解，以评估可能的生物学作用。 在应激或疾病条件下dORF失调的重要性。这一建议在概念上具有创新性 因为它是基于对一种新颖的、广泛而有效的翻译调节机制的探索， 在脊椎动物中是保守的。技术上，该提议将联合收割机组合基因组图谱（RNA-seq，核糖体 分析）;报告（细胞计数）;生物化学工具：RNA下拉，然后是蛋白质组学，CRISPR-Cas-9和 12 a（编辑）和我们的新Cas 13 d工具（在胚胎中敲低）;结合人类细胞和斑马鱼胚胎。 该项目的结果将有助于了解dORF如何翻译，塑造基因表达， 产生表型。核糖体的这种新功能增加了最近出现的调节作用， 基因表达上的翻译（例如uORF，密码子最优性）。了解dORF生物学将提供一个进入 点，甚至可能是一种诊断工具，将突变与人类疾病联系起来。识别分子 参与该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.