Conditional uORF-Dependent Translational Control of Plant Gene Expression

调节基因表达以产生适当水平的每种蛋白质，以应对不断变化的环境，对植物等无根生物来说是一个特别的挑战。发现基本的监管机制最终将使我们能够加强和操纵作物生长和生产力。我们对基因表达是如何在多个层面上受到调控的理解已经发生了几次革命，从DNA中的指令存储，到它们转录成mRNA中间体，再到它们被翻译成蛋白质。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和非编码rna的调节作用。该项目旨在了解一种新的调控形式，当mRNA被翻译成蛋白质时，它起作用。在所有真核生物中，mrna通常包含短编码区，位于指定预期蛋白产物的主编码区上游。大多数上游开放阅读帧（uorf）不具有进化守恒性。在植物中，大约100个uorf中的一小部分在数百万年的进化中显示出氨基酸的守恒——cpuorf。已知有几种cpuorf调节mRNA的蛋白质编码部分的翻译，因此蛋白质产物仅在某些情况下产生。这使得植物只有在特定的环境下才能快速制造特定的蛋白质。核糖体首先翻译CPuORF。然后，CPuORF肽与核糖体相互作用，导致下游蛋白质编码ORF的翻译停滞和失败。条件依赖于uorf的平移失速有两种工作方式。最常见的方法是当信号存在时，CPuORF肽导致停顿。当没有信号时，失速得到缓解。这样，当信号不存在时，主要的蛋白质产物就会产生，而当信号存在时，主要的蛋白质产物就不会产生。我们发现了三个以相反方式工作的cpuorf，这似乎很少见。在这种情况下，CPuORF肽导致核糖体在信号缺失和主要蛋白质产物不产生时停滞。然而，当信号存在时，失速被缓解，主要的蛋白质产物被制造出来。我们研究的三种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产生蛋白质。其中两种只有在植物缺水时才会产生蛋白质。第四种CPuORF只允许在特定化学物质作用于植物时产生蛋白质。这种新形式的调控很重要，因为它提供了了解基因表达基本原理的机会，也因为它在研究、合成生物学和农业方面的潜在应用。从长远来看，通过了解这种形式的调节是如何工作的，我们将能够设计出能够响应该领域特定应用化学品/条件的胃开关。为了达到这一阶段，我们首先需要了解使新生肽使核糖体停滞的分子机制，并允许在特定条件下释放停滞。这就要求我们观察肽脱离核糖体的过程，观察它们之间发生了怎样的接触，然后对这些预测进行检验。我们有办法做到这一点。利用冷冻电镜技术，我们可以获得含有和不含不同延迟肽的植物核糖体的高分辨率3D图像，使我们能够比较不同条件cpuorf之间的延迟机制。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中其他导致条件失速的cpuorf。最后，作为一个原理证明，我们将通过加热或化学处理来逆转核糖体的停滞，从而使植物随意开花。

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.