The proteins SFPQ (splicing Factor Proline/Glutamine rich), NONO (Non-POU domain-containing octamer-binding protein) and PSPC1 (paraspeckle protein component 1), are mammalian members of the Drosophila Behaviour/Human Splicing (DBHS) family. SFPQ, NONO and PSPC1 are involved in all steps of post-transcriptional gene regulation, as well as DNA repair and are primarily located in paraspeckles in mammals: liquid phase-separated, ribonucleoprotein sub-nuclear bodies templated by NEAT1 long non-coding RNA. Structured and low-complexity regions provide polyvalent interaction interfaces which facilitate homo- and heterodimerisation, polymerisation, interaction with RNA, and liquid phase-separation. The strength and competition of these interaction modes defines their ability to dissociate from paraspeckles to fulfil functional roles throughout the nucleus or the cytoplasm.
Firstly we address dimerization behaviour of DBHS proteins. We have previously characterised the crystal structures of all homo and heterodimers of the conserved regions the DBHS proteins, noting that heterodimers are preferred over homodimers, as demonstrated using in vitro partner-swapping experiments. To further explore this phenomenon we have generated a chimera of SFPQ and NONO which has a monomer-monomer interface with the properties of a heterodimer. This homodimeric protein, unlike SFPQ or NONO homodimers is resistant to partner swapping. The crystal structure of this chimera (PDB 7LRU) reveals a small number of amino acid substitutions that appear responsible for this behaviour.
Secondly we address nucleic acid binding by NONO. While the location of RNA-binding has been identified in previous studies, and RNA-binding is critical to cancer pathology of NONO, the binding site has long been considered “undruggable”. The recent discovery of thiol-reactive small molecules which stereospecifically target NONO has opened a new door to modulating gene regulatory activity through covalently bound small-molecules. We have determined the crystal structure of NONO bound covalently to compound SKBG-1 (PDB 9NZI) and demonstrated by microscale thermophoresis that SKBG-1 increases the affinity of NONO for RNA. [1].
[1] Lindsey, G. L., Hockley, T. K., Villa Gomez, A. V., Marshall, A. C., Brothers, W. R., Finney, C. T., Gross, J., Fox, A. H., Yeo, G. W., Melillo, B., Bond, C. S. & Cravatt, B. F. (2025). Cell Chemical Biology 33, 256