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，以响应不断变化的环境，对于固着生物如植物来说是一个特别的挑战。发现基础的调节机制将最终使我们能够提高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调节的理解中，在DNA中存储指令，将其转录成mRNA中间体以及将其翻译成蛋白质之间，已经发生了几次革命。例如，发现激活和抑制转录的因子，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。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，这种调控形式起作用。在所有真核生物中，mRNA通常包括短编码区，位于指定预期蛋白质产物的主要编码区的上游。这些上游开放阅读框（uORF）中的大多数没有显示进化保守性。在植物中，大约100个uORF的一个小子集在数百万年的进化中显示出氨基酸的保守性-CPuORF。已知几个CPuORF调节mRNA的蛋白质编码部分的翻译，使得蛋白质产物仅在某些情况下产生。这使得植物能够快速地制造特定的蛋白质，并且只在特定的情况下。核糖体首先翻译CPuORF。然后CPuORF肽与核糖体相互作用，导致停滞和下游蛋白质编码ORF翻译失败。条件性uORF依赖的翻译停滞以两种方式起作用。最常见的方式是当存在信号时，CPuORF肽引起停滞。当信号消失时失速现象减轻。通过这种方式，当信号不存在时产生的主要蛋白质产物和当信号存在时不产生的主要蛋白质产物。我们已经发现了三个以相反方式工作的CPuORF，这似乎更罕见。在这种情况下，当信号不存在并且主要蛋白质产物不产生时，CPuORF肽引起核糖体停滞。然而，当信号存在时，停滞被缓解，并产生主要的蛋白质产物。我们研究的三个CPuORF对农业中重要的环境信号做出了反应。一个CPuORF只允许蛋白质生产时，植物经历热。只有当植物经历水分限制时，两种才允许蛋白质生产。第四个CPuORF只允许蛋白质生产时，一个特定的化学品被施加到植物。这种新的形式的调控是重要的，因为它提供了机会，了解基因表达的基本原则，也因为它的潜力，被用于研究，合成生物学和农业。从长远来看，通过了解这种形式的调节是如何工作的，我们将能够设计出能够响应特定应用化学品/现场条件的pepto-switch。为了达到这一阶段，我们首先需要了解使新生肽停止核糖体的分子机制，允许在特定条件下释放停止。这需要我们观察离开核糖体的肽，看看有什么接触，然后测试这些预测。我们有这样做的工具。使用cryo-EM，我们可以获得具有和不具有不同失速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之间的失速机制。然后，我们可以在体外和植物体内测试结构预测。我们还将使用基因组工具来调查植物转录组中导致条件性停滞的其他CPuORF。最后，作为原理的证明，我们将通过加热或化学处理逆转核糖体的停滞，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.