Chloroplastidäre Ribonukleoproteine (cpRNPs): globale Stabilisatoren chloroplastidärer RNA-Reservoirs während der Stressantwort

叶绿体RNA的加工是由多种核编码的RNA结合蛋白介导的。尽管对涉及的因素和单个基因表达步骤的机制问题的了解越来越多，但我们仍然对叶绿体基因表达的真正调控步骤知之甚少。我们也不了解叶绿体基因是如何共同调节的，以及叶绿体基因的表达是如何适应不断变化的外部条件的。我们最近已经证明，叶绿体核糖核蛋白(CpRNPs)在全球范围内影响低温条件下叶绿体mRNA的积累和加工。未发表的实验进一步证明，cpRNPs在翻译胁迫条件下全局结合从叶绿体核糖体中分离出来的RNA。CpRNPs以依赖于光和温度的方式表达和磷酸化。综上所述，cpRNPs具有独特的潜力，可以作为叶绿体RNA池的外部信号的全球调节器。我们打算解决以下问题：1.cpRNPs和核糖体分别结合的RNA池在逆境驯化过程中如何相互对应？ii.磷酸化如何影响cpRNPs的作用，以及cpRNPs与外界信号之间的联系是什么？iii.cpRNPs如何介导拟南芥的抗寒性？IV.cpRNP作用的分子机制是什么，特别是叶绿体mRNAs上确切的结合位点是什么？我们将使用全基因组定量分析cpRNPs的RNA配体(RIP-Chip和iCLIP)来确定cpRNPs的作用及其已知的磷酸化位点。此外，我们将结合遗传和生物化学方法鉴定作用于cpRNPs的激酶。最后，将利用叶绿体基因表达的药理、遗传和生理扰动来确定在发育和逆境驯化过程中的什么时间cpRNPs对于为叶绿体mRNAs提供稳定性是重要的。总之，我们的分析旨在揭示cpRNPs如何在全球范围内控制和执行叶绿体RNA代谢，以响应与叶绿体功能和植物生存相关的逆境。

Chloroplast RNA processing is mediated by a multitude of nuclear-encoded RNA binding proteins. Despite growing knowledge on the factors involved and mechanistic questions of individual gene expression steps being addressed, we still know little about the truly regulatory steps of chloroplast gene expression. Nor do we understand how chloroplast genes are co-regulated and how chloroplast gene expression is acclimated to changing external conditions. We have recently demonstrated that chloroplast ribonucleoproteins (cpRNPs) impact chloroplast mRNA accumulation and processing under low temperature conditions on a global scale. Unpublished experiments furthermore demonstrate that cpRNPs globally bind RNAs freed from chloroplast ribosomes under conditions of translational stress. cpRNPs are expressed and phosphorylated in a light and temperature-dependent manner. Taken together, cpRNPs have the unique potential to act as global modulators of external signals towards chloroplast RNA pools. We propose to address the following questions:I. How do RNA-pools bound by cpRNPs and by ribosomes, respectively, correspond to each other during stress acclimation?II. How does phosphorylation impact the role of cpRNPs and what are the kinases linking cpRNPs to external signals?III. How do cpRNPs mediate cold-resistance in Arabidopsis?IV. What is the molecular mechanism of cpRNP action, in particular, what are the exact binding sites on chloroplast mRNAs?We will use genome-wide quantitative profiling of RNA ligands of cpRNPs (RIP-Chip and iCLIP) to determine the role of cpRNPs and their known phosphorylation sites during acclimation processes. Furthermore, we will identify kinases acting upon cpRNPs using a combination of genetic and biochemical approaches. Finally, pharmacological, genetic and physiological perturbations of chloroplast gene expression will be harnessed to determine, at what time during development and stress acclimation cpRNPs are important to provide stability to chloroplast mRNAs. In sum, our analysis is designed to unravel, how chloroplast RNA metabolism is controlled and executed by cpRNPs on a global scale in response to stressors relevant for chloroplast function and plant survival.