Structure and function of a cytoplasmic mRNA-localization complex from yeast

真核细胞在主动运输过程的帮助下组织它们的内部生命。mRNA的不对称转运和定位翻译是基因表达时空调控的普遍机制。它用于细胞过程，例如不对称细胞分裂、细胞分化或神经元的突触可塑性。mRNA转运复合物通常由几种蛋白质和RNA组成。一个非常简单但研究得很好的模型是在芽殖酵母有丝分裂过程中ASH1 mRNA的不对称定位。在这里，所有的核心因素都是已知的，并已被研究，主要是遗传。第一个资助期的目标是了解ASH1 mRNA转运复合物中核心因子的相互作用，并启动高分辨率结构研究。基于这些结果，我们现在能够在体外重建由多达四种蛋白质和一种RNA组成的转运核心复合物及其子复合物。在下一个资助期内，我们希望使用冷冻EM和X射线晶体学结合生物化学方法来解决整个复杂的结构。我们还将通过结构分析评估不同的亚复合物，并定量研究它们的组装。该项目旨在对这些分子机器进行第一次结构到功能评估。

Eukaryotic cells organize their inner life with the help of active transport processes. The asymmetric transport and localized translation of mRNA is a widespread mechanism for temporal and spatial control of gene expression. It is used during cellular processes such as asymmetric cell division, cell differentiation, or synaptic plasticity of neurons. mRNAtransport complexes usually consist of several proteins and RNAs. A comparably simple but well-studied model is the asymmetric localization of ASH1 mRNA during mitosis of the budding yeast. Here, all core factors are known and have been studied mostly genetically. The goal of the first funding period was to understand interactions of the core factors in the ASH1 mRNA transport complex and to initiate high-resolution structural studies. Based on these results, we are now able to in-vitro reconstitute transport-core complexes, consisting of up to four proteins and one RNA, as well as sub-complexes thereof. In the next funding period, we wish to use cryo-EM and X-ray crystallography in combination with biochemical approaches to solve the structure of the entire complex. We will also assess different subcomplexes by structural analysis and study their assembly quantitatively. This project is designed to yield a first structure-to-functional assessment of these molecular machines.