Combination of transcriptome-wide and structural approaches to study SHE binding to RNA localization elements in budding yeast

结合全转录组和结构方法来研究芽殖酵母中 SHE 与 RNA 定位元件的结合

• 批准号: 420693300
• 负责人: Dr. Julian König, Ph.D.
• 金额: --
• 依托单位: Institut für Molekulare Biologie gGmbH (IMB)
• 依托单位国家: 德国
• 项目类别: Research Units
• 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/420693300?language=en
• 关键词: Combination; transcriptome; wide; structural; approaches

In Saccharomyces cerevisiae, ASH1 mRNA and further transcripts are actively transported to the growing bud to support asymmetric cell division. A central player in this paradigm example of RNA localization is the RNA-binding protein (RBP) She2p. As part of the SHE machinery, She2p associates with its cargo mRNAs in the nucleus and promotes their subcellular localization in the cytoplasm. During this process, She2p engages into subcomplexes with different adapter proteins, namely Loc1p in the nucleus and She3p in the cytoplasm, which stabilize its binding to the RNA localization elements (LEs). In the first funding period, we could unravel how She2p and She3p jointly bind to an ASH1 LE RNA. Combining crystallography and chemical probing, we could directly visualize how the RNA adopts a distinct fold upon association of the adapter protein She3p. Based on the detailed structural information, we computationally derived a unifying descriptor, which allowed to predict in silico the location of SHE binding sites in known target mRNAs. Notably, we could validate in vitro She2p/She3p binding to newly predicted LEs, underlining that we indeed captured major molecular features of SHE recognition.In the second funding period, we propose to take our analysis to a transcriptome-wide scale to investigate how LEs are specifically recognized by the SHE machinery in the nucleus and the cytoplasm. Combining transcriptome-wide in vitro and in vivo RBP binding maps with structural biology and bioinformatics, we will address (i) which LEs are recognized by the SHE machinery in the cytoplasm, (ii) how SHE binding changes during nucleo-cytoplasmic remodeling of the transport complexes, and (iii) which RNA features are critical to define LEs. Our combined approach ranges from global binding patterns to high-resolution binding confirmations and will enable us to disentangle the combinatorial contribution of multiple proteins to SHE binding and mRNA localization this important model system.

在酿酒酵母（Saccharomyces cerevisiae）中，ASH 1 mRNA和其他转录物被主动转运到生长的芽中以支持不对称细胞分裂。RNA定位的这个范例中的核心参与者是RNA结合蛋白（RBP）She 2 p。作为SHE机制的一部分，She 2 p与其在细胞核中的货物mRNA相关联，并促进其在细胞质中的亚细胞定位。在此过程中，She 2 p与不同的衔接蛋白（即细胞核中的Loc 1 p和细胞质中的She 3 p）形成亚复合物，从而稳定其与RNA定位元件（LE）的结合。在第一个资助期，我们可以解开She 2 p和She 3 p如何共同结合到ASH 1 LE RNA。结合晶体学和化学探测，我们可以直接可视化RNA如何在与接头蛋白She 3 p结合时采用不同的折叠。基于详细的结构信息，我们通过计算得出了一个统一的描述符，该描述符允许通过计算机预测已知靶mRNA中SHE结合位点的位置。值得注意的是，我们可以在体外验证She 2 p/She 3 p结合到新预测的LEs，强调我们确实捕获了SHE recognition.In第二个资金周期的主要分子特征，我们建议采取我们的分析，以转录组范围内的规模，调查如何在细胞核和细胞质中的SHE机制，特别是识别LE。结合转录组范围内的体外和体内RBP结合图谱与结构生物学和生物信息学，我们将解决（i）哪些LE被细胞质中的SHE机制识别，（ii）SHE结合在转运复合物的核质重塑过程中如何变化，以及（iii）哪些RNA特征对定义LE至关重要。我们的结合方法范围从全球结合模式到高分辨率结合确认，将使我们能够解开多种蛋白质对SHE结合和mRNA定位这一重要模型系统的组合贡献。