Protein translation control of organism development

胚胎发育需要数千种蛋白质的精确时空合成。相应地，mRNAs的翻译控制对于正常的胚胎发生是必不可少的。我们研究计划的长期愿景是探索一种新颖的、潜在的广泛参与胚胎发育过程中翻译调控的因子-上游开放阅读框架(UORF)所起的作用。真核生物中的大多数转录本在其5‘非翻译区(5’UTRs)上都有uORFs，它们招募翻译核糖体，并将翻译机制与主要编码序列(MCDs)隔离开来。这会抑制蛋白质的合成。因此，尽管uORFs可能代表着广泛的翻译调节因子，但很少有人描述它们在任何生物学背景下的作用或影响。基本原理和初步数据：在果蝇胚胎中确定了22,000个核糖核酸序列，通过发育翻译uORF；其中许多水平高于它们下游的MCDs水平。我们对这些数据集的分析发现，在果蝇胚胎发生过程中，有305个转录因子转录本具有高uORF翻译率。为了测试这些uORFs的作用，我们通过体外翻译实验和体内转基因报告分析，发现转录因子颗粒头部(GRH)中的5‘UTRuORFs抑制和空间调节GRH MCDs的翻译。我们的短期目标是开始对转录因子uORFs进行系统的突变分析，以测试它们在调节胚胎发育所需的蛋白质合成的速度和时空模式中的作用。我们假设转录因子的翻译受转录产物5‘UTR中的uORFs调控。我们预测，uORF起始密码子的突变--阻止uORF翻译--将导致转录因子的异位、早熟和/或上调表达，导致胚胎发育的严重缺陷。目的1.转录因子翻译和发育功能中uORF功能的遗传分析。根据高uORF翻译和该基因在胚胎发育中的作用，将优先考虑用于分析的转录本。将评估uORF突变的影响：(I)我们将使用转基因记者筛选转录本，以确定抑制主要编码序列翻译的uORF。(Ii)抑制的uORF将通过基因组编辑中断，然后评估转录因子的表达和对胚胎发育的影响。目的2.uORF活性的体外翻译实验。我们将系统地筛选抑制主要编码序列翻译的uORF的转录本。对于具有多个uORF的转录本，我们将探索每个uORF对翻译抑制的相对贡献。意义重大。我们的创新目标将提供突破性证据，证明uORF在胚胎发育所需的翻译控制中发挥重要作用。这为我们的长期愿景奠定了基础，以检查uORF功能在发育中的许多作用和机制。

Embryonic development requires precise spatiotemporal synthesis of thousands of proteins. Correspondingly, translational control of mRNAs is essential for proper embryogenesis. The long-term vision of our research program is to explore the roles played by a novel, potentially widespread contributor to translation regulation during embryogenesis, upstream open reading frames (uORFs). Most transcripts in eukaryotes have uORFs in their 5'untranslated regions (5'UTRs) that recruit translating ribosomes and sequester translation machinery away from the main coding sequence (mCDS). This suppresses protein synthesis. In spite of the potential that uORFs thereby represent as widespread translational regulators, there are very few descriptions of their role or impact in any biological context. Rationale and Preliminary data: Ribo-seq of Drosophila embryos identified >22,000 translating uORFs through development; many at levels higher than their downstream mCDS. Our analysis of these datasets identified 305 transcription factor transcripts with high uORF translation rates during Drosophila embryogenesis. To test a role for these uORFs, using in vitro translation assays and in vivo transgenic reporter analysis, we found that 5'UTR uORFs within the transcription factor grainy head (grh), suppress and spatially regulate translation of grh mCDS. Our short-term objective is to start systematic mutant analysis of transcription factor uORFs to test their role in regulating rates and spatiotemporal patterns of protein synthesis required for embryogenesis. We hypothesize that transcription factor translation is regulated by uORFs in the 5'UTR of their transcripts. We predict that mutation of uORF start codons - to prevent uORF translation - will induce ectopic, precocious and/or upregulated expression of transcription factor, causing severe defects in embryonic development. Aim 1. Genetic analysis of uORF function in transcription factor translation and developmental function. Transcripts for analysis will be prioritized, based on high uORF translation and the gene's role in embryogenesis. The impact of uORF mutagenesis will be assessed: (i) We will screen transcripts using transgenic reporters to identify uORFs that suppress main coding sequence translation. (ii) Suppressive uORFs will be disrupted by genome editing, followed by assessment of transcription factor expression and impact on embryogenesis. Aim 2. In vitro translation assays of uORF activity. We will systematically screen transcripts for uORFs that suppress main coding sequence translation. For transcripts with multiple uORFs, we will explore the relative contribution of each uORF to translation suppression. Significance. Our innovative objectives will provide ground-breaking evidence that uORFs play essential roles in translation control required for embryogenesis. This sets the stage for our long-term vision to examine the many roles and mechanisms underlying uORF function in development.