MRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5′ to the terminal exon–exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as “premature” if it is located greater than 50–54 nucleotides 5′ to the final exon-exon junction. We have described a set of naturally occurring human β-globin gene mutations that apparently contradict this rule. The corresponding β-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type β-globin (β^WT) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between β^WT mRNA and β-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; β39). Functional analyses of these mRNAs with 5′-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5′ terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the “50–54 nt boundary rule.” These observations impact on current models of NMD.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3′–5′ linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Comprehension of the mechanisms by which ectotherms, such as fish, respond to thermal stress is paramount for understanding the threats that environmental changes may pose to wild populations. Heat shock proteins are molecular chaperones with an important role in several stress conditions such as high temperatures. In the Iberian Peninsula, particularly in Portugal, freshwater fish of the genus Squalius are subject to daily and seasonal temperature variations. To examine the extent to which different thermal regimes influence the expression patterns of hsp70 and hsc70 transcripts we exposed two species of Squalius (S. torgalensis and S. carolitertii) to different temperatures (20, 25, 30 and 35 °C). At 35 °C, there was a significant increase in the expression of hsp70 and hsc70 in the southern species, S. torgalensis, while the northern species, S. carolitertii, showed no increase in the expression of these genes; however, some individuals of the latter species died when exposed to 35 °C. These results suggest that S. torgalensis may cope better with harsher temperatures that are characteristic of this species natural environment; S. carolitertii, on the other hand, may be unable to deal with the extreme temperatures faced by the southern species.