Abstract
Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.
Highlights
It has been estimated that one third of hereditary genetic diseases, as well as many forms of cancer, are caused by mutations that lead to the generation of transcripts bearing a premature translation-termination codon (PTC)
The recognition of a stop codon as a PTC depends on the physical distance between the PTC and the cytoplasmic poly(A)-binding protein 1 (PABPC1), as PABPC1 and UPF1 both compete for the interaction with the eRF3 at the terminating ribosome – if PABPC1 is in close proximity to the PTC, it seems to function as an nonsense-mediated mRNA decay (NMD) repressor; on the other hand, when the interaction between PABPC1 and the termination complex is not favorable, UPF1 can interact with eRF3 in the termination complex to induce NMD [10,11,12,13,14]
To determine which poly(A) signals are active in the human HFE transcript 39-end processing, we carried out 39 rapid amplification of cDNA ends (39-RACE) experiments (Fig. 1) using nested forward primers located at HFE exons six and seven (Fig. 1A)
Summary
It has been estimated that one third of hereditary genetic diseases, as well as many forms of cancer, are caused by mutations that lead to the generation of transcripts bearing a premature translation-termination codon (PTC) Most of these PTC-containing mRNAs are targets for the nonsense-mediated mRNA decay (NMD) pathway [1,2,3]. If an mRNA contains a PTC located more than 50–54 nts upstream of at least one exon-exon junction, the ribosome will fail to displace these distal EJC(s) In this case, when the ribosome reaches the PTC, the eukaryotic translation release factors eRF1 and eRF3 at the PTC interact in cis with the retained EJC(s) via a multiprotein bridge [9]. The recognition of a stop codon as a PTC depends on the physical distance between the PTC and the cytoplasmic poly(A)-binding protein 1 (PABPC1), as PABPC1 and UPF1 both compete for the interaction with the eRF3 at the terminating ribosome – if PABPC1 is in close proximity to the PTC, it seems to function as an NMD repressor; on the other hand, when the interaction between PABPC1 and the termination complex is not favorable, UPF1 can interact with eRF3 in the termination complex to induce NMD [10,11,12,13,14]
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