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Binary specification of nonsense codons by splicing and cytoplasmic translation

Binary specification of nonsense codons by splicing and cytoplasmic translation The EMBO Journal Vol.17 No.12 pp.3484–3494, 1998 Binary specification of nonsense codons by splicing and cytoplasmic translation Different PTCs located in exons 1 and 2 direct the affected Rolf Thermann, Gabriele Neu-Yilik, mRNAs to NMD (Baserga and Benz, 1988; Enssle et al., Andrea Deters, Ute Frede, Kristina Wehr , 1,2 1993), which is associated with the lack of clinical Christian Hagemeier, Matthias W.Hentze symptoms in heterozygotes. In contrast, NMD does not and Andreas E.Kulozik occur when PTCs are located in the final exon 3. In these cases, heterozygous patients are clinically affected by an Department of Pediatrics, Charite´-Virchow Medical Center, Augustenburger Platz 1, Humboldt University, D-13353 Berlin and unusual dominant form of β-thalassemia (Thein et al., Gene Expression Programme, EMBL, Meyerhofstrasse 1, 1990; Hall and Thein, 1994). D-69117 Heidelberg, Germany In mammals, the mechanism(s) by which PTC-mutated Corresponding authors mRNAs are specifically recognized and targeted for decay is largely unknown, and both nuclear and cytoplasmic R.Thermann and G.Neu-Yilik contributed equally to this work mechanisms have been implicated to be involved in NMD. A nuclear mechanism is suggested by the following Premature translation termination codons resulting observations. (i) PTC-mutated mRNA but not pre-mRNA from nonsense or frameshift mutations are common is less abundant than wild-type mRNA in nucleus-associ- causes of genetic disorders. Complications arising from ated fractions of transfected cells, in the bone marrow of the synthesis of C-terminally truncated polypeptides patients with β-thalassemia or in fibroblasts of patients can be avoided by ‘nonsense-mediated decay’ of the with triose phosphate isomerase (TPI) deficiency (Maquat mutant mRNAs. Premature termination codons in the et al., 1981; Daar and Maquat, 1988; Belgrader et al., β-globin mRNA cause the common recessive form of 1993, 1994; Simpson and Stoltzfus, 1994; Kugler et al., β-thalassemia when the affected mRNA is degraded, 1995; Carter et al., 1996; Kessler et al., 1996). (ii) The but the more severe dominant form when the mRNA fate of PTC-mutated β-globin mRNA can be influenced escapes nonsense-mediated decay. We demonstrate that by the promoter from which it is expressed (Enssle et al., cells distinguish a premature termination codon within 1993). (iii) In some cases, PTCs have been reported to the β-globin mRNA from the physiological translation induce alternative splicing events (Naeger et al., 1992; termination codon by a two-step specification mechan- Dietz et al., 1993; Dietz and Kendzior, 1994; Lozano ism. According to the binary specification model pro- et al., 1994; Dietz, 1997). (iv) Intronic sequences have posed here, the positions of splice junctions are first been implicated to play a role in NMD of mutant TPI tagged during splicing in the nucleus, defining a stop mRNAs (Cheng et al., 1994). (v) The relative position of codon operationally as a premature termination codon a PTC with regard to the exon/intron structure of the by the presence of a 3 splicing tag. In the second step, unspliced pre-mRNA has been shown to play a crucial cytoplasmic translation is required to validate the role. mRNAs with a PTC in the last exon are usually not 3 splicing tag for decay of the mRNA. This model subject to NMD (Baumann et al., 1985; Baserga and explains nonsense-mediated decay on the basis of con- Benz, 1988; Urlaub et al., 1989; Thein et al., 1990; ventional molecular mechanisms and allows us to Mashima et al., 1992; Enssle et al., 1993; Belgrader and propose a common principle for nonsense-mediated Maquat, 1994; Cheng et al., 1994; Carter et al., 1996). (vi) decay from yeast to man. The stability of wild-type and PTC-mutated cytoplasmic Keywords: dominant β-thalassemia/mRNA stability/ mRNA was reported to be similar in transfected cells nonsense-mediated decay/translation/3-UTR splicing (Humphries et al., 1984; Baserga and Benz, 1992; Cheng and Maquat, 1993; Lozano et al., 1994). On the other hand, there are many lines of evidence that implicate a cytoplasmic mechanism in NMD. (i) In Introduction transgenic mice with a PTC-mutated human β-globin gene, Nonsense and frameshift mutations introduce premature RNA degradation products were found in the cytoplasm of translation termination codons (PTCs) into the open read- erythroid bone marrow cells (Lim and Maquat, 1992; Lim ing frames (ORFs) of the affected mRNAs and are common et al., 1992). (ii) A translational ORF starting with an causes of genetic disorders. Surprisingly, PTCs usually AUG is necessary for NMD (Naeger et al., 1992; Simpson direct the affected mRNAs to rapid degradation, a process and Stoltzfus, 1994). (iii) NMD can be suppressed by termed nonsense-mediated mRNA decay (NMD). The co-transfecting appropriate suppressor tRNAs (Takeshita physiological importance of NMD is related to the reduc- et al., 1984; Belgrader et al., 1993) and (iv) by inhibiting tion in the synthesis of C-terminally truncated proteins, global translation (Qian et al., 1993; Lozano et al., 1994; thus avoiding dominant-negative effects of non-functional Menon and Neufeld, 1994; Carter et al., 1995), or (v) by polypeptides (Maquat, 1995). alterating the structure of the 5-untranslated region PTCs in human β-globin mRNA represent a clinically (5-UTR) of a PTC-containing mRNA to inhibit translation well-documented example of this beneficial NMD effect. in cis (Belgrader et al., 1993; Kugler et al., 1995). 