Tropomyosin 1 and End-binding protein 1 in mRNA transport

mRNA 转运中的原肌球蛋白 1 和末端结合蛋白 1

• 批准号: 313646688
• 负责人: Dr. Anne Ephrussi, Ph.D.
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313646688?language=en
• 关键词: Tropomyosin; End; binding; protein; mRNA

During the first funding period we were able to show that many of the recently discovered novel RNA binding proteins (RBPs), which do not comprise any classical RNA binding domain are indeed able to bind RNA in vitro. Furthermore, we could demonstrate that nuclear magnetic resonance spectroscopy is an excellent method to validate RNA binding proteins and would advice a thorough in vitro validation of RNA binding proteins before starting a new project on novel RNA binding proteins. Two of the novel RNA binding proteins, Tropomyosin 1 and End-binding protein 1 (EB1) we could verify to be intrinsic RNA binding proteins. Tropomyosin 1 is essential for RNA transport as an adaptor protein for RNA to be transported to its final destination in the cell. Here, RNA binds via Tropomyosin 1 to Kinesin, which moves along microtubules. During the first funding period, we could solve the first high-resolution structure of a Kinesin-adaptor (Tropomyosin 1) protein complex via X-ray crystallography, which provides tremendous insights into the transport mechanism. Additionally, we could identify the RNA binding region of Tropomyosin 1. This enables us to pursue targeted functional studies in fruit flies, to understand the entire mechanism of RNA transport in molecular detail. Also, we aim to determine a ternary complex structure of Kinesin, Tropomyosin and RNA.Regarding EB1, we could not only verify its RNA binding, but also determine the exact region of binding on the protein surface. This overlaps with the binding site to microtubules. Moreover, the linker within EB1 interacts via a threonine with its N-terminal domain. This threonine is known to be phosphorylated, which might be an activity switch of EB1. In the second funding period we will follow this up with detailed mutational analyses in vitro and in vivo, to understand the influence of phosphorylation and RNA binding of EB1 on microtubule assembly.

在第一个资助期内，我们能够证明许多最近发现的不包含任何经典RNA结合结构域的新型RNA结合蛋白（RBP）确实能够在体外结合RNA。此外，我们可以证明，核磁共振光谱是一种很好的方法来验证RNA结合蛋白，并建议在开始一个新的项目之前，在体外进行彻底的验证RNA结合蛋白的新的RNA结合蛋白。其中两个新的RNA结合蛋白原肌球蛋白1和末端结合蛋白1（EB 1），我们可以证实是固有的RNA结合蛋白。原肌球蛋白1是RNA转运所必需的，作为RNA转运到细胞中最终目的地的衔接蛋白。在这里，RNA通过原肌球蛋白1与驱动蛋白结合，驱动蛋白沿着沿着微管移动。在第一个资助期间，我们可以通过X射线晶体学解决驱动蛋白-适配器（原肌球蛋白1）蛋白复合物的第一个高分辨率结构，这为运输机制提供了巨大的见解。此外，我们可以确定原肌球蛋白1的RNA结合区域。这使我们能够在果蝇中进行有针对性的功能研究，以了解RNA转运的分子细节的整个机制。另外，我们的目标是确定驱动蛋白，原肌球蛋白和RNA的三元复合物结构。对于EB 1，我们不仅可以验证其RNA结合，而且还可以确定其在蛋白质表面上的确切结合区域。这与微管的结合位点重叠。此外，EB 1内的接头通过苏氨酸与其N-末端结构域相互作用。已知该苏氨酸被磷酸化，这可能是EB 1的活性开关。在第二个资助期，我们将在体外和体内进行详细的突变分析，以了解EB 1的磷酸化和RNA结合对微管组装的影响。