A Defective Splicing Machinery Promotes Senescence through MDM4 Alternative Splicing

Defects in the splicing machinery have been implicated in various diseases, including cancer. We identified a general reduction in the expression spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced and telomere uncapping-induced senescence. Supporting the notion that defective splicing contributes to senescence, we showed that the splicing inhibitors herboxidiene and pladienolide B induce senescence both in normal and cancer cell lines. Furthermore, the individual depletion of several spliceosome components also promoted senescence. We noted an alternative splicing shift in MDM4, from the full-length MDM4-FL variant to MDM4-S during replicative and stress-induced senescence. This shift was replicated by splicing inhibition and the depletion of individual spliceosome components. Importantly, decreasing the level of MDM4-FL promoted senescence, while cell survival was improved both by reducing the level of MDM4-FL and by overexpressing MDM4-S. Overall, our work establishes that defects in the splicing machinery alter the alternative splicing of MDM4 to promote senescence. The BJ cell line was cultured until reaching 63 population doubling. At this point they were identified as undergoing replicative senescence using common senescence markers. Replicative senescent BJ cells and their young counterpart were then plated in triplicate, 24h before RNA extraction. After dosing and QC, only the best 2 of the 3, for each cell population, were kept for poly-A RNA sequencing (2 experimental samples in duplicate (4 samples total)).