3484 © Oxford University Press Binary specification of nonsense codons Although in principle, both nuclear and cytoplasmic mechanisms could cooperate in NMD, the difficulties in reconciling this complex set of findings have recently led to contradictory models for NMD in mammals. Some of these models involve unorthodox features of gene expression such as nuclear scanning of the ORF (Urlaub et al., 1989; Dietz et al., 1993; Aoufouchi et al., 1996; Carter et al., 1996; Li et al., 1997). In this report, we have analyzed NMD of human β-globin mRNA. Exploiting specific informative manipulations of mRNAs with and without PTCs, we show that nuclear splicing and cyto- plasmic translation co-operate to enact a mechanism that distinguishes physiological from premature translation termination codons. The proposed binary specification model not only explains the findings described here, but can reconcile most experimental data on NMD in mammalian cells on the basis of molecular mechanisms that are consistent with the conventional understanding of the gene expression pathway. Results Sequence context is not sufficient for PTC versus physiological translation termination codon definition Both translation initiation and physiological translation termination (Ter) codons display typical sequence contexts that affect the efficiency of their function (Kozak, 1986; McCaughan et al., 1995). We first examined whether PTCs were distinguished from Ter by their direct sequence context or by the position of the translation termination codon relative to other cis-acting elements. The first set of constructs was designed to analyze the role of the Ter sequence context (Figure 1A). A comparison of the wild-type β-globin gene with that bearing a naturally Fig. 1. Sequence context does not exert a dominant influence to occurring nonsense mutation at position 39 (PTC 39) was distinguish operationally a premature from a physiological termination codon. (A) Human β-globin gene constructs used for transfection. The used to document NMD in our HeLa cell transfection structural modifications and the nomenclature of the constructs are system. Relative to wild-type, PTC 39 was expressed shown diagrammatically. The ORF is represented by boxes, and the typically at a 4- to 5-fold lower level (Figure 1B, lanes 1 untranslated regions and introns by lines (see Materials and methods and 2). When PTC 39 was replaced by the physiological for details). (B) Northern blot of cytoplasmic HeLa cell RNA and β-globin termination codon Ter including its direct hybridization with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after sequence context of 15 flanking nucleotides on both sides normalization for transfection efficiency. (PTC 39-Ter; Figure 1A), NMD was observed, albeit to a somewhat lesser extent (Figure 1B, compare lanes 1 and 3). Replacement of PTC 39 by the physiological stop 1), suggesting that the position of PTC/Ter within the codon and its 30 immediately flanking nucleotides is thus penultimate exon may play an important role. not sufficient to bypass NMD. When the exon 2 sequences 3 of PTC 39 were replaced by the entire exon 3 to A minimal distance of a PTC mutation from the transfer a wider Ter sequence context into the vicinity of final intron is critical for nonsense-mediated decay PTC 39 (Figure 1A, PTC 39-E2/3), mRNA expression In most cases, PTCs within the last exon fail to specify remained low and was comparable with the level for PTC NMD. A distinct positional boundary for NMD at ~50 39-Ter (Figure 1B, compare lanes 1 and 4). Therefore, nucleotides upstream of the final intron previously has exon 3 does not contain dominant sequences that are been established for PTCs in TPI mRNA. If a PTC is capable of redefining PTC 39 as a bona fide Ter, or of located upstream of that boundary, the affected mRNA is redirecting the mRNA to a non-NMD pathway. Construct degraded. If the PTC is closer to the intron, the mRNA WT-E3-E3-Ter and the control WT-E3-E3-CAA remains stable (Cheng et al., 1990, 1994; Cheng and (Figure 1A) were designed to examine the effect of Ter Maquat, 1993). In contrast, the T-cell receptor-β (TCR-β) in its physiological exonic context but in an upstream gene does not exhibit a similar boundary: PTCs as close position. The accuracy of the predicted splice events was as eight nucleotides upstream from the last intron specify confirmed by cDNA sequencing of RT–PCR products NMD (Carter et al., 1996). The second set of constructs (data not shown). Both mRNAs were expressed at high with nested mutations (Figure 2A) was therefore designed levels (Figure 1A, compare lanes 5 and 6 with lane to define whether the known positional effects of PTC 3485 R.Thermann et al. Fig. 2. The distance of the translation termination codon from the final splice junction is critical for NMD. (A) Schematic representation of human β-globin gene constructs with the position of nested PTC mutations. (B) Northern blot of cytoplasmic RNA of HeLa cells transfected with the constructs shown in (A) and hybridization with β-globin and CAT cRNA probes. (C) The column diagram summarizes the results of three independent transfection experiments. Boxes indicate the mean and bars the maximum and minimum values obtained after normalization for transfection efficiency. (D) In-frame deletion and insertion constructs shifting the position of PTC 82 towards (PTC 82Δ) and PTC 88 away from the splice junction (PTC 88). Construct WT controls for destabilizing elements within the 27 nucleotide insertion. (E) Northern blot of cytoplasmic RNA of transfected HeLa cells and hybridization with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after normalization for transfection efficiency. mutations in the human β-globin gene (Baserga and Benz, are all expressed at much higher or normal levels. PTCs 1988; Thein et al., 1990; Enssle et al., 1993) are of the in the first and in the 5 region of the second exon thus TPI or the TCR-β type. direct mRNAs to the NMD pathway, whereas PTCs β-Globin mRNAs with a PTC at codon 26 (in exon 1) towards the 3 end of the second exon and in the third and at codons 39, 75 and 82 within the 5 two-thirds of exon are associated with high level mRNA expression. exon 2 result in NMD, and are expressed at low levels This polarity exhibits a clear boundary between 48 and (Figure 2B and C, compare lane 1 with lanes 2–5). In 66 nucleotides upstream of intron 2 (Figure 2), suggesting contrast, mRNAs bearing PTCs towards the 3 end of that the distance between the PTC and the exon 2–intron 2 exon 2 at codons 88, 91, 95, 98 and 103 (Figure 2B and splice junction may be important. This was tested directly C, lanes 6–10) and those with PTCs in exon 3 at codons with a set of deletion and insertion constructs (Figure 2D) 106, 107, 114 and 121 (Figure 2B and C, lanes 11–14) shifting the PTC towards or away from the boundary. 