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

PhD project strategic theme: Understanding the rules of life

Subcellular 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.