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

在酿酒酵母中，ASH1 mRNA和进一步的转录本被积极地运输到生长的芽中，以支持细胞的不对称分裂。在这种RNA定位的范例中，一个核心角色是RNA结合蛋白（RBP） She2p。作为SHE机制的一部分，She2p与其核中的货物mrna结合，并促进其在细胞质中的亚细胞定位。在这一过程中，She2p与不同的连接蛋白结合，即细胞核中的Loc1p和细胞质中的She3p，从而稳定其与RNA定位元件（RNA localization elements, LEs）的结合。在第一个资助期，我们可以揭示She2p和She3p如何联合结合到ASH1 LE RNA上。结合晶体学和化学探针，我们可以直接看到RNA在与适配器蛋白She3p结合时如何采用独特的折叠。基于详细的结构信息，我们计算得出了一个统一的描述符，它允许在计算机上预测已知目标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，以研究细胞核和细胞质中的SHE机制如何特异性识别LEs。将体外和体内全转录组RBP结合图与结构生物学和生物信息学相结合，我们将解决(i)细胞质中的SHE机制识别哪些LEs， （ii）转运复合体的核胞质重塑过程中SHE结合如何变化，以及（iii）哪些RNA特征对定义LEs至关重要。我们的联合方法范围从全局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。

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.