Single mRNA interactome capture in Arabidopsis - RAPping rhythms

像大多数生物一样，高等植物已经获得了一个内源性计时器——生物钟，为地球自转引起的昼夜循环做准备。生物钟使许多过程以24小时的节奏发生，例如光合作用活动、生长和对环境的反应。时钟的重要性是突出的增强适应性的有机体与功能时钟。生理节律的基础是大部分转录组的周期性变化。虽然节律转录被认为是驱动mRNA节律的主要机制，但只有当mRNA的半衰期足够短时，才能观察到昼夜节律转录振荡。因此，RNA水平的调控塑造了昼夜转录组。从合成到衰变，大量rbp与mRNA相关，以协调mRNA的加工和功能。我们的目标是全面描述参与昼夜转录振荡的rna结合蛋白。以拟南芥富含甘氨酸的rna结合蛋白7 （AtGRP7）的转录本为例。这是最丰富的转录本之一，其振荡幅度非常大。数学模型表明，除了启动子的时钟调节外，转录后调节对高振幅振荡也很重要。为了恢复AtGRP7转录物和体内相互作用的蛋白质，我们将使用长生物素化的反义寡核苷酸来捕获单个mRNA。rna -蛋白复合物将通过在体内与紫外光交联来稳定。具有不同序列标签的基因组AtGRP7构建体将用于针对标签序列的头部偶联寡核苷酸的下拉。此外，我们将使用寡核苷酸对抗内源性AtGRP7。这些方法将根据AtGRP7转录物的富集和核糖体RNA的消耗，以及已知AtGRP7相互作用蛋白的富集和不相关蛋白的消耗来进行优化。执行最佳的程序将按比例扩大。从捕获的AtGRP7 RNA中洗脱的蛋白质将通过质谱法进行鉴定。选择的rna结合蛋白在塑造AtGRP7振荡中的功能将使用突变体进行研究。为了全面了解rbp的调控环境，将使用iCLIP确定其结合位点和直接体内靶点的宽度。在这些目标中，我们将检查时钟基因和节律输出基因。总的来说，该项目将为阐明协调昼夜节律的转录后调控和理解决定昼夜节律rna命运的因素提供一个入口，并将为细胞中转录后网络的潜在机制提供新的见解。

Higher plants like most organisms have acquired an endogenous timekeeper, the circadian clock, to prepare for the day-night-cycle caused by the Earth´s rotation. The circadian clock causes many processes to occur with a 24-h rhythm, e.g. photosynthetic activity, growth and responses to the environment. The importance of the clock is highlighted by enhanced fitness of organisms with a functional clock. Underlying the physiological rhythms are periodic changes in a large fraction of the transcriptome. While rhythmic transcription is considered the prime mechanism driving mRNA rhythms, circadian transcript oscillations are only observed if an mRNA has a sufficiently short half-life. Thus, regulation at the RNA level shapes the circadian transcriptome. From synthesis to decay, a plethora of RBPs associate with mRNAs to coordinate mRNA processing and function.Our goal is a comprehensive description of RNA-binding proteins involved in circadian tran-script oscillations. As a paradigm serves the transcript encoding Arabidopsis thaliana glycine-rich RNA-binding protein 7 (AtGRP7). This is one of the most highly abundant transcripts, and its oscillations show a very large amplitude. Mathematical modelling has shown that posttranscriptional regulation in addition to clock regulation of the promoter is important for high amplitude oscillations.To recover the AtGRP7 transcript and proteins interacting in vivo we will work toward single mRNA capture using long biotinylated antisense oligonucleotides. RNA-protein complexes will be stabilized by in vivo crosslinking with UV light. Genomic AtGRP7 constructs with different sequence tags will be used for pulldown with bead-coupled oligonucleotides directed against the tag sequences. In addition, we will use oligonucleotides against endogenous AtGRP7.These methods will be optimized based on the enrichment of the AtGRP7 transcript and de-pletion of ribosomal RNA, and enrichment of known AtGRP7 interacting proteins and depletion of unrelated proteins. The procedure performing best will be scaled up. The proteins eluted from the captured AtGRP7 RNA will be identified via mass spectrometry. A function of selected RNA-binding proteins in shaping AtGRP7 oscillations will be investigated using mutants. To obtain global insights into the regulatory milieu of the RBPs their binding sites and the breadth of their direct in vivo targets will be determined using iCLIP. Among these targets we will check for clock genes and rhythmic output genes. Overall, the project will provide an entré to elucidating posttranscriptional regulons coordinating circadian rhythms and understanding what determines the fate of circadian RNAs and will deliver new insight into mechanisms underlying posttranscriptional networks in the cell.