agreement with this finding, ELISA analysis revealed a significant increase in p53 levels in livers of fS6/CRE+ mice compared to fS6/CRE- mice post PH. More strikingly, the expression of p53 targets was induced even before PH, suggesting that stress induced by impaired ribosome biogenesis was sufficient to induce p53 [34]. Consistent with the in vivo data, depletion of RPS6 in cultured human A549 cells with siRNA also led to a robust induction of p53 and p21. The p53 response was not specific to loss of RPS6, as depletion of RPS23, another essential component of the 40S subunit, or of RPL7a, a 60S subunit ribosomal protein, also led to increased expression of p53 and p21, indicating this phenomenon was a general response to inhibition of ribosome biogenesis. Incorporation of BrdU, which was reduced by 60% upon RPS6 depletion, was reversed by co-depletion of RPS6 and p53 [34]. Previous studies have proposed that inhibition of rRNA synthesis disrupts the structure of the nucleolus, releasing RPL11, an essential RP of the 60S subunit, into the nucleoplasm where it interacts with MDM2, inhibiting its E3-ligase activity towards p53 [35,36]. However, stabilization of p53 in cells depleted of RPS6 does not depend on nucleolar disruption. Immunofluorescent staining of two nucleolus markers, NHP2 and nucleolin, and the 60S subunit protein RPL7a confirmed that the nucleolar structure was intact in cells depleted of RPS6 [34], suggesting that the p53 induction upon impaired 40S biogenesis does not require nucleolar disruption. Interestingly, co-depletion of RPL11 together with RPS6 was able to abolish the induction of p53 and rescue the S-phase entry defect observed in cells depleted of RPS6 alone. Moreover, co-depletion of RPL11 with either RPS23 or RPL7a suppressed the accumulation of p53 and p21 induced by RPS23 or RPL7a depletion alone [34]. In support of the essential role of RPL11 in the p53 response, significantly more RPL11 co-immunoprecipitated with MDM2 upon depletion of RPS6 or RPL7a compared to control non-silencing siRNA treated cells. Thus, cells are able to sense impairment of ribosome biogenesis and invoke a p53 response in an RPL11dependent manner. If RPL11 is required for the p53 response, and impaired ribosome biogenesis occurs in the absence of nucleolar disruption, by what mechanism is free RPL11 available to bind to MDM2? Cells depleted of an RP of the large subunit, such as RPL7a, have impaired 60S ribosomal biogenesis; RPL11 which is not incorporated into the pre-60S subunit is likely to accumulate as ribosome-free RPL11. However, 60S subunit production is unperturbed upon depletion of a RP of the small subunit, such as RPS6, so that simple accumulation of ribosome-free RPL11 is unlikely. This suggests that a mechanism to increase levels of RPL11 must exist when 40S ribosome biogenesis is impaired. Polysome analysis of RPL11 transcripts revealed that more RPL11 mRNA transcripts were recruited to actively translating polysomes in RPS6 depleted cells, compared to control cells. Moreover, western blot analysis also showed a significant increase in total RPL11 protein level upon RPS6 depletion. The translational up-regulation of RPL11 is a general response to defective 40S biogenesis, as RPL11 transcripts shifted to heavier polysome in cells depleted of a second RP of the small subunit, RPS23, but not in cells depleted of the large subunit RP, RPL7a [34]. Interestingly, other RP transcripts including RPS8, RPS16 and RPL26 were also translationally upregulated upon RPS6 depletion [34]. Translation of RP mRNAs is
HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz
121