Molecular mechanisms activated in response to ribosomal stress

The ribosome biogenesis is an energetic expensive process that is finely regulated according to the growth conditions and to the cellular stress. Any defect in this process induces in the cell a ribosomal stress, generally leading to the inhibition of cell proliferation and the activation of apoptosis mostly through a p53-dependent mechanism (Fumagalli et al., 2009; Pestov et al., 2001). Some genetic diseases, such as Diamond Blackfan Anemia, have been shown to be associated to defective ribosome biogenesis and therefore have been named ribosomopathies. To study the molecular mechanisms activated in response to ribosomal stress, we altered the production of one or more ribosomal proteins in different cell lines by siRNA-mediated down regulation. In the first part of the project we analyzed the signaling pathways activated in response to ribosomal stress. In particular we selected cellular components known to be involved in ribosome synthesis: 1) mTORC1 2) mTORC2 and 3) AMPK. Analysis of RPS19-depleted prostate cancer cell line PC3 showed that the phosphorylation status of mTORC1 downstream targets S6 kinase 1 (S6K1) and 4E-BP1 was not altered after ribosomal stress. Then we analyzed the serine/threonine kinase AKT, because of its role in the mTORC2-ribosome pathway (Zinzalla et al., 2011). Western blot analysis revealed that the AKT phosphorylation was decreased after depletion of RPS19 in three different prostate cancer cell lines: 22RV1, DU145 and PC3. The same phenomenon was observed in PC3 cells after depletion of other ribosomal proteins (RPS6 and RPS7). Therefore, consistent with Zinzalla et al., we can conclude that mTORC2 is probably involved in the response to ribosomal stress. The last pathway that we analyzed was the AMP-kinase (AMPK), a kinase activated under conditions of metabolic stress. The depletion of different ribosomal proteins (RPS19, RPS6 and RPS7) showed that there was an increase in the phosphorylation level of AMPK indicating its activation in the different prostate cancer cell lines analyzed. This result was consistent with Danilova and colleagues that observed an activation of AMPK in ribosomal protein-depleted zebrafish model (Danilova et al., 2014). In addition, we addressed the phosphorylation status of eukaryotic elongation factor 2 (eEF2) as a downstream target of AMPK. We found that in 22RV1, DU145 and PC3 cells depletion of RPS6, RPS19, RPS7 induced an increase of eEF2 phosphorylation. This could explain the translation elongation inhibition recently reported by our group (Gismondi et al., 2014). In the second part of the project we analyzed the effect of the depletion of RPS19 or other ribosomal proteins on rRNA synthesis. Consistent with a number of publications (Badhai et al., 2009; Choesmel et al., 2007), we observed an alteration of rRNA maturation at the level of 21S precursor. In addition, we observed that RPS19 depletion caused a reduction of the 47S rRNA level in K562C, 22RV1 and HEK293 cells. The same effect was observed after depletion of RPS6 and RPL11. Since the analysis of 47S rRNA stability did not show any alteration (unpublished data), we addressed RNA Polymerase I (Pol I) activity. For this purpose we performed ChIP assay in HEK293 cells after depletion of RPS19. We found that ribosomal stress caused a decrease of Pol I recruitment on two different portions of ribosomal DNA (rDNA). Moreover we observed alteration of the binding of Pol I transcription factor UBF. Therefore, these results indicated that ribosomal stress altered not only Pol I recruitment but also the assembly/activity of Pol I complex. Finally, in order to investigate the signaling pathway responsible for the decrease of Pol I activity, we analyzed AKT and AMPK, both known to be involved in the regulation of Pol I (Chan et al., 2011). The overexpression of a constitutively active form of AKT in HEK293 cells shown that this kinase was not involved in the decrease of rRNA synthesis during ribosomal stress. Similarly, inhibition of AMPK by RNA interference or Compound C in 22RV1 cells revealed that the downregulation of this kinase was not sufficient to rescue rRNA synthesis after ribosomal stress. Therefore, the signaling pathways that could mediate the downregulation of rRNA synthesis after ribosomal stress remains to be addressed.