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的RNA结合蛋白(RBPs)确实能够在体外与RNA结合，这些蛋白不包含任何经典的RNA结合域。此外，我们可以证明核磁共振波谱是验证RNA结合蛋白的一种很好的方法，并建议在启动新的RNA结合蛋白的新项目之前，对RNA结合蛋白进行彻底的体外验证。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(EB1)，我们可以证实是内源性的RNA结合蛋白。原肌球蛋白1是RNA转运所必需的，是RNA转运到细胞内最终目的地的适配蛋白。在这里，RNA通过原肌球蛋白1与沿微管移动的动蛋白结合。在第一个资助期，我们可以通过X射线结晶学解决第一个高分辨率的动蛋白适配器(原肌球蛋白1)蛋白质复合体的结构，这为运输机制提供了巨大的洞察力。此外，我们还可以确定原肌球蛋白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运输的整个机制。此外，我们的目标是确定一个由动蛋白、原肌球蛋白和RNA组成的三元复合体结构。对于EB1，我们不仅可以验证其与RNA的结合，还可以确定蛋白质表面的确切结合区域。这与微管的结合部位重叠。此外，EB1内的连接子通过苏氨酸与其N-末端结构域相互作用。这种苏氨酸是已知的被磷酸化的，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的磷酸化和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.