Onyango David O, Lee Gabriella, Stark Jeremy M
Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, CA, USA.
Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USA.
Oncotarget. 2017 Oct 6;8(55):93319-93337. doi: 10.18632/oncotarget.21555. eCollection 2017 Nov 7.
Disruption of RNA splicing causes genome instability, which could contribute to cancer etiology. Furthermore, RNA splicing is an emerging anti-cancer target. Thus, we have evaluated the influence of the spliceosome factor PRPF8 and the splicing inhibitor Pladienolide B (PlaB) on homologous recombination (HR). We find that PRPF8 depletion and PlaB treatment cause a specific defect in homology-directed repair (HDR), and single strand annealing (SSA), which share end resection as a common intermediate, and BRCA1 as a required factor. Furthermore, PRPF8 depletion and PlaB treatment cause reduced end resection detected as chromatin-bound RPA, BRCA1 foci in response to damage, and histone acetylation marks that are associated with BRCA1-mediated HR. We also identified distinctions between PlaB and PRPF8 depletion, in that PlaB also reduces 53BP1 foci, and BRCA1 expression. Furthermore loss of 53BP1, which rescues SSA in BRCA1 depleted cells, and partially rescues SSA in PRPF8 depleted cells, has no effect on SSA in PlaB treated cells. Finally, while PRPF8 depletion has no obvious effect on the integrity of interchromatin granules, PlaB disrupts these structures. These findings indicate that PRPF8 is important for BRCA1-mediated HR, whereas PlaB also has a more general effect on the DNA damage response and nuclear organization.
RNA剪接的破坏会导致基因组不稳定，这可能促成癌症的病因。此外，RNA剪接是一个新兴的抗癌靶点。因此，我们评估了剪接体因子PRPF8和剪接抑制剂普拉地诺醇B（PlaB）对同源重组（HR）的影响。我们发现，PRPF8缺失和PlaB处理会在同源定向修复（HDR）和单链退火（SSA）中导致特定缺陷，HDR和SSA都将末端切除作为共同中间体，并且需要BRCA1作为一个因子。此外，PRPF8缺失和PlaB处理会导致末端切除减少，表现为染色质结合的RPA、损伤后出现的BRCA1焦点以及与BRCA1介导的HR相关的组蛋白乙酰化标记减少。我们还发现了PlaB和PRPF8缺失之间的区别，即PlaB还会减少53BP1焦点和BRCA1表达。此外，53BP1的缺失在BRCA1缺失的细胞中能挽救SSA，在PRPF8缺失的细胞中能部分挽救SSA，但对PlaB处理的细胞中的SSA没有影响。最后，虽然PRPF8缺失对染色质间颗粒的完整性没有明显影响，但PlaB会破坏这些结构。这些发现表明，PRPF8对BRCA1介导的HR很重要，而PlaB对DNA损伤反应和核组织也有更广泛的影响。
