Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the link between translation initiation and premature stop codon definition

The nonsense-mediated decay (NMD) is a surveillance mechanism that recognizes and rapidly degrades transcripts bearing a premature termination codon (PTC). Although in mammals the location of a PTC more than 50 nucleotides upstream the terminal exonexon junction has been pointed as a mark for NMD, it is now known that the physical distance between the PTC and cytoplasmic poly(A)-binding protein 1 (PABPC1) is a crucial determinant in PTC definition. We have previously reported that mRNAs carrying an AUG-proximal PTC evade NMD although apparently fulfilling the defined criteria for PTC definition. This unexpected NMD-evasion reflects an AUG-proximity effect. To test the hypothesis that the AUG-proximity effect on NMD resistance could be the result of a positional effect of PABPC1 relative to the PTC due to the inherent nature of the process of short ORF translation process, the PABPC1/eRF3 interaction was inhibited by RNA interference. Under conditions of overexpression of a PABPC1 mutant lacking the eRF3-interaction domain (PABPC1DelC), levels of beta15 transcripts were quantified by RT-qPCR and showed to be significantly decreased. The reciprocal experiment, i.e. conditions of overexpression of an eRF3 mutant lacking the PABPC1-interaction domain (eRF3DelN) was also performed. Results showed that the NMD-resistance of an AUG-proximal nonsense-mutated mRNA can be converted to NMD-sensitive when PABPC1/eRF3 interaction is impaired. Moreover, inhibition of PABPC1-eIF4G interaction by overexpression of PAIP2 protein, as well as the knockdown of the initiation factor eIF3h and eIF3f subunit, targets these transcripts for NMD. Our findings support a role for PABPC1 and associated initiation factors during translation in NMD-resistance of AUG-proximal nonsense-mutated transcripts, providing evidence for a link between translation initiation and PTC definition