Two of the four human ubiquitin-encoding genes express ubiquitin as an N-terminal fusion precursor polypeptide, with either ribosomal protein (RP) RPS27a or RPL40 at the C-terminus. RPS27a and RPL40 have been proposed to be important for the induction of the tumour suppressor p53 in response to defects in ribosome biogenesis, suggesting that they may play a role in the coordination of ribosome production, ubiquitin levels and p53 signalling. Here, we report that RPS27a is cleaved from the ubiquitin-RP precursor in a process that appears independent of ribosome biogenesis. In contrast to other RPs, the knockdown of either RPS27a or RPL40 did not stabilise the tumour suppressor p53 in U2OS cells. Knockdown of neither protein blocked p53 stabilisation following inhibition of ribosome biogenesis by actinomycin D, indicating that they are not needed for p53 signalling in these cells. However, the knockdown of both RPS27a and RPL40 in MCF7 and LNCaP cells robustly induced p53, consistent with observations made with the majority of other RPs. Importantly, RPS27a and RPL40 are needed for rRNA production in all cell lines tested. Our data suggest that the role of RPS27a and RPL40 in p53 signalling, but not their importance in ribosome biogenesis, differs between cell types.