Protein translation control of organism development

生物体发育的蛋白质翻译控制

• 批准号: RGPIN-2020-06195
• 负责人: Allan, Douglas
• 金额: $ 3.06万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746602
• 关键词: Protein; translation; control; organism; development

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.

胚胎发育需要精确的时空合成成千上万的蛋白质。相应地，mRNA的翻译控制对于正确的胚胎发生是必不可少的。我们的研究计划的长期愿景是探索一个新的，潜在的广泛的贡献者在胚胎发育过程中的翻译调控，上游开放阅读框架（uORF）所发挥的作用。真核生物中的大多数转录物在其5 '非翻译区（5' UTR）中具有uORF，其募集翻译核糖体并将翻译机器与主要编码序列（mCDS）隔离。这会抑制蛋白质合成。尽管uORF因此代表作为广泛的翻译调节因子的潜力，但在任何生物学背景下对其作用或影响的描述很少。原理和初步数据：果蝇胚胎的Ribo-seq鉴定了超过22，000个在发育过程中翻译的uORF;许多水平高于其下游mCDS。我们对这些数据集的分析确定了305个转录因子转录本在果蝇胚胎发育过程中具有高uORF翻译率。为了测试这些uORF的作用，使用体外翻译测定和体内转基因报告基因分析，我们发现转录因子颗粒头（grh）内的5 'UTR uORF抑制和空间调节grh mCDS的翻译。 我们的短期目标是开始系统的突变体分析的转录因子uORF，以测试其在调控率和时空模式的蛋白质合成胚胎发育所需的作用。我们推测转录因子的翻译受转录本5 'UTR中uORF的调控。我们预测uORF起始密码子的突变-以阻止uORF翻译-将诱导转录因子的异位、早熟和/或上调表达，导致胚胎发育的严重缺陷。目标1。uORF在转录因子翻译和发育功能中的遗传分析。基于高uORF翻译和基因在胚胎发生中的作用，将优先考虑用于分析的转录本。将评估uORF诱变的影响：（i）我们将使用转基因报告基因筛选转录物以鉴定抑制主要编码序列翻译的uORF。(ii)通过基因组编辑破坏抑制性uORF，然后评估转录因子表达和对胚胎发生的影响。目标2. uORF活性的体外翻译测定。我们将系统地筛选抑制主要编码序列翻译的uORF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。意义我们的创新目标将提供突破性的证据，证明uORF在胚胎发生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，以研究uORF在发育中的许多作用和机制。