The NONO protein regulates nonclassical DNA structure: effects on circadian genes and DNA damage. The NONO protein regulates nonclassical DNA structure: effects on circadian genes and DNA damage

The Drosophila behaviour/human splicing (DBHS) protein family of NONO, PSPC1, and SFPQ has been suggested to regulate diverse aspects of RNA metabolism, with implications for the circadian clock, metabolism, brain function, and cancer. Whether these proteins share similar functions and involve a common molecular mechanism is currently unknown. First, using mouse embryonic fibroblasts (MEFs), we showed that 20% of genes are expressed in a circadian manner, peaking at two distinct clock phases, about 12 hours apart. We then discovered that in both NONO- and PSPC1-deficient MEFs, around 2000 circadian and non-circadian genes are differentially expressed, with only 35% of them commonly regulated. Considering specifically circadian genes, up- or downregulation by NONO and PSPC1 is mainly dependent upon gene phase. Examining NONO-deficient cells in more detail, we observed genome-wide effects upon both transcription and downstream steps of RNA metabolism, such as alternative splicing. We then postulated a regulatory role of NONO on R-loops homeostasis as a common mechanism underlying the diverse circadian and non-circadian effects. Indeed, our analysis showed that genome-wide R-loops distribution is markedly altered in NONO-depleted cells. Interestingly, these alterations occur in time-of-day dependent manner among the circadian genes. Furthermore, we have shown here that NONO regulates cellular response to DNA damage through R-loops. Overall, the R-loops modulation by NONO can potentially induce corresponding effects upon RNA metabolism at diverse phases of both clock and transcription cycles, and might explain the widely diverse reported effects of NONO via a common molecular process.