3486 Binary specification of nonsense codons Construct PTC 82Δ differs from PTC 82 by a 27 nucleotide in-frame deletion, and PTC 88 differs from PTC 88 by a 27 nucleotide in-frame insertion. In PTC 82Δ, the distance between PTC 82 and the splice junction was thus reduced from 66 to 39 nucleotides, which results in a switch from low to high mRNA expression (Figure 2E, compare lanes 3 and 4). In PTC 88, the distance between PTC 88 and the splice junction was increased from 48 to 75 nucleotides, which redirects this mRNA to the NMD pathway (Figure 2E, compare lanes 5 and 6). The wild- type control with the same insertion (WT) is expressed at normal levels (Figure 2E, lane 7), demonstrating that the 27 nucleotide insert per se does not harbor destabilizing elements. The distance between the PTC and the final intron thus represents the reference point for a distinct positional polarity of the TPI type. Splicing in the 3-UTR subverts a wild-type mRNA to nonsense-mediated decay The data described above and previous findings (Carter et al., 1996) indicate the importance of splicing 3 of premature translation termination codons for the NMD pathway. They also suggest the provocative possibility that a wild-type mRNA could be subverted to the NMD pathway by the introduction of a spliceable intron at sufficient distance downstream from Ter within the 3-UTR. The constructs shown in Figure 3A were designed to test this hypothesis directly. Construct WT-SP-Ter contains a heterologous intron plus 15 nucleotides of flanking sequences at both sides 62 nucleotides down- stream of Ter. Importantly, and in contrast to previously reported experimental designs (Carter et al., 1996; Li et al., 1997), the ORF was not altered by this manipulation, and specific cis-acting intronic effects such as those reported for TPI RNA (Cheng et al., 1994) were excluded here. The controls contain either just the two 15 nucleotides flanking sequences (WT-spf and PTC 39-spf) or the Fig. 3. Splicing in the 3-UTR directs a wild-type mRNA to the NMD identical intron without splice donor and acceptor signals pathway. (A) Human β-globin gene constructs with modifications of the 3-UTR. The presence or absence of GU...AG motifs indicates (WT-sp). Analysis of these constructs (Figure 3B) reveals whether or not the intron was furnished with a splice donor and that: (i) controls WT-spf and PTC 39-spf are comparable acceptor site (see Materials and methods for details). (B) The Northern with wild-type and PTC 39, respectively (Figure 3B, blot of cytoplasmic RNA of transfected HeLa cells was hybridized to compare lanes 1 and 2); (ii) the presence of an unspliceable β-globin and CAT cRNA probes. The percentage values refer to the intron in construct WT-sp does not influence the level of mean of three independent experiments after normalization for transfection efficiency. wild-type mRNA expression (Figure 3B, compare lanes 1 and 6; note the slower migration of WT-sp mRNA); and (iii) the spliceable intron of construct WT-SP-Ter is intron, i.e. 3 of the positional boundary defined in Figure 2 removed from the mature mRNA (Figure 3B, compare (Figure 3A). Neither mRNA is subject to NMD (Figure 3B, lanes 3 and 6), resulting in NMD of the WT-SP-Ter compare lane 1 with lanes 4 and 5), demonstrating that: mRNA, i.e. the physiological stop codon is operationally (i) the positional boundary established for the natural final redefined as a PTC by the presence of the downstream intron of the β-globin gene (Figure 2) can be translocated intron, and an mRNA with a wild-type ORF is directed when the exon–intron structure of the gene is altered; (ii) to the NMD pathway. Note that WT-spf and WT-SP-Ter the same positional requirements appear to apply to 3- encode identical mature mRNAs, with the latter but UTR introns; and (iii) a 3-UTR intron must be spliceable not the former being subject to NMD. The functional to subvert a wild-type mRNA to NMD. In conclusion, relationship between the position of Ter and the 3 splice NMD depends in cis on the position of the premature or event was examined further: (i) with construct WT- physiological translation termination codon relative to the SP-CAA that differs from WT-SP-Ter by a Ter→Gln most 3 spliceable intron. These findings also unequivoc- (TAA→CAA) mutation, thus removing the translational ally identify a nuclear component of the NMD pathway. termination signal 5 of the intron inserted into the 3-UTR; and (ii) with construct WT-SP-CAA-Ter that Nonsense-mediated decay of mutant mRNAs differs from WT-SP-CAA by a novel Ter 18 nucleotides requires their cytoplasmic translation downstream of the CAA, thus repositioning the translation Findings from several laboratories have implicated a role termination codon to only 44 nucleotides 5 of the novel for translation or ‘translation-like’ mechanisms in NMD 3487 R.Thermann et al. (see above). In studies employing general translation of human β-globin genes (constructs shown in Figure 4C) inhibitors (Qian et al., 1993; Lozano et al., 1994; Menon thus allows the role of cytoplasmic translation in NMD and Neufeld, 1994; Carter et al., 1995) or suppressor to be probed specifically. A wild-type β-globin mRNA tRNAs (Takeshita et al., 1984; Belgrader et al., 1993), bearing an IRE within its 5-UTR (WT-IRE) is stable possible pleiotropic effects of these approaches complicate regardless of the availability of iron (Figure 4D, lanes 1 the interpretation of the data. Furthermore, these and other and 3). In the presence of iron, translation is readily studies using specific translational inhibitory alterations detectable by immunoblotting (Figure 4D, lane 