| Summary: | Nonsense-mediated mRNA decay (NMD) was firstly described as a surveillance pathway that recognizes and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs) resulted from mutations or errors in RNA processing. Many transcriptome-wide studies demonstrated that NMD also targets mRNAs transcribed from a large subset of wild-type genes, thus arising as a post-transcriptional regulatory mechanism of gene expression. Hence, NMD contributes to the regulation of many essential biological processes, including stress responses. Recent reports revealed that NMD is capable of regulating the Unfolded Protein Response (UPR) that is induced in conditions of endoplasmic reticulum (ER) stress. This is achieved, at least in part, by the degradation of the IRE1α mRNA, a natural NMD-target and one of the three primary factors of the UPR. The other two factors are ATF6 and PERK, and there are evidences suggesting that the PERK-branch can also be involved in the NMD-mediated regulation of the UPR. In this work, we intended to test if the PERK mRNA is a natural NMD-target and, if so, determine the role and relevance of NMD to the PERK-mediated response during the UPR. By using 5’/3’ rapid amplification of cDNA ends (RACE) we have confirmed the sequences and lengths of the annotated 5’ and 3’ untranslated regions (UTRs) of the PERK mRNA. This allowed us to confirm the presence of two possible NMD-inducing features: upstream open reading frames (uORFs) in the 5’UTR and a long 3’UTR (~100bp). To test if PERK mRNA is a direct NMD-target, we have assessed its mRNA levels and stability in conditions of impaired NMD by UPF1 (NMD central factor) siRNA-mediated knockdown in HeLa cells. Surprisingly, the UPF1 knockdown did not induce the upregulation of PERK mRNA and neither stabilize it, suggesting that PERK mRNA is not a natural NMD-target. Our data suggests that NMD does not regulate the UPR through degradation of the PERK mRNA as it does with the IRE1α mRNA. However, we cannot rule out the hypothesis of NMD acting in the PERK-branch, for instance, by degrading downstream targets of PERK.
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