Spatiotemporal analysis of in vivo RNA-protein interaction

体内 RNA-蛋白质相互作用的时空分析

• 批准号: 422321065
• 负责人: Professorin Dr. Dorothee Staiger
• 金额: --
• 依托单位: Arbeitsgruppe RNA Biologie und Molekulare Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422321065?language=en
• 关键词: Spatiotemporal; analysis; vivo; RNA; protein

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.

从合成到降解，RNA与细胞中的RNA结合蛋白（RBP）动态相互作用，以在前体mRNA剪接、转运、翻译和RNA降解水平上协调转录组。缺乏RBP体内结合靶点和结合景观的全基因组视图代表了理解植物RBP作用模式的差距。在我们的实验室中建立的植物中的单个核苷酸分辨率交联免疫沉淀（iCLIP）使我们能够获得RBP AtGRP 7（拟南芥富含甘氨酸的RNA结合蛋白7）的体内结合景观的全局图像。在这里，用紫外线照射植物以稳定RNA-蛋白质复合物并保持体内状态。将复合物免疫沉淀，并从共沉淀的RNA产生文库用于高通量测序。由于逆转录酶在交联位点处保留的肽处停止，因此使用生物信息学流水线通过核苷酸分辨率来确定结合位点。AtGRP 7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点。此外，AtGRP 7穿梭于细胞核和细胞质之间，因此可能在两个亚细胞区室中发挥不同的调节功能。 为了进一步分析AtGRP 7在亚细胞区室中的功能，我们将分别确定其在细胞质和细胞核中的靶转录物。我们已经表明，AtGRP 7结合到转录已知的调节下冷应力和AtGRP 7影响选择性剪接在低温下。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。 这些数据的分析将揭示哪些转录本优先在任一隔室中相互作用。在两个区室中结合的转录物可以表现出在区室之间的结合位点（非翻译区、编码序列和内含子）的位置的移位，暗示结合事件的不同调节结果。此外，我们将确定AtGRP 7的结合响应于低温的变化。随后，我们将通过测试对细胞质和细胞核中结合靶点的RNA稳定性、选择性剪接或选择性多聚腺苷酸化的影响来揭示AtGRP 7如何影响其靶点。此外，我们将通过评估AtGRP 7表达改变的植物的冷适应来评估AtGRP 7对植物对冷暴露的生理反应的影响。与此同时，我们将致力于改善生物信息学，以确定从拟南芥中产生的iCLIP数据的结合位点，并检测定量变化。总的来说，本项目将为研究转录后网络的动态及其与拟南芥低温反应的相关性铺平道路。