Structural plasticity of the coiled-coil interactions in human SFPQ

The proteins SFPQ (splicing Factor Proline/Glutamine rich) and NONO (non-POU domain-containing octamer-binding protein) are mammalian members of the Drosophila Behaviour/Human Splicing (DBHS) protein family, which share 76% sequence identity in their conserved 320 amino acid DBHS domain. SFPQ and NONO are involved in all steps of post-transcriptional regulation and are primarily located in mammalian paraspeckles: liquid phase-separated, ribonucleoprotein sub-nuclear bodies templated by NEAT1 long non-coding RNA. A combination of structured and low-complexity regions provide polyvalent interaction interfaces that facilitate homo- and heterodimerisation, polymerisation, interactions with oligonucleotides, mRNA, long non-coding RNA, and liquid phase-separation, all of which have been implicated in cellular homeostasis and neurological diseases including neuroblastoma. The strength and competition of these interaction modes define the ability of DBHS proteins to dissociate from paraspeckles to fulfil functional roles throughout the nucleus or the cytoplasm. In this study, we define and dissect the coiled-coil interactions which promote the polymerisation of DBHS proteins, using a crystal structure of an SFPQ/NONO heterodimer which reveals a flexible coiled-coil interaction interface which differs from previous studies. We support this through extensive solution small-angle X-ray scattering experiments using a panel of SFPQ/NONO heterodimer variants which are capable of tetramerisation to varying extents. The QM mutant displayed a negligible amount of tetramerisation (quadruple loss of function coiled-coil mutant L535A/L539A/L546A/M549A), the Charged Single Alpha Helix (ΔCSAH) variant displayed a dimer-tetramer equilibrium interaction, and the disulfide-forming variant displayed constitutive tetramerisation (R542C which mimics the pathological Drosophila nonAdiss allele). We demonstrate that newly characterised coiled-coil interfaces play a role in the polymerisation of DBHS proteins in addition to the previously described canonical coiled-coil interface. The detail of these interactions provides insight into a process critical for the assembly of paraspeckles as well as the behaviour of SFPQ as a transcription factor, and general multipurpose auxiliary protein with functions essential to mammalian life. Our understanding of the coiled coil behaviour of SFPQ also enhances the explanatory power of mutations (often disease-associated) observed in the DBHS family, potentially allowing for the development of future medical options such as targeted gene therapy.