Abstract
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.
Highlights
Factors involved in RNA splicing have been linked to tumor suppression, and are emerging as cancer therapeutic targets
We sought to examine the influence of PRPF8 on chromosomal double strand break (DSB) repair, based on findings that PRPF8/PRP8 forms a conserved complex with XAB2/SYF1 [19], which has been shown to be important for the end resection step of homologous recombination (HR) in human cells [14]
To investigate the links between RNA splicing factors and genome stability, we have examined the effects of PRPF8 depletion and Pladienolide B (PlaB) treatment on HR repair of chromosomal breaks (Figure 9)
Summary
Factors involved in RNA splicing have been linked to tumor suppression, and are emerging as cancer therapeutic targets. The role of splicing factors in tumor suppression and as therapeutic targets likely includes their central role in shaping the transcriptome, and proper regulation of gene expression [1, 5]. Disruption of RNA splicing factors have been shown to cause genome instability [6, 7], which is both a hallmark of cancer, as well as a contributor to the therapeutic response to clastogenic anti-cancer agents [8]. Understanding the links between RNA splicing and genome stability will provide insight into cancer etiology and development of therapeutic targets
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