Abstract
Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.
Highlights
Since p53 is negatively regulated by murine double minute 2 (MDM2) and murine double minute 4 (MDM4, known as MDMX) [7], inhibition of MDM2 and MDM4 results in p53mediated antiproliferative activity, including apoptosis and cellular senescence [8, 9]
Alternative splicing of MDM4 mRNA is regulated by serine/arginine-rich splicing factor 3 (SRSF3), which belongs to the SR protein family of splicing factors [12]
Consistent with the RNA-seq analysis, RT-PCR analysis revealed that pre-mRNA processing factor 19 (PRPF19) knockdown caused exon 6 skipping in the MDM4 mRNA, as indicated by downregulation of the MDM4-FL isoform and upregulation of the MDM4-S isoform (Fig. 5C, upper panel)
Summary
In addition to cell cycle arrest, cellular senescence is characterized by morphological changes, high β-galactosidase activity, and chromatin remodeling [3,4,5] Senescent cells with these phenotypic alterations are observed in the tumor environment, embryonic development, and tissue repair [6], suggesting that cellular senescence plays important roles in multiple aspects of cellular and tissue homeostasis. Since MDM4 is more highly expressed in cancer tissues than in normal tissues and dampens the tumorsuppressive functions of p53 [13,14,15,16], alternative splicing of MDM4 mRNA is considered to be a critical step in cancer progression [17]. The DDR alters the expression of splicing factors and the alternative splicing of mRNAs involved in the cell cycle and apoptosis [23]. Our findings provide new insights into the roles of splicing factors in the regulation of cellular senescence
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