Investigating the p21 Ubiquitin-Independent Degron Reveals a Dual Degron Module Regulating p21 Degradation and Function

A group of intrinsically disordered proteins (IDPs) are subject to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDPs/IDRs are targeted to the 20S proteasome via interaction with the C-terminus of the PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using a split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interactions with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.