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

在酿酒酵母中，ASH1mRNA和更多的转录本被积极地运输到生长的芽中，以支持不对称的细胞分裂。在这种RNA本地化的范例中，一个核心角色是RNA结合蛋白(RBP)She2p。作为SHE机制的一部分，She2p与其在细胞核中的货运mRNAs结合，并促进它们在细胞质中的亚细胞定位。在这个过程中，She2p与不同的适配蛋白形成亚复合体，即细胞核中的Loc1p和细胞质中的She3p，这稳定了它与RNA定位元件(LES)的结合。在第一个资助期，我们可以解开She2p和She3p是如何与ASH1 le RNA结合的。结合结晶学和化学探测，我们可以直接可视化RNA如何在适配蛋白She3p结合时采用不同的折叠。基于详细的结构信息，我们通过计算得到了一个统一的描述符，该描述符可以用计算机预测已知靶标mRNAs中SHE结合位点的位置。值得注意的是，我们可以在体外验证She2p/She3p与新预测的LES的结合，强调我们确实捕获了SHE识别的主要分子特征。在第二个资助期，我们建议将我们的分析扩展到转录组范围，以研究LES是如何被细胞核和细胞质中的SHE机制特异性识别的。结合体外和体内转录组范围的RBP结合图以及结构生物学和生物信息学，我们将解决(I)细胞质中的SHE机制识别哪些LES，(Ii)SHE结合如何在运输复合体的核质重塑过程中发生变化，以及(Iii)哪些RNA特征是定义LES的关键。我们的组合方法范围从全局结合模式到高分辨率结合确认，并将使我们能够解开多个蛋白质对SHE结合和mRNA定位这一重要模型系统的组合贡献。

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.