Structural insights into DBHS protein dimerisation and nucleic acid binding

This thesis describes the structural and functional characterisation of the Drosophila behaviour/human splicing (DBHS) protein family in a study that combines X-ray crystallography with solution scattering and complementary biophysical characterisation. The DBHS protein family is something of an enigma, with reported functions in almost every step of gene regulation. Humans possess three DBHS paralogues; non-POU domain-containing octamer binding protein (NONO), paraspeckle protein component 1 (PSPC1) and splicing factor proline/glutamine rich (SFPQ), all of which share a highly conserved domain architecture. Since their discovery over two decades ago, the biological and clinical relevance of the family has undergone rapid development. Perturbing DBHS protein structure or abundance is strongly associated with the malignant progression of cancers such as neuroblastoma, intellectual disability in humans and broad transcriptional and post-transcriptional defects. As such, the DBHS proteins are a viable therapeutic target and/or prognostic marker. However, the mechanistic details of their function are unknown. Towards developing a molecular understanding of DBHS protein function, this study aimed to solve DBHS protein structures and complement the structures with an investigation into their RNA binding mechanisms. The literature surrounding DBHS protein function has not been comprehensively reviewed in light of recent structural advances for well over a decade. Thus, the first part of this study presents a detailed literature review. Through an in-depth foray into the literature, the family are identified as dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions. As such, we describe them as a multipurpose molecular scaffold. In spite of recent structural advances, the role of combinatorial dimerisation and mechanisms of nucleic acid binding are unclear and often overlooked in the literature. To explore the molecular origins of DBHS proteins and firmly place the role of dimerisation and RNA binding in function, the first set of experimental results detail the X-ray crystal structure of NONO-1, an ancestral DBHS protein from Caenorhabditis elegans, which was solved and complemented with a thorough bioinformatic analysis. The second part of this study focuses on the challenges of characterising the previously uncharacterised human NONO homodimer. Here, the pursuit of stable NONO protein and high quality X-ray diffraction produced a case study that describes methods for overcoming problems with protein stability, data collection and non-crystallographic symmetry. Finally, the novel X-ray crystal structures of both the NONO and PSPC1 homodimers are analysed in detail. The RNA binding ability of NONO is described using microscale thermophoresis, isothermal titration calorimetry and X-ray solution scattering, providing the first detailed molecular insights into the association of nucleic acid with a DBHS dimer. Moving forward, the data presented within this thesis provide a sound structural framework to reliably investigate this remarkably adaptable and versatile protein family. The novel data on RNA binding mechanisms and the dynamic of dimerisation makes a significant contribution to our understanding of DBHS molecular mechanisms. The insights provided by this work are an important first step towards potentially developing therapeutics targeting DBHS proteins.