Award details

A novel eukaryotic RNA thermoswitch: molecular function and biotech applications

Reference	BB/V006096/1
Principal Investigator / Supervisor	Dr Betty Chung
Co-Investigators / Co-Supervisors	Professor George Lomonossoff
Institution	University of Cambridge
Department	Pathology
Funding type	Research
Value (£)	739,245
Status	Current
Type	Research Grant
Start date	01/04/2021
End date	31/03/2024
Duration	36 months

Abstract

RNA is the central molecule in gene expression in all extant life, serving as the messenger for protein synthesis. It is also central to biocatalysis, seen dramatically in the ribosome but also in ribozymes and RNAPzymes such as telomerase. Ultimately, RNA function depends on its structure - it has a seemingly limitless variety of structures that allow many diverse functions. We have recently identified a new class of regulatory RNA thermoswitches in the 5'UTRs of a subset of genes in the model flowering plant A. thaliana, that directly regulate translation in response to temperature (Chung* et al 2020 Nature Plants, with accompanying News and Views highlight). The discovery of the plant RNA ThermoSwitch (pRTS) will enable the following two objectives to be achieved. We will: 1. Decipher pRTS-mediated translation dynamics. This information will further our understanding of inherent differences between the plant and animal translation machinery and will provide a molecular explanation for the more thermo-responsive nature of plants. 1a. Determine whether pRTSs are phylogenetically conserved within the plant kingdom. 1b. Dissect the molecular dynamics of pRTS-dependent translation by mapping all translation complexes (scanning, initiating, elongating and terminating complexes) before and after pRTS activation. 2. Capitalise on the temperature-inducible nature of pRTSs to utilise them as biobricks for an energy-efficient, inducible high-yield heterologous gene expression system. 2a. Identify optimal features of pRTSs for thermo-induced protein synthesis 2b. Develop a thermo-inducible high-yield gene expression system that can be utilised by the biopharma industry 2c. Proof of concept strategy: To confirm efficiency, yield and efficacy in planta with the system developed in 2b. Together these will elucidate the molecular mechanism of the newly discovered ThermoSwitch in plants and lay the groundwork for its application in biopharming.

Summary

Responsiveness to environmental stresses, including temperature, is a crucial feature for sessile organisms such as plants so they can adjust their growth and development accordingly. We have recently discovered a novel plant RNA ThermoSwitch (pRTS) that is responsible for day-time rhythmic growth of the model flowering plant Arabidopsis (Chung* et al, May 2020, Nature Plants, with accompanying News and Views highlight). In contrast to more established protein-based thermo-sensors, the pRTS is an RNA element, which directly regulates protein synthesis through rapid conformation changes in response to temperature fluctuations. Through a combination of computation and experimental assays, we have demonstrated that the pRTS drives the protein synthesis of several transcription factors such PIF7, WRKY22 and HSFA2, that control the expression of many proteins, within minutes after the temperature changes from 17C to 27C. Further investigation on the role of PIF7 revealed that it drives transcription for several growth regulators, resulting in day-time growth of Arabidopsis. However, this previous work just scratched the surface of understanding the biological function of pRTS. Many very important questions remained unanswered, especially as to how pRTSs manipulate translation dynamics to achieved enhanced protein synthesis within such a short timeframe. Further, the newly discovered pRTSs have great biotechnological potential as energy-efficient rapid-inducers for heterologous protein expression for molecular pharming - an appealing system for high throughput production in response to immediate needs such as vaccine production during outbreaks and pandemics. We will address these two questions through the following objectives: 1. We will decipher pRTS-mediated translation dynamics. This information will further our understanding of inherent differences between the plant and animal translation machinery and will provide a molecular explanation for the more thermo-responsive nature of plants. 1a. We will determine whether pRTSs are phylogenetically conserved within the plant kingdom. 1b. We will dissect the molecular dynamics of pRTS-dependent translation by mapping all translation complexes (scanning, initiating, elongating and terminating complexes) before and after pRTS activation. 2. We will develop an energy-efficient thermo-inducible high-level expression system for molecular pharming. 2a. We will identify optimal features of pRTSs for rapid thermo-induced protein synthesis 2b. We will develop a thermo-inducible high-yield heterologous gene expression system that can be utilised by the biopharma industry 2c. Proof of concept strategy: To confirm efficiency, yield and efficacy in planta with the system developed in 2b. This research is exciting for several reasons. The discovery of plant ThermoSwitches opens up a whole new avenue for adaptive response to temperature changes in plants at the level of protein synthesis. We are ideally poised to exploit this new research direction. Understanding the translation dynamics driven by the plant ThermoSwitch may also help explain why translation machinery in the animal kingdom is less responsive to temperature change. Importantly, biotechnological utilisation of pRTSs could provide an ideal system for rapid vaccine production in an economical manner when demand is urgent.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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