A novel eukaryotic RNA thermoswitch: molecular function and biotech applications

对包括温度在内的环境压力的反应是植物等固着生物的一个重要特征，因此它们可以相应地调整其生长和发育。我们最近发现了一种新的植物RNA ThermoSwitch（pRTS），它负责模式开花植物拟南芥的日间节律生长（Chung* et al，May 2020，Nature Plants，with accompanying News and Views highlight）。与更成熟的基于蛋白质的热传感器相比，pRTS是RNA元件，其通过响应于温度波动的快速构象变化直接调节蛋白质合成。通过计算和实验测定的组合，我们已经证明了pRTS驱动几种转录因子如PIF 7、WRKY 22和HSFA 2的蛋白质合成，这些转录因子在温度从17 ℃变化到27 ℃后的几分钟内控制许多蛋白质的表达。对PIF 7作用的进一步研究表明，PIF 7可驱动多种生长调节因子的转录，从而导致拟南芥的日间生长。许多非常重要的问题仍然没有得到解答，特别是关于pRTSs如何操纵翻译动力学以在如此短的时间内实现增强的蛋白质合成。此外，新发现的pRTSs具有巨大的生物技术潜力，作为能量高效的快速诱导剂，用于异源蛋白质表达，用于分子嫁接-一种有吸引力的系统，用于高通量生产，以响应诸如爆发和流行期间的疫苗生产的迫切需要。我们将通过以下目标来解决这两个问题：1.我们将破译pRTS介导的翻译动力学。这些信息将进一步加深我们对植物和动物翻译机制之间固有差异的理解，并将为植物更具热响应性的性质提供分子解释。1a.我们将确定pRTSs是否在植物界中是遗传上保守的。我们将通过绘制pRTS激活前后的所有翻译复合物（扫描、起始、延伸和终止复合物）来剖析pRTS依赖性翻译的分子动力学。我们将开发一个节能的热诱导高水平表达系统的分子药物。我们将确定快速热诱导蛋白质合成的pRTS的最佳特征2b。我们将开发一种可用于生物制药行业的热诱导高产异源基因表达系统2c。概念验证策略：用2b中开发的系统在植物中确认效率、产量和功效。这项研究令人兴奋的原因有几个。植物温控开关的发现为植物在蛋白质合成水平上对温度变化的适应性反应开辟了一条全新的途径。我们已经做好了充分准备来开拓这一新的研究方向。理解植物热开关驱动的翻译动力学也可能有助于解释为什么动物王国的翻译机制对温度变化的反应较小。重要的是，pRTSs的生物技术利用可以在需求紧迫时以经济的方式提供快速疫苗生产的理想系统。

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 synthesis2b. We will develop a thermo-inducible high-yield heterologous gene expression system that can be utilised by the biopharma industry2c. 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.