5), whereas of the 5-UTRs of PTC mRNAs (Belgrader et al., 1993; hardly any protein can be identified in iron-deficient cells Kugler et al., 1995) could not distinguish unambiguously (Figure 4D, lane 2). The iron-dependent regulation of between conventional cytoplasmic translation and the less translation is lost when a point-mutated non-functional orthodox nuclear translation-like mechanisms that are a IRE is introduced into the β-globin 5-UTR: the mRNA critical feature of the nuclear scanning model (Carter is translated constitutively (Figure 4C, construct WT- et al., 1996; Li et al., 1997). IREΔC, Figure 4E lanes 4 and 7) and also is stable The interaction between iron-responsive elements (Figure 4D, lanes 5 and 7). In contrast, PTC 39 mRNA (IREs) and iron-regulatory proteins (IRPs) enables the bearing a functional IRE (Figure 4C, construct PTC 39- specific translational regulation of mRNAs bearing IREs IRE) is subject to NMD when translation is enabled, but in their 5-UTRs (Klausner et al., 1993; Hentze and Kuhn, escapes NMD in iron-deficient cells where IRP binding 1996). In iron-deficient cells, IRP binding to an IRE inhibits the cytoplasmic translation of the mutated mRNA specifically inhibits the formation of a 48S pre-initiation (Figure 4). The NMD escape is due to translational complex at the 5 end of the mRNA, thus blocking an inhibition rather than to iron deficiency per se, because a early step of translation (Gray and Hentze, 1994). Elevation PTC 39 mRNA bearing a non-functional IRE (Figure 4C, of intracellular iron levels inactivates IRP binding and constructs WT-IREΔC and PTC 39-IREΔC) is subject to thus permits the translation of the mRNA. IRP-1 (98 kDa) NMD in both iron-replete and iron-deficient cells and IRP-2 (105 kDa) are expected to be expressed exclus- (Figure 4D, compare lanes 3 with 4, 5 with 6 and 7 with 8). ively in the cytoplasm, because they are too large to The β-globin antibodies fail to detect any polypeptide traverse the nuclear membrane passively and they do not expressed from the PTC 39 mRNAs, hence it was not contain a known nuclear localization signal for active possible formally to demonstrate translational control of nuclear import. This spatial restriction was assessed dir- these mRNAs by immunoblotting (Figure 4E, lanes 1, 3, ectly by immunofluorescence studies of cells with an IRP-1- 6 and 8). We therefore analyzed the polysome association specific antibody (Pantopoulos et al., 1995) that show a (Figure 5) of the mRNAs expressed from the constructs cytoplasmic signal excluding nuclei (Figure 4A). Although shown in Figure 4C. This analysis confirms that: (i) wild- IRP-1 is the predominant IRP in mammalian cells, this type β-globin mRNAs are associated preferentially with analysis does not exclude the presence of IRP-2 in the heavier, and PTC 39 mRNAs with lighter polysomes when nucleus. We therefore performed gel retardation experi- translation is enabled, reflecting the differences in length ments with nuclear and serial dilutions of cytoplasmic of the respective ORFs (Figure 5A, C and D); and (ii) the extracts from HeLa cells with an IRE probe (Pantopoulos IRE–IRP interaction results in an accumulation of the et al., 1995) that forms electrophoretically co-migrating wild-type and the PTC 39 mRNAs at the top of the gradient, complexes with IRP-1 and IRP-2 with equal affinity showing that they are not associated with ribosomes (Hentze and Ku¨hn, 1996). Binding activity was readily (Figure 5B). We conclude that specific inhibition of detectable with as little as 0.07 μg of cytoplasmic protein ribosome association and cytoplasmic translation in cis (Figure 4B, lanes 1–8). In contrast, no signal was detectable prevents the degradation of PTC 39 mRNA. Thus, cyto- in the nuclear fraction, even when 25 μg of nuclear protein plasmic translation of the mutated mRNA is necessary (i.e. a 350-fold higher amount than 0.07 μg) was for NMD. used (Figure 4B, lanes 12–14). To exclude non-specific inhibitory effects on complex formation exerted by com- Discussion ponents in the nuclear extract, we compared complex formation between the IRE probe and recombinant human Cells have evolved mechanisms to distinguish wild-type IRP-1 in assays with or without 25 μg nuclear protein from mutated mRNAs, and to degrade the latter. These (Figure 4B, lanes 15 and 16). Taken together, these data mechanisms can operate when PTCs, but not missense show that IRP-1 and IRP-2 can be identified readily in mutations, disrupt the ORF. Nonsense-mediated decay is the cytoplasm and that neither appears to be present in an ill-understood, yet biologically and medically import- the nucleus. The introduction of an IRE into the 5-UTR ant, mechanism as demonstrated by mutations in the smg Fig. 4. Cytoplasmic translation is necessary to direct a PTC-mutated mRNA to the NMD pathway. (A) Immunofluorescence of B6 cells with pre- immune serum (left) and IRP-1-specific antibody (anti-IRP; right). (B) Gel retardation analyses with an IRE probe and serial dilutions of cytoplasmic (lanes 1–11) and nuclear (lanes 12–14) protein from fractionated HeLa cells and recombinant human IRP-1 together with (lane 15) or without (lane 16) nuclear protein. The IRE probe forms co-migrating complexes (arrow) with IRP-1 and IRP-2. (C) Wild-type and PTC 39-mutated human β-globin gene constructs with functional (IRE, bold stem loop) and non-functional (IREΔC, thin stem loop) IREs in the 5-UTR (see Materials and methods for details). (D) Northern blot of cytoplasmic RNA of transfected HeLa cells that were grown in medium either supplemented (iron)or not (iron–) with heme arginate as an iron source. Translation of the constructs containing a functional IRE could thus be regulated specifically (translation– or translation). Hybridization was performed with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after normalization for transfection efficiency. (E) Immunoblot of total protein of HeLa cells transfected with the constructs shown in (C). The medium was either supplemented (iron) or not (iron–) with heme-arginate. Hybridization was performed with human β-globin- and vigilin-specific antibodies. Vigilin is a protein that is expressed independently of the availability of iron and served as a control for equal loading of the lanes. 