Increased expression of NONO, a multifunctional nuclear RNA and DNA binding protein, is highly associated with poor patient survival in neuroblastoma, however its oncogenic regulatory role in this disease is not fully understood.
Initially we used CRISPR-cas9 to insert GFP into the endogenous NONO gene to make a GFP fusion in various cell lines, including HeLa and the neuroblastoma lines Kelly and BE2C. In contrast to HeLa, the neuroblastoma cell lines showed fewer paraspeckles and much more numerous and fine ‘non-paraspeckle’ nuclear distributed NONO protein.
To investigate the nature of the distributed NONO nuclear signal, we incubated cells with 1,6-hexanediol, a compound known to disrupt liquid-phase transition condensates, and observed dramatically abolished NONO foci in Kelly cells. This suggests that, similar to paraspeckles, the nuclear distributed NONO is built into phase-separated structures. In addition, nuclease digestion with either RNase or DNase also completely eradicated the nuclear NONO in Kelly cells, suggesting both RNA and DNA are essential structural components of this NONO distribution. This is in contrast to paraspeckle-associated NONO, which is solely reliant on RNA, and not DNA, for its localization.
CLIP-seq analysis of NONO in neuroblastoma showed extensive binding to several important oncogenic pre-mRNAs, including a 5kb downstream region of HAND2, the first several introns of GATA2 and the first introns of DAZAP1 and KCND2. NONO RIP assays followed by quantification of mRNAs validated these observations. Using siRNA-resistant plasmid system, we showed NONO mutant plasmid lacking RRM1 no longer enriched these pre- and mature mRNAs.
These results lead us to a hypothesis in which the RRM1 region of NONO is required for its extensive binding to oncogenic pre-mRNAs at their transcription sites, forming phase separated condensates. These insights suggest both the RNA and DNA binding capacity of NONO might be involved in its oncogenic gene-regulatory mechanism of neuroblastoma.