Abstract High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA binding protein, NONO, in neuroblastoma is associated with poor patient outcome, however there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biophysical and molecular analysis to reveal complex NONO-dependent regulation of gene expression, finding that NONO forms RNA- and DNA-tethered phase-separated condensates throughout the nucleus. CLIP analyses show that NONO mainly binds to the 5’ end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA binding activity. NONO preferentially regulates super enhancer-associated genes, including HAND2 and GATA2. In the absence of functional NONO-RNA condensates, inefficient pre-mRNA processing at these loci leads to decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA binding activity of NONO may have therapeutic potential in this cancer context.

RNA binding is essential for NONO condensates to modulate pre-mRNA processing of super enhancer-associated genes in neuroblastoma