3488 Binary specification of nonsense codons 3489 R.Thermann et al. Fig. 5. Translation-competent wild-type and PTC 39 β-globin mRNAs are associated with polysomes. The top panel shows representative gradient profiles of HeLa cells transiently transfected with the constructs described in Figure 4C. The number of the fractions that were used for Northern blotting are indicated below the gradient profiles. The individual panels show Northern blots of the different fractions after hybridization with β-globin- or β-actin-specific probes. The left side represents cells that were transfected with wild-type and the right side those transfectd with a PTC 39-mutated construct. The table indicates the modification of the 5-UTR, the iron status of the cells and the specific translational competence of the transfected β-globin gene. (a) Functional IRE with iron supplementation resulting in translational competence. (b) Functional IRE without iron supplementation resulting in translational incompetence. (c) Non-functional IRE with iron supplementation. (d) Non-functional IRE without iron supplementation. (e) Representative blots hybridized with a β-actin-specific probe controlling for the quality of the gradient and general translational competence of the transfected cells. genes that are required for NMD in Caenorhabditis PTC-mutated mRNA degradation is independent of the elegans, and display synthetic lethality with nonsense recognition of translational sense within the nucleus but mutations in the unc-54 myosin heavy chain B gene (Pulak occurs while the mRNA is still physically associated with and Anderson, 1993), or by forms of β-thalassemia with the nucleus. Those PTC-mutated mRNA molecules that symptomatic heterozygotes and a dominant mode of escape the nucleus-associated degradation mechanism are inheritance (Thein et al., 1990; Hall and Thein, 1994). stable once they have reached the cytoplasm (Maquat, NMD involves different phases: discrimination of a 1995). In contrast, different variations of ‘nuclear scan- PTC from the physiological stop codon, commitment of ning’ models postulate the existence of a (ribosome- the mutated mRNA to NMD, and nucleolytic degradation like) structure that scans nuclear (pre-)mRNAs for their of the mRNA. Much work has focused on the early phases translational sense and induces either altered splicing to of NMD. A complex array of findings obtained from avoid the PTC or the degradation of PTC-mutated mRNAs various mammalian systems has led to the formulation of within the nucleus (Urlaub et al., 1989; Dietz et al., 1993; several models. In spite of significant progress to distin- Dietz and Kendzior, 1994; Aoufouchi et al., 1996; Carter guish between these models, at least two mutually exclus- et al., 1996; Li et al., 1997). ive models remain (Urlaub et al., 1989; Maquat, 1995; Our strategy based on PTC mutations of the β-globin Carter et al., 1996). Neither of the two models can fully gene aimed to unambiguously identify necessary and account for the available data (see below). One central sufficient components during the discrimination and com- issue revolves around the question of whether NMD is a mitment phase of NMD. The first set of experiments nuclear, a nucleus-associated or a cytoplasmic mechanism. addresses the role of splicing as a nuclear component of The analyses of the TPI gene suggested that PTC recogni- the NMD pathway. In the TPI gene, a sequence element tion takes place after splicing and that intronic sequences in the final intron was implicated in marking the mRNAs provide a mark that translating ribosomes must approxi- in cis. NMD depends on the distance between this sequence mate or traverse in order to confer mRNA stability element and the PTC mutation, but not on splicing per se (Maquat, 1995). However, the inhibition of translation (Cheng et al., 1994). In contrast, an analysis of the TCR-β was shown not to destabilize the mRNA (Belgrader et al., gene implicated a functional role for splicing but no distinct 1993). According to the co-translational export model, positional effect (Carter et al., 1996). The interpretation of 3490 Binary specification of nonsense codons the TCR-β data is complicated by two aspects; the tion is blocked, the tags are not encountered and the duplication or deletion of exonic and intronic sequences mRNA remains stable (Figure 6, pathway b). If the tags of that particular gene significantly alters the ORFs of the are encountered by elongating ribosomes within the ORF different TCR-β-related mRNAs, and the use of a TCR- (as would happen with tags representing all former introns β intron rather than a heterologous one. It is difficult, upstream of termination codons), the mRNA is not valid- therefore, to exclude a possible effect of specific cis-acting ated for the NMD pathway. In this case, the tags may intronic or exonic sequences that have been implicated in remain ‘neutral’, could be ‘approved’ or removed, and affecting mRNA stability in other mammalian systems the mRNA is not degraded (Figure 6, pathway a). If (Cheng et al., 1994; Liu and Mertz, 1996). mRNA translation occurs and a tag 3 of the translation In the experiments shown in Figure 3, a spliceable stop codon is identified (as would happen with PTCs in intron was inserted into the 3-UTR of the human all but the final exon and with 3-UTR intron tags), the β-globin mRNA without changing the structure of the tag is validated for NMD and the mRNA targeted for mature mRNA (compare WT-spf with WT-SP-Ter). More- degradation (Figure 6, pathway c). If PTC mutations are over, the intron used is unrelated to the β-globin gene, located at an insufficient distance from the final tag avoiding the inadvertant inclusion of confounding homo- (Figure 2) (Baumann et al., 1985; Urlaub et al., 1989; logous cis-acting elements. The results demonstrate that Thein et al., 