Spatiotemporal analysis of in vivo RNA-protein interaction

从合成到衰变，RNA与细胞中的RNA结合蛋白(RBP)动态相互作用，在前mRNA剪接、运输、翻译和RNA衰变的水平上协调转录组。缺乏对活体RBP结合靶标和结合景观的全基因组观点，代表着对植物限制性商业惯例作用模式的理解存在差距。我们实验室在植物中建立的单个核苷酸分辨交联免疫沉淀(ICLIP)使我们能够获得RBP AtGRP7(拟南芥富含甘氨酸的RNA结合蛋白7)在体内结合的全球图谱。在这里，植物受到紫外线的照射，以稳定RNA-蛋白质复合体，并保持体内的状况。这些复合体是免疫共沉淀的，并从共沉淀的RNA中产生文库用于高通量测序。当逆转录酶停滞在交联点的多肽时，结合位置通过使用生物信息学管道的核苷酸解析来确定。AtGRP7与非翻译区、编码序列和内含子结合，表明它在RNA成熟的多个步骤中调节其靶标。此外，AtGRP7穿梭于胞核和胞浆之间，因此可能在两个亚细胞间发挥不同的调节功能。为了进一步剖析AtGRP7在亚细胞室中的功能，我们将分别在细胞质和细胞核中确定其靶向转录本。我们已经证明AtGRP7与已知在冷应激下调节的转录本结合，并且AtGRP7影响低温反应的选择性剪接。因此，我们将对暴露于4°C的植物和保持在20°C的对照植物的细胞核和细胞质部分进行iCLIP实验。对这些数据的分析将揭示哪些转录本在这两个隔室中优先相互作用。在两个隔间结合的转录本可能显示出隔间之间结合位点(非翻译区、编码序列和内含子)的位置移动，暗示结合事件的不同调控结果。此外，我们还将确定结合的AtGRP7在低温响应下的变化。随后，我们将通过测试对RNA稳定性的影响、细胞质和细胞核中结合靶的选择性剪接或选择性多聚腺苷基化来揭示AtGRP7是如何影响其靶标的。此外，我们将通过评估AtGRP7表达改变的植物的冷适应来评估AtGRP7对植物冷暴露的生理反应的影响。同时，我们将致力于改进生物信息学，从拟南芥产生的iCLIP数据中确定结合位点，并检测数量变化。总体而言，该项目将为研究转录后网络的动力学及其与拟南芥低温反应的相关性铺平道路。

From synthesis to decay, RNAs dynamically interact with RNA-binding proteins (RBPs) in the cell to orchestrate the transcriptome at the level of pre-mRNA splicing, transport, translation, and RNA decay. The lack of a genome-wide view on RBP in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RBPs. Establishment of individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) in plants in our laboratory has enabled us to obtain a global picture of the in vivo binding landscape of the RBP AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7). Here, plants are irradiated with UV light to stabilize RNA-protein complexes and preserve the in vivo situation. The complexes are immunoprecipitated and libraries are generated from the co-precipitated RNAs for high-throughput sequencing. As reverse transcriptase stalls at the peptide remaining at the crosslink site, binding sites are determined with nucleotide resolution using bioinformatics pipelines. AtGRP7 binds to untranslated regions, coding sequences, and introns, indicating that it regulates its targets at multiple steps of RNA maturation. Furthermore, AtGRP7 shuttles between the nucleus and cytoplasm and thus may exert different regulatory functions in the two subcellular compartments. To further dissect the functions of AtGRP7 in the subcellular compartments, we will determine its target transcripts in the cytoplasm and the nucleus separately. We have shown that AtGRP7 binds to transcripts known to be regulated under cold stress and that AtGRP7 affects alternative splicing in response to low temperature. Therefore, we will perform the iCLIP experiments on the nuclear and the cytoplasmic fractions for plants that are exposed to 4 °C and for control plants kept at 20 °C. Analysis of these data will reveal which transcripts interact preferentially in either compartment. Transcripts bound in both compartments may exhibit a shift in the location of the binding site (untranslated regions, coding sequence, and introns) between the compartments, hinting at different regulatory outcomes of the binding events. Furthermore, we will identify changes of AtGRP7 of binding in response to low temperature. Subsequently, we will unravel how AtGRP7 impacts its targets by testing for an influence on RNA stability, alternative splicing or alternative polyadenylation of the binding targets in the cytoplasm and the nucleus. Furthermore, we will assess the impact of AtGRP7 on physiological responses of the plant to cold exposure by assessing cold adaptation in plants with altered AtGRP7 expression. In parallel, we will work towards improving the bioinformatics to determine binding sites from iCLIP data generated in Arabidopsis and to detect quantitative changes. Overall, this project will pave the way to investigate the dynamics of posttranscriptional networks and their relevance for the low temperature response of Arabidopsis.