Structure of Dazl based translation control complexes

Human fertility depends upon the production of mature sperm and eggs, and defects in the control of gene expression during this process contribute to infertility, a problem affecting 12-15% of couples worldwide. Deletions on the Y chromosome in infertile men suggested a fertility factor at this location and subsequent mapping identified the Deleted in AZoospermia (DAZ) gene. Members of the DAZ gene family are required for fertility in species across evolution. Mouse Dazl, a member of the DAZ gene family, is an RNA binding protein. Dazl recognises specific sequences on certain messenger RNAs in developing germ cells and recruits protein cofactors in order to regulate how much protein is produced from that mRNA, dictating expression of that gene. We aim to determine the three-dimensional structure of the Dazl protein bound to RNA or the protein cofactors. This will allow us to visualise the components of this system and how they interact at very high resolution, demonstrating how they normally function in controlling gene expression.

Dazl is an RNA binding protein that is critical for correct post-transcriptional control of gene expression during meiosis and gameteogenesis. We have identified RNA binding sites for Dazl in genes regulated by Dazl in vivo, established that Dazl binds these sequences at high affinity in vitro, identified protein cofactors recruited to Dazl in order to control translation, and produced crystals of the Dazl RRM domain. For the proposed work, we wish to: 1) Express and purify each of the protein components of Dazl based translation control complexes: Dazl (subdomains and full length protein), PABP (C-terminal domain), and Pum2 (Puf domain). We will purchase synthetic RNA ligands. 2) Crystallise and solve the structure of: Dazl protein, Dazl:RNA complexes, the Dazl:PABP complex, the Dazl:Pum2 complex with and without RNA. 3) Analyse specific interactions within each of these complexes and relate this to translational control.