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Conditional uORF-Dependent Translational Control of Plant Gene Expression

植物基因表达的条件性 uORF 依赖性翻译控制

基本信息

批准号：
BB/T006072/1
负责人：
Brendan Davies
金额：
$ 91.92万
依托单位：
University of Leeds
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT006072%2F1
关键词：
Conditional uORF Dependent Translational Control

项目摘要

Regulating gene expression to produce the appropriate level of each protein, in response to an ever-changing environment, is a particular challenge for sessile organisms such as plants. Discovering the underpinning regulatory mechanisms will ultimately allow us to enhance and manipulate crop growth and productivity. There have been several revolutions in our understanding of how gene expression is regulated at multiple levels, between the storage of instructions in DNA, their transcription into an mRNA intermediate and their translation to make proteins. For example, the discovery of factors that activate and repress transcription, epigenetic mechanisms that control the availability of the information and the regulatory roles of non-coding RNAs. This project aims to understand a novel form of regulation, which acts when the mRNA is translated to make proteins. In all eukaryotes, it is common for mRNAs to include short coding regions, upstream of the main coding region that specifies the intended protein product. The majority of these upstream open reading frames (uORFs) show no evolutionary conservation. In plants, a tiny subset of about 100 uORFs show amino acid conservation over many millions of years of evolution - the CPuORFs. Several CPuORFs are known to regulate translation of the protein-coding part of the mRNA, so that the protein product is only made in certain circumstances. This allows the plant to make specific proteins rapidly and only in set circumstances. Ribosomes start by translating the CPuORF. The CPuORF peptide then interacts with the ribosome, causing stalling and a failure to translate the downstream protein-coding ORF. Conditional uORF-dependent translational stalling works in two ways. The most common way is where the CPuORF peptide causes stalling when a signal is present. Stalling is alleviated when the signal is absent. In this way, the main protein product made when the signal is absent and not made when the signal is present. We have discovered three CPuORFs that work in the opposite way, which seems to be rarer. In this case, the CPuORF peptides cause ribosome stalling when the signal is absent and the main protein product is not made. However, when the signal is present, stalling is alleviated and the main protein product is made. The three CPuORFs that we have studied respond to environmental signals that are important in agriculture. One CPuORF only permits protein production when the plant experiences heat. Two only permit protein production when the plant experiences water restriction. A fourth CPuORF only permits protein production when a specific chemical is applied to the plant.This novel form of regulation is important because of the opportunity it affords to understand a fundamental principle of gene expression and also because of its potential to be used in research, synthetic biology and agriculture. In the long term, by understanding how this form of regulation works, we will be able to design pepto-switches capable of responding to specific applied chemicals/conditions in the field. To reach this stage we first need to understand the molecular mechanism that enables nascent peptides to stall the ribosome, allowing stalling to be released under specific conditions. This requires us to watch the peptides exiting the ribosome, to see what contacts are made and, subsequently, to test those predictions. We have the tools to do this. Using cryo-EM we can obtain high-resolution 3D images of the plant ribosome with and without the different stalling peptides, allowing us to compare stalling mechanisms between different conditional CPuORFs. We can then test structural predictions in vitro and in planta. We will also use genomic tools to survey the plant transcriptome for other CPuORFs that cause conditional stalling. Finally, as a proof of principle, we will engineer plants to flower at will, on reversal of the ribosome stalling by heat or chemical treatment.

对于植物等固着生物来说，调节基因表达以产生每种蛋白质的适当水平，以应对不断变化的环境，是一个特别的挑战。发现支撑调控的机制最终将使我们能够增强和操纵作物生长和生产力。在我们对基因表达如何在多个水平上进行调控的理解中，已经发生了几次革命，从指令存储在DNA中，它们转录成mRNA中间体，再到它们翻译成蛋白质。例如，激活和抑制转录的因子的发现，控制信息可用性的表观遗传机制，以及非编码RNA的调节作用。这个项目旨在了解一种新的调控形式，当信使核糖核酸被翻译成蛋白质时，它就会起作用。在所有真核生物中，mRNAs通常包括短编码区，位于指定目的蛋白产物的主编码区的上游。这些上游开放阅读框架(UORF)中的大多数没有显示出进化保守性。在植物中，大约100个uORF的一个很小的子集显示出在数百万年的进化过程中氨基酸的守恒--CpuORF。已知有几个CpuORFs调节mRNA的蛋白质编码部分的翻译，因此只有在特定情况下才能合成蛋白质产品。这使得植物只在特定的环境下才能快速制造特定的蛋白质。核糖体从翻译CpuORF开始。然后，CPuORF多肽与核糖体相互作用，导致停滞和无法翻译下游编码蛋白质的ORF。条件uORF依赖的翻译拖延以两种方式起作用。最常见的方式是CPuORF多肽在信号存在时导致失速。当没有信号时，失速现象会得到缓解。通过这种方式，主要的蛋白质产物是在没有信号时产生的，而不是在信号存在时产生的。我们已经发现了三个CPuORF以相反的方式工作，这似乎更罕见。在这种情况下，CPuORF多肽导致核糖体停滞，当信号缺失时，主要蛋白质产物也没有产生。然而，当信号存在时，停滞就会减轻，主要蛋白质产物就会产生。我们研究的三个CpuORF对农业中重要的环境信号做出反应。一个CPuORF只允许在植物受热时生产蛋白质。其中两种只允许在植物受到水分限制时生产蛋白质。第四个CpuORF只允许在植物上应用特定的化学物质时产生蛋白质。这种新的调节形式很重要，因为它提供了理解基因表达的基本原理的机会，也因为它有可能用于研究、合成生物学和农业。从长远来看，通过了解这种形式的监管是如何工作的，我们将能够设计能够对领域中特定应用的化学品/条件做出反应的Pepto开关。为了达到这一阶段，我们首先需要了解使新生多肽能够阻止核糖体的分子机制，从而允许在特定条件下释放失速。这需要我们观察离开核糖体的多肽，看看是什么联系在一起，并随后测试这些预测。我们有工具来做到这一点。使用低温电子显微镜，我们可以获得植物核糖体的高分辨率3D图像，包括不同的停滞肽，使我们能够比较不同条件CPuORF之间的停滞机制。然后，我们可以在体外和植物中测试结构预测。我们还将使用基因组工具来调查植物转录组，以寻找其他导致条件性停滞的CPuORF。最后，作为原则的证明，我们将在通过加热或化学处理逆转核糖体停滞的情况下，使植物随意开花。