Ribosome-free L3 targets E2F pathway: a new p53 independent mechanism linking ribosomal stress to cancer cell proliferation

Our previous data demonstrated that ribosomal stress induced by anticancer drugs in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 as inhibitor of cell cycle. In particular, ribosomal stress induces L3 expression and promotes its nucleolar exit. To understand the molecular mechanism underlying this effect, HCT 116p53-/- cells were treated with Actinomycin D to induce ribosomal stress and with cycloheximide to block NMD pathway. Results showed that the delocalization of L3 during ribosomal stress was associated with an increase of alternative L3 pre-mRNA, substrate of the NMD. Next, we quantified known cell cycle-associated E2F target genes as CycD1, CycE1 and CDK1 by quantitative RT-PCR in HCT 116p53-/- and L3∆HCT 116p53-/- cells, in which L3 is stably silenced. We detected a significant up-regulation of E2F target genes associated with L3 knockdown. Results from reporter gene assays in presence and in absence of L3 upon drug-induced ribosomal stress demonstrated that L3 acts as negative regulator of E2F transcriptional activity. In conclusion, L3 regulates cell proliferation independently of p53 by targeting E2F and inhibiting its function.