Single mRNA interactome capture in Arabidopsis - RAPping rhythms

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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.