Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2α

The PITSLRE kinases belong to the large family of cyclin-dependent protein kinases. Their function has been related to cellcycle regulation, splicing and apoptosis. We have previously shown that the open reading frame of the p110 transcript contains an IRES (internal ribosome entry site) that allows the expression of a smaller p58 isoform during the G2/M stage of the cell cycle. In the present study we investigated further the role of cisand trans-acting factors in the regulation of the PITSLRE IRES. Progressive deletion analysis showed that both a purinerich sequence and a Unr (upstream of N-ras) consensus binding site are essential for PITSLRE IRES activity. In line with these observations, we demonstrate that the PITSLRE IRES interacts with the Unr protein, which is more prominently expressed at the G2/M stage of the cell cycle. We also show that phosphorylation of theα-subunit of the canonical initiation factor eIF-2 is increased at G2/M. Interestingly, phosphorylation of eIF-2α has a permissive effect on the efficiency of both the PITSLRE IRES and the ornithine decarboxylase IRES, two cell cycle-dependent IRESs, in mediating internal initiation of translation, whereas this was not observed with the viral EMCV (encephalomyocarditis virus) and HRV (human rhinovirus) IRESs.