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 59 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 ␤-globin gene mutations that apparently contradict this rule. The corresponding ␤-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type ␤-globin (␤ WT ) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between ␤WT mRNA and ␤-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; ␤39). 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) 1 is an mRNA surveillance mechanism that rapidly degrades mRNAs carrying premature translation termination codons (1). Nonsense-containing mRNAs targeted by NMD can be generated by naturally occurring frameshift and nonsense mutations, splicing errors, leaky 40 S scanning, and utilization of minor AUG initiation sites (2, 3). A major function of the NMD pathway is to block the synthesis of truncated proteins that could have dominant negative effects on cell function (2, 4).Recent studies have shown that the NMD pathway in mammalian cells is linked to splicing-dependent deposition of a protein complex 20 -24 nucleotides (nt) 5Ј of each exon-exon junction (exon-junction complex; EJC). The EJC contains the general splicing activator RNPS1, the RNA export factor Aly/ REF, the shuttling protein Y14, the nuclear matrix-localized serine-arginine-containing protein SRm160, the oncoprotein DEK, and the Y14 binding protein magoh. The interaction of magoh with Y14 may have a role in cytoplasmic localization of mRNAs and in anchoring the NMD-specific factors Upf3 and Upf2 to the mRNA (5-18). Previous published data have shown that Upf3 and Upf2 join the EJC in different subcellular compartments: Upf3 (Upf3a and Upf3b) is loaded onto mRNAs in the nucleus during splicing via interactions with components of the EJC. In contrast, Upf2 joins the complex soon after cytoplasmic export is initiated (14,19,20). According to the present models, translating ribosomes displace EJCs from the open reading frame (ORF) during the "pioneer" round of cytoplasmic translation (21). If, however, the mRNA contains a pr...

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.

Hierarchical epigenetic alterations occur at transition points during colorectal neoplastic progression. These cumulative changes are closely correlated with a gain of DNMT3B expression, suggesting a causal relationship.