Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

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 beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 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 decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position

mRNA for glutathione peroxidase 1 (GPx1) is subject to cytoplasmic nonsense-mediated decay (NMD) when the UGA selenocysteine (Sec) codon is recognized as nonsense. Here, we demonstrate by moving the sole intron of the GPx1 gene that either the Sec codon or a TAA codon in its place elicits NMD when located >/=59 bp but not 相似文献   

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

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.     

RNA splicing promotes translation and RNA surveillance

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.     

Context analysis of termination codons in mRNA that are recognized by plant NMD

The nonsense-mediated mRNA decay (NMD) system is an RNA surveillance system that degrades mRNAs possessing premature translation termination codons (PTCs). Although NMD factors are well conserved in eukaryotes, it is speculated that the contexts of those termination codons that are subject to NMD are different depending on the organism. Context analysis of termination codons that are recognized by the plant NMD system would clarify NMD target mRNAs in plants, and contribute to our understanding of its biological relevance in plants. In the present study we analyzed the positions of termination codons that were recognized as PTCs using an Agrobacterium transient expression assay, i.e. the accumulation of a series of plant mRNAs with nonsense mutations in different contexts was tested in plants. The results indicated that termination codons that are located distant from the mRNA 3' termini or >50 nucleotides upstream of the 3'-most exon-exon junction are recognized as substrates for NMD.     

Evidence that the Upf1-related molecular motor scans the 3'-UTR to ensure mRNA integrity

Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon-junction complex (EJC), which is deposited at exon-exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon-exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 3'-untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 5'-side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation-termination codon and a downstream exon-exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 3'-UTR in the 5'-to-3' direction for the mRNA-binding factors including EJCs to ensure mRNA integrity.     

Intron function in the nonsense-mediated decay of beta-globin mRNA: indications that pre-mRNA splicing in the nucleus can influence mRNA translation in the cytoplasm.

Generally, mRNAs that prematurely terminate translation are abnormally low in abundance. In the case of mammalian cells, nonsense codons most often mediate a reduction in the abundance of newly synthesized, nucleus-associated mRNA by a mechanism that is not well understood. With the aim of defining cis-acting sequences that are important to the reduction process, the effects of particular beta-globin gene rearrangements on the metabolism of beta-globin mRNAs harboring one of a series of nonsense codons have been assessed. Results indicate that nonsense codons located 54 bp or more upstream of the 3'-most intron, intron 2, reduce the abundance of nucleus-associated mRNA to 10-15% of normal without altering the level of either of the two introns within pre-mRNA. The level of cytoplasmic mRNA is also reduced to 10-15% of normal, indicating that decay does not take place once the mRNA is released from an association with nuclei into the cytoplasm. A nonsense codon within exon 2 that does not reduce mRNA abundance can be converted to the type that does by (1) inserting a sufficiently large in-frame sequence immediately upstream of intron 2 or (2) deleting and reinserting intron 2 a sufficient distance downstream of its usual position. These findings indicate that only those nonsense codons located more than 54 bp upstream of the 3'-most intron reduce beta-globin mRNA abundance, which is remarkably consistent with which nonsense codons within the triosephosphate isomerase (TPI) gene reduce TPI mRNA abundance. We propose that the 3'-most exon-exon junction of beta-globin mRNA and, possibly, most mRNAs is marked by the removal of the 3'-most intron during pre-mRNA splicing and that the "mark" accompanies mRNA during transport to the cytoplasm. When cytoplasmic ribosomes terminate translation more than 54 nt upstream of the mark during or immediately after transport, the mRNA is subjected to nonsense-mediated decay. The finding that deletion of beta-globin intron 2 does not appreciably alter the effect of any nonsense codon on beta-globin mRNA abundance suggests that another cis-acting sequence functions in nonsense-mediated decay comparably to intron 2, at least in the absence of intron 2, possibly as a fail-safe mechanism. The analysis of deletions and insertions indicates that this sequence resides within the coding region and can be functionally substituted by intron 2.     

A new function for nonsense-mediated mRNA-decay factors

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?     

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.