1990; Mashima et al., 1992; Enssle et al., a structurally wild-type mRNA without any changes in its 1993; Belgrader and Maquat, 1994; Cheng et al., 1994), ORF is directed to the NMD pathway by a splicing event this may be (falsely) ‘approved’ or removed by the leading in the 3-UTR (Figure 3). Downstream splicing can edge of the elongating ribosome, which thus plays an therefore serve as a sufficient means for redefining the mRNA protective role. The factor or the complex that physiological translation stop codon Ter as a PTC. The validates downstream tags for NMD remains to be identi- operational specification of a PTC includes the requirement fied, but could well represent (components of) a ribosomal for a minimal distance between the termination codon and post-termination complex (Ruiz-Echevarria et al., 1998). the 3-splicing event (Figure 2). This position dependence It is possible that such a complex could contain homologs displays a clear boundary similar to that described for the of the UPF proteins (Applequist et al., 1997) which are TPI gene (Cheng et al., 1994). The potential mechanistic required for NMD in yeast and, interestingly, have been basis of the boundary effect is discussed below. shown to be ribosome associated (Ruiz-Echevarria The other issue that we directly addressed is the et al., 1996). specific role of translation in the cytoplasm. Whereas the This binary specification model can explain or account introduction of hairpins into the 5-UTR or the use of for many previous, seemingly contradictory findings that suppressor tRNAs (Takeshita et al., 1984; Belgrader et al., were decribed above. Our model can also explain a recent 1993) may interfere with the function of the notional report demonstrating that translation re-initiation after a nuclear ribosome-like structure that represents a critical premature stop codon results in the escape of TPI mRNA feature of the nuclear scanning model (Carter et al., 1996; from the NMD pathway (Zhang and Maquat, 1997). One Li et al., 1997), the IRE/IRP system specifically regulates of the most puzzling aspects of NMD has been the nuclear mRNA translation in the cytoplasm (Figure 4A and B; or cytoplasmic site of PTC identification and the frequently Klausner et al., 1993; Hentze and Kuhn, 1996). The design nucleus-associated site of mRNA degradation. Our find- of the experiments shown in Figures 4 and 5 also excludes ings functionally assign the relevant translational activity possible pleiotropic effects of general inhibitors of protein to the cytoplasm, and thus do not support some of the synthesis. Moreover, our strategy exploits the functional unconventional aspects of the nuclear scanning model. (IRE/IRP) rather than preparative (subcellular fractiona- The nuclear association of PTC mRNA degradation in tion) definition of the cytoplasmic compartment. This some (but not all) cases of NMD is consistent with the allows us to distinguish whether the influence of transla- binary specification model. We suggest that some mRNPs tional features on the NMD pathway is conferred by the undergoing nuclear export have an inherent transient conventional cytoplasmic translation apparatus or by a affinity for the preparatively defined ‘nuclear fraction’ notional translation-like nuclear mechanism (Carter et al., while being accessible to the cytoplasmic translation 1996; Li et al., 1997) and also avoids uncertainties apparatus on the cytoplasmic side of the nuclear envelope. about the purity of nuclear preparations. We show that The residual levels of PTC-mutated mRNAs of com- conventional cytoplasmic translation is a necessary com- monly 10–30% of the wild-type levels may be explained ponent to validate a PTC-mutated β-globin mRNA for the by a 100% efficiency of tagging and/or by tag dissoci- NMD pathway (Figures 4 and 5). ation. The normal cytoplasmic stability of the remaining Our results suggest a binary specification model for the 10–30% PTC mRNAs that bear no tag is predicted by the discrimination and commitment phase of NMD (Figure 6). binary specification model. An observation that can be In the first step, splicing induces the tagging of the position explained by neither the binary specification nor by any of (former) introns. It is conceivable that such a tag could of the other models is the effect of PTC mutations on be a spliceosome-associated protein that remains bound splice-site choice (Dietz et al., 1993; Dietz and Kendzior, to, and escorts the mRNA into the cytoplasm (Visa et al., 1994; Dietz, 1997) or the splicing process per se (Lozano 1996). In the exceptional cases where additional specific et al., 1994; Aoufouchi et al., 1996), which may be cis-acting sequences (Cheng et al., 1994) can substitute induced by another PTC-dependent mechanism. for the presence of a spliceable downstream intron (such In yeast, NMD has been perceived to be enacted by a as the TPI mRNA), the tag (protein) may be recruited by mechanism which is profoundly different from that used these specific sequences. During or after nuclear export, in multicellular eukaryotes. NMD in yeast is splicing the second step requires cytoplasmic translation. If transla- independent, cytoplasmic and apparently dependent on a 3491 R.Thermann et al. Fig. 6. The binary specification model of NMD. The details of the model are described in the text. at the 3 end. In construct WT-E3-E3-Ter, the entire exon 2 is replaced specific ‘downstream sequence element’ (DSE) (Ruiz- in-frame by a PCR fragment containing the entire coding sequence of Echevarria et al., 1996). Interestingly, the DSE is thought exon 3, termed exon 3*. Construct WT-E3-E3-CAA differs from WT- to require recognition by a ribosomal post-termination E3-E3-Ter by a TAA→CAA mutation of the translation termination complex (Ruiz-Echevarria et al., 1998). The binary speci- codon of exon 3*. Constructs PTC 26, PTC 75, PTC 82, PTC 91, PTC 95, PTC 98, PTC fication model provides a conceptional bridge between 103, PTC 106, PTC 107, PTC 114 and PTC 121 were created by NMD in yeast and mammals. Hypothetically, the mammal- site-directed mutagenesis (Hagemeier, 1996). Construct PTC 88 was ian splicing tag could be substituted by the DSE (or a generated by replacing the sequences in the wild-type construct by a DSE-binding protein) in Saccharomyces cerevisiae, where PCR fragment derived from a proband with heterozygous β-thalassemia introns are rare. All subsequent steps of NMD in yeast carrying a ΔG frameshift mutation at codon 82/83, which results in a TGA PTC at codon 88 (Vetter et al., 1997). and man could then conceivably be alike. Further charac- Constructs PTC 82Δ and PTC 88 were generated by site-directed terization of the splicing tag and of the function of the mutagenesis (Hagemeier, 1996). Construct PTC 82Δ differs from con- DSE represent experimental tests of this hypothetical struct PTC 82 by a 27 bp deletion of codons 91–99. In construct PTC prediction. 