CacyBP/SIP - RPL6 interaction: potential influence on ribosome function.

Previously, we have shown that CacyBP/SIP interacts with NPM1, a protein involved in ribosome biogenesis. In this work, we extended our previous studies to look for the potential impact of CacyBP/SIP on ribosome biogenesis and/or function. Using mass spectrometry analysis, we have found that several RPs could be potential CacyBP/SIP targets. Since RPL6 was one of the proteins with the best quality scores identified in this analysis we focused on the possible interaction between CacyBP/SIP and RPL6. By applying various biochemical methods, we confirmed this interaction and showed that it was direct. Moreover, in silico analysis allowed us to establish the domains/fragments of both proteins involved in the binding. To further explore the possible role of CacyBP/SIP in ribosome function we performed several analyses using neuroblastoma NB2a cell line with stably silenced CacyBP/SIP expression. We have found, by applying OPP (O-propargyl-puromycin), which labels nascent polypeptides, that the number of cells with enhanced staining in the perinuclear area, reminiscent of rough ER localization, was significantly lower in the cell line with diminished CacyBP/SIP level. To verify the influence of CacyBP/SIP on the efficiency of protein synthesis we investigated the level of Hsp70, a stress-inducible protein, in NB2a cells subjected to heat shock. The results, showing markedly higher Hsp70 production in control cells, indicate that CacyBP/SIP, most probably through interaction with RPL6 and/or other RPs, may have some influence on ribosome function and, possibly, on protein synthesis in the cell.