Single mRNA interactome capture in Arabidopsis - RAPping rhythms

高等植物和大多数生物一样，已经获得了一个内源性的计时器--生物钟，为地球自转引起的昼夜循环做准备。生物钟导致许多过程以24小时的节奏发生，例如光合作用活性，生长和对环境的反应。生物钟的重要性通过增强具有功能时钟的生物体的适应性而突出。生理节律的基础是大部分转录组的周期性变化。虽然有节奏的转录被认为是驱动mRNA节奏的主要机制，但只有在mRNA具有足够短的半衰期时才能观察到昼夜节律的转录振荡。因此，RNA水平的调节塑造了昼夜节律转录组。从合成到衰变，大量的RBP与mRNA结合以协调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。作为范例，提供了编码拟南芥富含甘氨酸的RNA结合蛋白7（AtGRP7）的转录物。这是最丰富的转录本之一，其振荡显示出非常大的振幅。数学建模表明，转录后调节除了时钟调节的启动子是重要的高振幅oscillation.To恢复AtGRP7转录和蛋白质在体内相互作用，我们将努力对单一的mRNA捕获使用长生物素化的反义寡核苷酸。RNA-蛋白质复合物将通过用UV光进行体内交联来稳定。具有不同序列标签的基因组AtGRP7构建体将用于与针对标签序列的珠偶联寡核苷酸的下拉。此外，我们还将利用针对内源性AtGRP7的寡核苷酸，通过富集AtGRP7转录本和去除核糖体RNA，富集已知的AtGRP7相互作用蛋白和去除不相关蛋白，对这些方法进行优化。表现最好的程序将扩大规模。将通过质谱法鉴定从捕获的AtGRP7 RNA洗脱的蛋白质。选定的RNA结合蛋白在塑造AtGRP7振荡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确定其结合位点及其直接体内靶标的宽度。在这些目标中，我们将检查时钟基因和节奏输出基因。总的来说，该项目将提供一个阐明转录后调节子协调昼夜节律和理解是什么决定了昼夜节律RNA的命运的切入点，并将对细胞中转录后网络的机制提供新的见解。

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.