CAP-Independent Translation of PUMA-α in Apoptotic Response to DNA Damage
DNA damage induces the expression of p53-upregulated-mediator-of-apoptosis (PUMA-α) to activate the intrinsic apoptosis pathway. Our lab has shown that cisplatin induces PUMA-α mRNA without increasing PUMA-α protein or apoptosis in renal epithelial cells of mice with germline mutations to block nuclear import of Abl-tyrosine kinase. This finding suggests that PUMA-α translation is also regulated in DNA damage response. Inspection of PUMA-α sequence reveals an upstream open reading frame (uORF), which if translated, may interfere with translation from the downstream AUG of the PUMA-α ORF. To determine if this uORF is translated, I inserted a Venus_p2a_Neo ORF using CRISPR/Cas9 technology into PUMA-α Exon-1a to increase the uORF coding capacity from 41 aa to a 577 aa. Following selection for neomycin-resistance and single-cell cloning, I detected Venus expression by fluorescence microscopy and Neo expression by immunoblotting in several edited cell clones, supporting translation of the uORF and suggesting PUMA-α is translated from an internal AUG, which can occur through Leaky Scanning (CAP-dependent) or Re-initiation (CAP-independent). To distinguish between these two mechanisms, I treated cells with 4EGI-1, an inhibitor of CAP-dependent translation and a DNA damaging agent separately or in combination, and found that 4EGI-1 reduced p53 and p21CIP1 but not PUMA-α protein levels. Together, the findings that the uORF is translated and that 4EGI-1 does not reduce PUMA-α protein strongly suggest that PUMA-α translation is driven by CAP-independent re-initiation and support future investigation to solve the nuclear Abl-dependent translation re-initiation mechanism for PUMA-α expression.