EXTRA-RIBOSOMAL ROLE OF RPS 19 IN NORMAL AND TUMOR CELL LINES EXPOSED TO IONIZING RADIATIONS

Abstract

Eukaryotic ribosome biogenesis is a highly regulated process that takes place into nucleolus and requires the coordinated activity of all the three RNA-polymerases (Pol). Any alteration in such process induces a cellular condition named “Ribosomal Stress” which is associated to rare genetic disorder named “Ribosomopathies” (Freed et al., 2010). The known diseases are 8 and one of these is the Diamond-Blackfan Anemia (DBA), a congenital erythroid aplasia characterized by defective erythropoiesis and macrocytic anemia associated to growth retardation, physical abnormalities and cancer. About 25% of case are associate with mutations in the gene encoding for RPS19 and up to 50% of DBA patients display mutations in other RPs genes (Ball 2011). Ribosomal stress condition promotes the activation of tumor suppressor p53, which in turn activates downstream proteins, such as p21/ CDKN1A (p21) able to check cell cycle (Quin et al., 2014). p53 is also a factor involved in DNA Damage Response (DDR). Ionizing Radiations (IR) or radiomimetic drugs lead to Double Strand Breaks (DSBs) that can be resolved through two pathways activated in relation to cell cycle: Non Homologous End Joining (NHEJ) and Homologous Recombination (HR) (Sancar et al., 2004). Key player of DDR is ATM kinase which is activated via auto-phosphorylation and consequent monomerizzation (Vignar et al., 2013). Once activated, ATM phosphorylates a plethora of downstream proteins including H2AX, 53BP1, CHK2, p53, RAD51, RAD52 whose effects are to induce the cells toward DNA repair, cell cycle arrest or apoptosis. In the last few years many evidences have been gained about the extra-ribosomal functions of Ribosome Proteins (RPs). In fact, beside their primary function in the translation machinery, RPs seem to take part in several biological processes as regulation of cell cycle progress, apoptosis, tumorigenesis; however no data have been yet reported about RPs and DNA damage caused by IR. Based on data reporting an increased radiosensitivity in DBA patients (Van Diemen et al., 1997), our aim was to investigate the relationship between Ribosomal Stress conditions and the activation of DDR; in particular to identify the extra-ribosomal role of RPS19 in DNA damage caused by IR exposure. We evaluated cellular effects related to cell cycle and DNA repair in normal human primary fibroblasts MRC-5, glioblastoma U251-MG and colon cancer HCT116 cells which have been subjected to RPS19 siRNA knockdown and subsequently exposed to IR. Ribosomal stress condition reduced protein synthesis process by the block of the elongation phase of translation, as described by the inhibitory level of phosphorylation of eEF2. In addition, X-Rays caused the early phosphorylation of eEF2 than control counterpart cells. One of the main factors altered by the ribosomal stress condition is p53, which was stabilized mainly 16 hours after IR. Moreover p53 activation, described by its molecular target p21, was observed as long term response to damage. As possible consequence of p21 activation, the number of RPS19-depleted HCT116 and MRC-5 cells able to enter in S phase after X-Ray exposure was reduced. IR treatment activates DDR pathways, thus we focused on analysis of factors involved in HR ad NHEJ repair mechanisms. RPS19-depleted and irradiated cells displayed alteration in number of γ-H2AX and 53BP1 foci (DSBs markers), mainly at early time post irradiation. Also the activation of ATM kinase was impaired by RPS19 knockdown. Despite ATM total protein level was unchanged, its phosphorylation was deregulated in iRPS19 irradiated cells. Indeed, in each iRPS19 cell lines, p-Ser1981-ATM protein slowed down to return basal, in fact it was detectable till long time after IR. Once activated, the kinase ATM directly phosphorylates downstream factor, including CHK2 on Thr68 and p53 on Ser15. Only HCT116 iRPS19 irradiated cells described alteration of CHK2 phosphorylation, as well as p-Ser1981-ATM. Indeed, p-Thr68- CHK2 protein was rapidly activated and was recorded till last time point analyzed. On the other hand p Ser15-p53 level was altered only in U251-MG iRPS19 cells. As well as p-Ser1981-ATM, p53 protein phosphorylation on Ser15 was enhanced and longer activated than counterpart cells. However, fibroblasts and colon cancer cells did not describe p-Ser15-p53 level alteration as response to IR, both in presence and in absence of RPS19 meaning a different mechanism of regulation of p53. HR repair pathway requires the activity of RAD51 protein, able to start strand invasion and repair of DNA lesions. Nevertheless, RAD51 transcription level did not change, ribosomal stress condition reduced RAD51 protein level. In RPS19-depleted cells RAD51 total protein level was further reduced after IR and, as consequence, also its redistribution on DNA lesions was impaired. Moreover, such protein reduction was not dependent by physical interaction with RPS19 and was not correlated to the chaperone HSP90 (RAD51 interactor). Finally, the complex MRN was not affected by ribosomal stress condition alone and in combination to IR, as well as KU80 protein key factor in NHEJ repair mechanism. In conclusion, data collected have demonstrated that RPS19 depletion affects some factors involved in DDR, in particular RAD51, key player in HR repair pathway. Thus, RPS19 depletion could induce not only sensitivity to IR but specially inability to resolve IR-induced DSBs by HR pathway.