Conditional uORF-Dependent Translational Control of Plant Gene Expression

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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.