Characterisation and chemical inhibition of a novel regulatory module controlling chloroplast protein translation

NOT 4 E3连接酶在真核生物中是保守的，由独特的RING和RNA识别基序（RRM）结构域配对组成，其功能位于蛋白水解和RNA生物学的界面。与酵母和动物基因组中发现的单一变体相比，植物基因组编码多个NOT 4旁系同源物，这表明该蛋白质在植物王国中的功能多样化。我们最近表明，3种拟南芥属N 0 T4同源物之一N 0 T4 A通过促进叶绿体靶向蛋白PGR 3的表达而在调节叶绿体功能中发挥关键作用（Bailey等人，2021 Nature Communications）。PGR 3是几种叶绿体RNA的稳定和翻译所必需的，包括编码核心光合组分和质体核糖体亚基的那些。因此，N 0 T4 A-PGR 3信号传导模块对于通过影响质体核糖体生物发生和类囊体中光合机制的有效组装来确保叶绿体蛋白质稳定是重要的。尽管确定了这个NOT 4A-PGR 3信号传导模块，我们仍然不知道将NOT 4A活性与PGR 3产生联系起来的潜在机制。在这里，我们将使用一系列分子，细胞生物学和遗传学方法来破译NOT 4A和PGR 3之间的分子联系。我们将确定NOT 4A如何确保有效的PGR 3表达，并探讨这如何有助于在生长，发育和应激反应过程中的叶绿体功能的动态。此外，我们将与工业合作伙伴先正达（Syngenta）一起探索该模块是否可以化学靶向破坏叶绿体蛋白生物合成。因此，该项目将为调节叶绿体形式和功能的机制提供新的思路，并确定这种机制是否可以用于识别新的除草剂候选物。

The NOT4 E3 ligase is conserved across eukaryotes and consists of a unique RING and RNA Recognition Motif (RRM) domain pairing that places its function at the interface of proteolysis and RNA biology. Plant genomes encode for multiple NOT4 paralogs, compared to single variants found in yeast and animal genomes, which indicates diversification of function for this protein in the plant kingdom. We recently showed that one of the 3 Arabidopsis NOT4 homologs, NOT4A, has diverged towards a key role in regulating chloroplast function, through promoting the expression of the chloroplast targeted protein PGR3 (Bailey et al 2021 Nature Communications). PGR3 is required for stabilisation and translation of several chloroplast RNAs, including those that encode for core photosynthetic components and subunits of the plastid ribosome. As such, the NOT4A-PGR3 signalling module is important for ensuring chloroplast proteostasis by influencing plastid ribosome biogenesis and effective assembly of photosynthetic machineries in the thylakoids. Despite identifying this NOT4A-PGR3 signalling module, we still do not know the underlying mechanisms that connect NOT4A activity to PGR3 production. Here we will use a range of molecular, cell biology and genetic approaches to decipher the molecular connection between NOT4A and PGR3. We will determine how NOT4A ensures efficient PGR3 expression and explore how this contributes to the dynamics of chloroplast function during growth, development and stress response. Furthermore, in conjunction with industrial partner Syngenta we will explore whether this module can be chemically targeted to disrupt chloroplast protein biogenesis. As such, this project will shed new light onto a mechanism regulating chloroplast form and function, and determine if this mechanism can be exploited for the identification of new herbicide candidates.