Rapid ribonucleoprotein-dependent recruitment of mRNA to ribosomes in the high light acclimation of Arabidopsis thaliana

该研究旨在了解叶绿体代谢状态对核糖核蛋白控制介导的胞质翻译的快速影响。该项目利用了低光到高光（L-H）转移的精确实验设计，并利用了翻译组的快速条件重组。它基于三个假设，即（i）叶绿体控制其细胞附近的核糖体的翻译活性，（ii）L-H转移后核糖体负载mRNA的大量重组取决于UTR和RNA结合蛋白（RBP）的特征，以及（iii）RBP的翻译后修饰决定了特定mRNA向核糖体的募集。这三个假设将在七个工作包中得到解决。应激相关的锌指蛋白SAP 3将被用作一个例子来测试三个序列基序的特异性和功能，这三个序列基序被发现在L-H转移后10分钟优先与多核糖体相关的272个转录物中的5-UTR中高度过度表达。使用RNaseq对多聚体RNA进行分析，我们将完善核糖体快速募集mRNA的数据集以及该调节过程的可逆性。通过使用转染的原生质体和稳定转化的拟南芥sap 2/sap 3-双突变体，将详细检查这三个基序以及5-UTR和3-UTR在H-L-转移中的意义。将鉴定与基序和UTR结合的RBP，并将鉴定低光和L-H样品的多核糖体蛋白质组之间的动态差异。将在体外和体内研究鉴定的RBP的分子和生理作用。为了完成对所涉及的途径和信号的理解，将使用确定的遗传突变体（例如mpk 6）来解决翻译L-H调节与MAP激酶途径的上游偶联。下游靶基因如σ因子5和小HSP的调节将通过敲除植物的生理分析来评估。该项目根据呼吁中定义的具体目标和工作计划纳入SPP 1935的框架，即澄清RNP的调控和动态，建立和应用特定RNP分离和鉴定技术，以及在高等植物光适应基本过程的背景下扩展包括上游和下游元素的特定调控网络。

The proposed research aims at understanding the extremely rapid effect of chloroplast metabolic state on cytosolic translation mediated by control of ribonucleoproteins. The project takes advantage of the precise experimental design of low light-to-high light- (L-H)-transfer and exploits the very fast conditional reorganization of the translatome. It is based on three hypotheses namely that (i) chloroplasts control the translational activity of ribosomes in their cellular vicinity, (ii) that the massive reorganization of ribosome-loaded mRNAs upon L-H-transfer depends on features of the UTR and RNA-binding proteins (RBPs) and (iii) that posttranslational modifications of RBPs determine the recruitment of specific mRNA to the ribosomes. The three hypotheses will be addressed in seven work packages. The stress associated Zinc-finger protein SAP3 will be used as an example to test the specificity and function of three sequence motifs found to be highly overrepresented in the 5-UTR among the 272 transcripts preferentially associated with the polysomes 10 min after L-H-transfer. Using RNAseq on polysomal RNAs we will refine the data set of rapid ribosome-recruited mRNAs and the reversibility of this regulatory process. The significance of the three motifs and the 5-UTR and 3-UTR in H-L-transfer will be scrutinized by using transfected protoplasts and stably transformed sap2/sap3-double mutants of Arabidopsis thaliana. RBPs will be identified that bind to the motifs and the UTRs and dynamic differences between the polysomal proteomes of low light and L-H-samples will be identified. The molecular and physiological role of identified RBPs will be investigated in vitro und in vivo. In order to complete the understanding of the involved pathways and signals, upstream coupling of the translational L-H-regulation to the MAP kinase pathway will be addressed using defined genetic mutants, e.g. mpk6. Regulation of downstream target genes like the sigma factor 5 and small HSPs will be assessed by physiological analysis of knock out plants. This project is integrated into the framework of SPP1935 according to the specific aims and work plan defined in the call, i.e. clarification of regulation and dynamics of RNPs, establishment and application of technologies for specific RNP isolation and identification, and expansion of the specific regulatory network including upstream and downstream elements in context of the fundamental process of light acclimation in higher plants.