Structural and RNA binding studies of Hrp48 – a regulator of translation in female Drosophila dosage compensation

Post-transcriptional gene control is essential in gene expression, and one major step is the regulation of translation, controlling functions in a plethora of biological processes. A well-studied example of translation regulation is the repression of msl-2 mRNA translation, a crucial step in the regulation of X-chromosome dosage compensation in females of Drosophila melanogaster. The repression is coordinated by the protein Sex-lethal (Sxl), which binds to both untranslated regions (UTRs) of the msl-2 mRNA. At the 3’ UTR Sxl recruits further RNA binding proteins, Unr and Hrp48 to form a ribonucleoprotein (RNP) complex which targets an early translation initiation step. Hrp48 directly interacts with the eIF3d subunit of the 43S preinitiation complex, but the detailed molecular role of Hrp48 during translational repression and its interaction with msl-2 is not well understood. Hrp48 consists of two N-terminal RNA recognition motifs (RRM) and in this work I solved the crystal structure of RRM1 at 1.2 Å resolution and validated the structure prediction model of RRM2 by NMR spectroscopy. In order to identify the RNA interaction site and binding affinities of Hrp48, I utilized NMR spectroscopy titrations as the differences in affinities were not resolved by isothermal titration calorimetry. The two RRM domains of Hrp48 bind the RNA simultaneously and synergistically forming a 1:1 complex in solution. Based on NMR relaxation and RDC experiments, the complex behaves very dynamically and the two RRMs remain flexible with respect to each other upon RNA- binding, suggesting a binding mode unusual for tandem-RRMs. The identified RNA-binding sites were corroborated by cellular assays performed by our collaborator. Studies directed to the understanding of the complex formation between Hrp48, Unr, Sxl and msl-2 suggest no interaction of the proteins in the absence of RNA, however the three proteins bind msl-2 simultaneously. I established a protocol to reproducibly form the quaternary complex of Sxl, Unr, Hrp48 and msl-2. It has been shown previously, that Sxl and Unr synergistically bind msl-2. My data shows that the incorporation of Hrp48 to the RNP complex occurs in a non-cooperative fashion