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

博士项目战略主题：了解mrna的亚细胞定位规则是控制蛋白质在细胞中运作的重要机制。这一过程对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定位中起关键作用。它们通过接头蛋白识别mRNA分子，并将其沿细胞骨架转运。转运机制如何识别特异性mrna并将其定位到不同的细胞内位点，目前尚不清楚。在真核细胞中，运动蛋白动力蛋白负责将mrna运送到微管的负端。对动力蛋白介导的mRNA转运系统了解最多的是遗传易感生物果蝇，该过程在卵母细胞、胚胎、神经母细胞和感觉神经元中起着重要作用。Bullock小组已经表明，长距离mRNA运输的最小组分是动力蛋白，其激活复合物动力蛋白，动力蛋白-动力蛋白适配器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结合域，并且Egl mRNA靶标中的茎环没有明显的序列相似性，因此该复合物如何识别货物的结构基础尚不清楚。在果蝇的卵发生过程中，编码轴决定因子的mrna转运到卵母细胞的不同位置需要dynein-dynactin-BicD-Egl复合物。转运机制的活性如何适应这些mRNA物种的不同定位也不清楚。我博士的第一个目标是阐明动力蛋白复合体识别mRNA的结构基础。这项工作将建立在结合不同mRNA定位信号的Egl-BicD复合物的高分辨率低温电镜结构上，该结构是最近与LMB的Andrew Carter小组合作产生的。我将使用微尺度热泳术研究Egl和RNA茎环的哪些结构特征介导识别。我将进一步评估特定RNA特征对运输的重要性，使用已建立的体外运动测定和荧光RNA显微注射到果蝇胚胎中。一旦了解了对活性至关重要的RNA特征，我将与生物信息学家合作，在果蝇基因组中寻找并验证新的定位信号。第二个相关目标是了解dynein-dynactin-BicD-Egl机制的活性如何适应于将不同mrna定位到不同的细胞内位置。在果蝇卵母细胞中，hnRNP Squid （Sqd）对于grk mRNA定位到背前区至关重要。Bullock小组的初步工作提出了一种可能性，即Sqd通过促进RNA聚合促进grk mRNA向背前区运输，从而增加动力蛋白拷贝数。我将通过体外和体内实验评估RNA和动力蛋白拷贝数对RNA运输的贡献，包括优化方法来可视化单个RNPs在卵母细胞中的运输。预计这项研究将进一步加深我们对微管马达如何识别特定mrna并将其分类到不同目的地的理解。

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.