| Description |
Many cancers are curative with surgery, chemotherapy, radiotherapy, or a combination thereof. However, a subset of patients' cancers return and form a refractory cancer, which is often resistant to therapies, difficult to treat, and create a poor prognosis for the patient. Therefore, it is necessary to develop therapies that target refractory cancers. To this end, we utilize the forward and reverse genetics of the zebrafish to identify and validate mutations in genes that sensitize cells to ionizing radiation (IR)- induced apoptosis. We determine that loss-of-function mutations in ribonucleic acid (RNA) splicing factors cause an increase in IR-induced apoptosis, specifically in the neural tissue. Curiously, despite the ubiquitous requirement of RNA splicing in all cells, the neural tissue is the most sensitive to disruption of RNA splicing. Previous studies in yeast and cell culture have shown that the disruption of RNA processing, including RNA splicing, can cause R-loops, which are RNA:DNA hybrids that form when the nascent RNA base pairs to the complementary DNA strand. Aberrant R-loop accumulation causes an increase in DNA double-strand breaks (DSBs), and can be resolved by RNaseH1, an enzyme that specifically cleaves the RNA in an RNA:DNA hybrid. Here, we show that disruption of RNA splicing in the zebrafish causes R-loops and DNA DSBs. Expression of a conditional RNaseH1 mitigates the increase in DNA DSBs and sensitivity to IRinduced apoptosis. While we hypothesized that radiosensitivity was through a mechanism iv of increased p53 expression, we show that the increase in p53, seen in most RNA splicing mutants, is neither necessary nor sufficient for radiosensitivity. Our study further emphasizes the link between RNA splicing and the DNA damage response pathway. Furthermore, whereas previous single-cell models of R-loop formation have been unable to recognize tissue-specificity, the zebrafish provides a robust model to study why the loss of a cell-essential gene causes tissue-specific IRsensitivity. Future studies will be necessary to determine mechanisms of tissue-specific radiosensitivity, as well as determining whether targeting RNA splicing factors can be of clinical therapeutic value. |