88, a 27 bp fragment was inserted into construct PTC 88 in-frame at codon 99. Construct WT differs from PTC 88 by the absence of the In conclusion, we present specific evidence that the PTC 88 mutation. discrimination and commitment to degrade PTC-mutated In the constructs shown in Figure 3A, the ApaI and XhoI sites of the human β-globin mRNAs involves the co-operation of Bluescript polylinker were deleted. New ApaI and XhoI restriction sites conventional nuclear and cytoplasmic molecular mechan- were then inserted by site-directed mutagenesis into the 3-UTR at a isms. Our findings and their interpretation raise the pos- position 41 bp downstream of the physiological stop codon. Construct WT-SP-Ter was generated by introducing the MINX-intron (Zillmann sibility of a common principle for NMD from yeast to et al., 1988) with 30 bp flanking sequences including its splice donor man. Further implications of our findings relate to the and acceptor sites into the ApaI and XhoI sites in the 3-UTR. Construct common usage of introns in the 3-UTRs of expression WT-SP-CAA differs from WT-SP-Ter by a TAA→CAA mutation of Ter. vectors in molecular biology and gene therapy, which may The novel in-frame Ter of this construct is located 41 bp downstream of the inserted intron. Construct WT-SP-CAA-Ter differs from WT-SP- warrant re-evaluation. CAA by a new TAA that was created by site-directed mutagenesis at an in-frame position 18 bp downstream of the former Ter. Construct WT-sp differs from construct WT-SP-Ter by the absence of the intron flanking Materials and methods sequences including the splice donor and acceptor sites. Construct WT- Plasmid constructs spf differs from WT-SP-Ter by the absence of the intron, but retains the The constructs shown in Figures 1A–3A contain a 4.4 kb human intron flanking sequences in the 3-UTR. In construct PTC 39-spf, the β-globin gene with a linked SV40 enhancer inserted as a NotI fragment WT CAG at position 39 of construct WT-spf was replaced by a TAG PTC. into pBlueskriptSK II (Stratagene) (Enssle et al., 1993). The wild-type The constructs shown in Figure 4A contain the human β-globin gene and the PTC 39 constructs are derived from a healthy proband and from fragment as described above in a pGEM-5Zf () expression vector a patient with homozygous β-thalassemia, respectively. Construct PTC (Promega). In constructs WT-IRE and PTC 39-IRE, a 35 bp fragment 39 was modified by overlap extension PCR (Pogulis et al., 1996) to containing a functional IRE (Hentze et al., 1987) was inserted by site- generate construct PTC 39-Ter, in which the stop codon at position 39 directed mutagenesis (Hagemeier, 1996) into the 5-UTR 37 nucleotides (TAG) and the 15 bp flanking both sides were replaced by the physiologi- downstream of the cap structure of the wild-type and the PTC 39 cal stop codon (TAA) including 30 bp of its immediate flanking constructs (Figure 1A) (Goossen and Hentze, 1992). Constructs WT- sequences. In construct PTC 39-E2/3, exon 2 sequences 3 of PTC 39 IREΔC and PTC 39-IREΔC differ from the IRE constructs by a deletion were replaced in-frame by a PCR fragment containing exon 3 including of a single cytosine residue, which functionally inactivates the IRE the physiological translation termination codon and a novel splice donor (Hentze et al., 1988). 3492 Binary specification of nonsense codons The structure of all constructs was confirmed by DNA sequence was added to each fraction to a final concentration of 0.5%. Subsequently, analysis of the insert including the promotor and the entire transcribed fractions were subjected to phenol extraction. The extracted RNA was region from the cap site to the polyadenylation signal. analyzed by Northern blotting as described above. Transfection efficiency was determined by assaying for CAT activity. Cell culture and transfections HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) Immunoblotting under standard conditions. Cells were transiently transfected by calcium Cytoplasmic protein was extracted after lysis in cold lysis buffer plus phosphate precipitation (Ausubel et al., 1994) with 20 μgofthe 10 μg/ml PMSF as described for polysome gradients. Equal amounts of test constructs and 15 μgofan Escherichia coli chloramphenicol cells were analyzed on 5–15% SDS–polyacrylamide gradient gels. acetyltransferase (CAT) gene cloned into the multiple cloning site of the The proteins were electroblotted onto PVDF membranes (Immobilon, plasmid pSG5 (Green et al., 1988), which served as a control for Schleicher and Schuell) using a semi-dry apparatus. Membranes were transfection efficiency. Cells were washed after 20 h and harvested stained with Ponceau S to ensure that the transfer was complete and 24 h later. uniform, and then blocked with 5% skimmed milk in Tris-buffered saline For the experiments with the IRE-containing constructs, culture media (TBS) plus 0.05% Tween-20. The primary β-globin antibody was raised were supplemented with 100 μM of the iron source heme arginate in rabbits against the N-terminal 13 amino acids of human β-globin (Leiras, Turku, Finland) 8 h after washing. For iron starvation, it was (Eurogentec, Seraing, Belgium). Immunoblotting was performed using sufficient to grow