Conditional uORF-Dependent Translational Control of Plant Gene Expression

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences

Abstract

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.

Technical Summary

All cells face the challenge of producing the appropriate level of each protein, in response to changing internal and external conditions. Most studies have focused on transcriptional regulation, where excellent tools and resources exist. However, mRNA levels are often not a good proxy for protein levels. One reason for this is the newly emerging area of translational control, which has the advantage of responding rapidly to change, since it does not require new transcription. This proposal focuses on a novel form of translational regulation in which a short peptide, encoded by an open reading frame in the 5' UTR, causes the plant ribosome to stall before it translates the major protein product encoded by the mRNA (mORF). Crucially, in the cases that we plan to study, stalling is alleviated under specific stress conditions of agronomic importance. We have identified peptides that only permit mORF translation when the plant experiences heat, drought or thermospermine. Discovering this mechanism would open the door to engineering peptides that respond to environmental conditions, combinations of conditions or specific chemicals. We will use cryo-EM to deliver the first high-resolution 3D image of an Arabidopsis cytoplasmic ribosome, translating and stalled at each of these peptides. We will also discover more peptides that work in the same way and solve 2-4 further ribosome structures stalled at these peptides. We will use comparisons between these structures to determine the stalling mechanism, which we will test in vitro and/or in vivo. Finally, we will show that this system can be used as a conditional pepto-switch to engineer a developmental change in a plant. The peptides that show this remarkable property are highly conserved across the plant kingdom, from dicots to grasses, suggesting that discoveries made here can be translated into all crops.

Planned Impact

We propose to deliver the first molecular-level, mechanistic understanding of how plant Conserved Peptide Upstream Open Reading Frames (CPuORFs) act in cis to control protein production, in response to environmental conditions/treatments. We will use state-of-the-art technologies to discover how the nascent CPuORF-encoded peptides cause the plant ribosome to stall, in such a way that stalling is reversed when the plant experiences challenges of agricultural significance. We will also determine whether these regulatory modules can be customised and applied. We focus on three conditions, important for crop productivity, heat stress, water restriction and salt stress as well as a chemical trigger. CPuORFs were originally identified through their remarkable sequence conservation between Arabidopsis and rice, making it probable that these approaches will be directly applicable to any crop species. This project therefore aligns with the BBSRC's 'grand challenge' of delivering food security through a "fundamental understanding of biological processes and mechanisms". The unprecedented molecular level understanding that will emerge from our structural biology approach has the potential to permit the future design of pepto-switches capable of precise regulation of translation. This technology should have the advantage of lacking side effects, since current evidence suggests that the peptides are not active in trans, as they are trapped and only act in the ribosome exit tunnel during translation.
Fundamental research: This proposal seeks to understand the mechanisms used by CPuORFs to regulate translation conditionally. We will identify new conditions to which CPuORFs respond and new, responsive CPuORFs, to uncover their mechanism of action and explore how they can be customised. One further outcome will be the first solved structure for the Arabidopsis cytoplasmic ribosome, which we anticipate will be of benefit to the plant science community. The molecular switches that we characterise will be useful for fundamental research and synthetic biology. In addition, the large and comparable stress-induced ribosome footprint profiling datasets will provide useful resources.
Applied research: It is imperative that crop productivity is maintained or increased despite significant environmental challenges. Finding methods to improve the ability to respond to abiotic stress is an important challenge within plant sciences, in both the academic and commercial sectors. Regulation of translation is emerging as a prime target for manipulation, in order to allow rational design of future crops. There is already a simple example of modifying crops by targeting translational control exerted by uORFs1. In this case, the authors simply boosted expression of downstream mORFs by genome editing uORFs. In contrast, the long-term outcome of the research described in this proposal is the possibility to design novel inducible expression systems that respond to external or internal conditions, combinations of conditions or applied chemical triggers.
Training: Will be delivered by training the PDRA and technician in new areas of research. Although the PDRA already has experience in molecular genetics and biochemistry, this project allows him to acquire new skills in structural biology and ribosome profiling. We will also involve at least one PhD student, alongside a minimum of 6 undergraduate/masters students, as part of their research projects, in preparation for future employment.
Public engagement: Will be delivered through a range of individual activities in schools, higher education and popular science initiatives and via the university publicity office, allowing us to communicate a greater understanding of plant gene/environment interactions and the consequences for food production.
Reference:
1. Genome editing of upstream open reading frames enables translational control in plants. Zhang et al. Nat Biotechnol. (2018) 36:894.

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