Tropomyosin 1 and End-binding protein 1 in mRNA transport

在第一个资助期间，我们能够证明许多最近发现的新型RNA结合蛋白（rbp），它们不包含任何经典的RNA结合结构域，确实能够在体外结合RNA。此外，我们可以证明核磁共振波谱是验证RNA结合蛋白的一种极好的方法，并建议在开始新的RNA结合蛋白项目之前对RNA结合蛋白进行彻底的体外验证。两个新的RNA结合蛋白，原肌球蛋白1和末端结合蛋白1 (EB1)，我们可以证实是内在的RNA结合蛋白。原肌球蛋白1作为RNA转运到细胞内最终目的地的衔接蛋白，对RNA转运至关重要。在这里，RNA通过原肌球蛋白1与肌动蛋白结合，肌动蛋白沿着微管移动。在第一个资助期内，我们可以通过x射线晶体学解决第一个高分辨率结构的运动蛋白接头（原肌球蛋白1）蛋白复合体，这为运输机制提供了巨大的见解。此外，我们还鉴定出原肌球蛋白1的RNA结合区。这使我们能够在果蝇中进行有针对性的功能研究，从分子细节上了解RNA转运的整个机制。此外，我们的目标是确定一个三元复结构的运动蛋白，原肌球蛋白和RNA。对于EB1，我们不仅可以验证其与RNA的结合，还可以确定其在蛋白表面的确切结合区域。这与微管的结合位点重叠。此外，EB1内的连接子通过苏氨酸与其n端结构域相互作用。已知这种苏氨酸被磷酸化，这可能是EB1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EB1磷酸化和RNA结合对微管组装的影响。

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.