Abstract
The cyclin-dependent kinase inhibitor p21 is an important player in stress pathways exhibiting both tumor-suppressive and oncogenic functions. Thus, expression of p21 has to be tightly controlled, which is achieved by numerous mechanisms at the transcriptional, translational, and posttranslational level. Performing immunoprecipitation of bromouridine-labeled p21 mRNAs that had been incubated before with cytoplasmic extracts of untreated HCT116 colon carcinoma cells, we identified the DEAD-box RNA helicase DDX41 as a novel regulator of p21 expression. DDX41 specifically precipitates with the 3'UTR, but not with the 5'UTR, of p21 mRNA. Knockdown of DDX41 increases basal and γ irradiation-induced p21 protein levels without affecting p21 mRNA expression. Conversely, overexpression of DDX41 strongly inhibits expression of a FLAG-p21 and a luciferase construct, but only in the presence of the p21 3'UTR. Together, these data suggest that this helicase regulates p21 expression at the translational level independent of the transcriptional activity of p53. However, knockdown of DDX41 completely fails to increase p21 protein levels in p53-deficient HCT116 cells. Moreover, posttranslational up-regulation of p21 achieved in both p53+/+ and p53-/- HCT116 cells in response to pharmaceutical inhibition of the proteasome (by MG-132) or p90 ribosomal S6 kinases (by BI-D1870) is further increased by knockdown of DDX41 only in p53-proficient but not in p53-deficient cells. Although our data demonstrate that DDX41 suppresses p21 translation without disturbing the function of p53 to directly induce p21 mRNA expression, this process indirectly requires p53, perhaps in the form of another p53 target gene or as a still undefined posttranscriptional function of p53.