Elucidating mechanisms of cytoplasmic mRNA transport using in vitro and in vivo methods

博士项目战略主题：了解生命的规则mRNA的亚细胞定位是控制蛋白质在细胞中运作的重要机制。这一过程对于胚胎发育和包括神经元、肌肉和成纤维细胞在内的特化细胞的功能至关重要。分子马达在mRNA定位中起着关键作用。它们通过衔接蛋白识别mRNA分子，并将其沿着细胞骨架转运。具体的mRNA如何被运输机制识别并定位到不同的细胞内位点还知之甚少。在真核细胞中，动力蛋白负责将mRNA转运到微管的负端。动力蛋白介导的mRNA转运系统在遗传上易于处理的生物果蝇中得到了最好的理解，其中该过程在卵母细胞、胚胎、成神经细胞和感觉神经元中起着重要作用。布洛克小组已经证明，长距离mRNA运输的最小组分是动力蛋白、其活化复合物动力肌动蛋白、动力蛋白-动力肌动蛋白衔接子BicD、RNA结合蛋白Egl和双链mRNA定位信号。由于Egl缺乏典型的双链RNA结合结构域，并且Egl的mRNA靶标中的茎环不具有明显的序列相似性，因此复合物如何识别货物的结构基础尚不清楚。在果蝇卵子发生中，动力蛋白-动力肌动蛋白-BicD-Egl复合物是将编码轴决定簇的mRNA转运到卵母细胞中不同位点所必需的。运输机制的活性如何适应于差异地定位这些mRNA种类也是未知的。我博士学位的第一个目标是阐明动力蛋白复合物识别mRNA的结构基础。这项工作将建立在与不同mRNA定位信号结合的Egl-BicD复合物的高分辨率cryo-EM结构上，该结构是最近通过与LMB的Andrew Carter小组合作产生的。我将研究Egl和RNA茎环的结构特征介导的识别使用微型热泳。我将进一步评估的重要性，具体的RNA功能的运输使用一个既定的体外运动试验和显微注射荧光RNA到果蝇胚胎。一旦了解了对活动至关重要的RNA特征，我将与生物信息学家合作，在果蝇基因组中搜索和验证新的定位信号。第二个相关的目的是了解动力蛋白-动力蛋白-BicD-Egl机制的活性如何适应于将不同的mRNA定位于不同的细胞内位点。在果蝇卵母细胞中，hnRNP Squid（Sqd）对于grk mRNA定位于背前部区域至关重要。布洛克小组的初步工作提出了一种可能性，即Sqd通过促进RNA多聚化，从而增加动力蛋白拷贝数，从而促进grk mRNA向背前部区域的运输。我将评估RNA和动力蛋白拷贝数对RNA运输的贡献，使用体外和体内测定，包括优化方法，以可视化卵母细胞中单个RNP的运输。预计这项研究将进一步了解微管马达如何识别特定的mRNA并将其分类到不同的目的地。

PhD project strategic theme: Understanding the rules of lifeSubcellular localisation of mRNAs is an important mechanism for controlling where proteins operate in cells. This process is critical for embryonic development and for the function of specialised cells including neurons, muscles and fibroblasts. Molecular motors play a key role in mRNA localisation. They recognise mRNA molecules via adaptor proteins and translocate them along the cytoskeleton. How specific mRNAs are recognised by the transport machinery and localised to distinct intracellular sites is poorly understood. The motor protein dynein is responsible for transporting mRNAs to the minus ends of microtubules in eukaryotic cells. The best understood system for dynein-mediated mRNA transport is in the genetically tractable organism Drosophila, in which this process plays important roles in oocytes, embryos, neuroblasts and sensory neurons. The Bullock group has shown that the minimal components for long-distance mRNA transport are dynein, its activating complex dynactin, the dynein-dynactin adaptor BicD, the RNA-binding protein Egl and double-stranded mRNA localisation signals. As Egl lacks canonical double-stranded RNA binding domains and the stem-loops in Egl's mRNA targets do not share overt sequence similarity, the structural basis of how the complex recognises cargo is unclear. In Drosophila oogenesis, the dynein-dynactin-BicD-Egl complex is required for transport of mRNAs encoding axis determinants to different sites in the oocyte. How the activity of the transport machinery is adapted to localise these mRNA species differentially is also not known. The first objective of my PhD is to elucidate the structural basis of mRNA recognition by the dynein complex. This work will build on high-resolution cryo-EM structures of the Egl-BicD complex bound to different mRNA localisation signals, produced recently through a collaboration with Andrew Carter's group at LMB. I will investigate which structural features of Egl and the RNA stem loops mediate recognition using microscale thermophoresis. I will further assess the importance of specific RNA features for transport using an established in vitro motility assay and microinjection of fluorescent RNAs into Drosophila embryos. Once the RNA features that are critical for activity are understood, I will work with a bioinformatician to search for and validate new localisation signals in the Drosophila genome. The second, related objective is to understand how the activity of the dynein-dynactin-BicD-Egl machinery is adapted to localise different mRNAs to different intracellular sites. In the Drosophila oocyte, the hnRNP Squid (Sqd) is critical for the localisation of grk mRNA to the dorso-anterior region. Preliminary work in the Bullock group has raised the possibility that Sqd promotes trafficking of grk mRNA to the dorso-anterior region by promoting RNA multimerisation, which in turn increases dynein copy number. I will assess the contribution of RNA and dynein copy number on RNA trafficking using in vitro and in vivo assays, including optimising methods to visualise trafficking of individual RNPs in the oocyte. It is anticipated that this research will further our understanding of how microtubule motors recognises specific mRNAs and sorts them to different destinations.