the cells in unmodified medium that contained only 2 standard conditions and a 1:2000 dilution of the antibody. The vigilin 0.25 μmol of Fe . At 16 h after addition of heme arginate, i.e. 24 h antibody was a rabbit antiserum against the 15 C-terminal amino acids after transfection, the cells were harvested. of human vigilin and was diluted 1:6000. The secondary antibody (anti- rabbit IgG coupled to horseradish peroxidase; Dianova) was diluted Isolation and analysis of RNA 1:3000. Specific staining was detected using a bioluminescence kit Total cytoplasmic RNA was purifed from the supernatant of homogenized (Boehringer Mannheim) according to the manufacturer’s instructions. cells with TRIzol reagent (Gibco-BRL). Northern blot analysis was performed as previously described (Enssle et al., 1993) with 1–2 μgof total cytoplasmic RNA or, when fractions of polysome gradients were analyzed, with half of each fraction. Blots were hybridized using in vitro Acknowledgements transcribed P-labeled antisense cRNA β-globin and CAT probes. The template for the β-actin probe was a 383 bp human β-actin cDNA Heme arginate was generously provided by Leiras, Turku, Finland. We PCR fragment, which was subjected to single strand PCR in the presence thank Dr Kostas Pantopoulos (EMBL, Heidelberg) for performing the of [ P]dCTP (Konat et al., 1994). Hybridization was carried out at experiment shown in Figure 4A. This study was supported by the 65°C overnight. Deutsche Forschungsgemeinschaft (DFG). The signals were quantified by imaging in a GS-250 Molecular Imager (Bio-Rad). β-Globin mRNA levels are expressed as a percentage of wild-type after normalization for transfection efficiency. References Indirect immunofluorescence Aoufouchi,S., Yelamos,J. and Milstein,C. (1996) Nonsense mutations B6 cells were grown on coverslips in DMEM. Coverslips were washed inhibit RNA splicing in a cell-free system: recognition of mutant twice with phosphate-buffered saline (PBS), and cells were permeabilized codon is independent of protein synthesis. Cell, 85, 415–422. with 0.5% Triton X-100 in CKS buffer (100 mM NaCl, 300 mM sucrose, Applequist,S.E., Selg,M., Raman,C. and Jack,H.M. (1997) Cloning and 10 mM PIPES pH 6.8, 3 mM MgCl , 1 mM EGTA) containing 0.1 mM characterization of HUPF1, a human homolog of the Saccharomyces phenylmethylsulfonyl fluoride (PMSF) for 2 min on ice. Subsequently, cerevisiae nonsense mRNA-reducing UPF1 protein. Nucleic Acids cells were fixed with 3.7% paraformaldehyde in CKS buffer for 10 min Res., 25, 814–821. at room temparature, followed by three 5 min washes in PBS. Labeling Ausubel,F.M., Brent,R., Kingston,R.I., Moore,D.D., Seidman,J.G., with rabbit polyclonal antibodies raised against recombinant human Smith,J.A. and Struhl,K. (1994) Current Protocols in Molecular IRP-1 (Pantopoulos et al., 1995) or pre-immune serum as a specificity Biology. Vol. 1. John Wiley, New York. control was performed according to standard methods. Samples were Baserga,S.J. and Benz,E.J.,Jr (1988) Nonsense mutations in the human examined and photographed in a Zeiss Axioskop fluorescence β-globin gene affect mRNA metabolism. Proc. Natl Acad. Sci. USA, microscope. 85, 2056–2060. Baserga,S.J. and Benz,E.J.,Jr (1992) Beta-globin nonsense mutation: Gel retardation experiments deficient accumulation of mRNA occurs despite normal cytoplasmic These were performed exactly as previously described (Pantopoulos and stability. Proc. Natl Acad. Sci. USA, 89, 2935–2939. Hentze, 1995). Baumann,B., Potash,M.J. and Kohler,G. (1985) Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the Cell fractionation mouse. EMBO J., 4, 351–359. Cytoplasmic and nuclear fractions from HeLa cells used for IRE/IRP Belgrader,P. and Maquat,L.E. (1994) Nonsense but not missense bandshift analyses were prepared essentially as described (Kugler et al., mutations can decrease the abundance of nuclear mRNA for the 1995), with the following modifications: the lysis buffer contained 0.5% mouse major urinary protein, while both types of mutations can Triton X-100, 0.2% Na deoxycholate instead of 0.5% NP-40 and was facilitate exon skipping. Mol. Cell. Biol., 14, 6326–6336. supplemented with 1 mM dithiothreitol (DTT). Cells of a 10 cm dish Belgrader,P., Cheng,J. and Maquat,L.E. (1993) Evidence to implicate were lysed in 1 ml of lysis buffer. After cell lysis, the nuclei were translation by ribosomes in the mechanism by which nonsense codons pelleted and washed once with lysis buffer containing detergent and reduce the nuclear level of human triosephosphate isomerase mRNA. once with buffer without detergent. Nuclei were then centrifuged (15 000 Proc. Natl Acad. Sci. USA, 90, 482–486. g, 45 min, 4°C) through a 1.8 M sucrose cushion in reticulocyte standard Belgrader,P., Cheng,J., Zhou,X., Stephenson,L.S. and Maquat,L.E. (1994) buffer (Kugler et al., 1995). The pellet was resuspendend in 250 μlof Mammalian nonsense codons can be cis effectors of nuclear mRNA lysis buffer without detergent and lysed by freeze–thawing. half-life. Mol. Cell. Biol., 14, 8219–8228. 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The gradients were centrifuged for 2 h at 36 000 r.p.m. in an SW 40 abundance of nuclear mRNA without affecting the abundance of pre- rotor at 4°C. Thirteen fractions were collected from the top of the mRNA or the half-life of cytoplasmic mRNA. Mol. Cell. Biol., 13, gradient. UV absorbance at 254 nm was measured continously. SDS 1892–1902. 3493 R.Thermann et al. Cheng,J., Fogel Petrovic,M. and Maquat,L.E. (1990) Translation to near codons in human β-globin mRNA result in the production of mRNA the distal end of the penultimate exon is required for normal levels degradation products. Mol. Cell. Biol., 12, 1149–1161. of spliced triosephosphate isomerase mRNA. Mol. Cell. Biol., 10, Liu,X. and Mertz,J.E. (1996) Sequence of the polypyrimidine tract of 5215–5225. the 3-terminal 3 splicing signal can affect intron-dependent pre- Cheng,J., Belgrader,P., Zhou,X. and Maquat,L.E. (1994) Introns are cis mRNA processing in vivo. 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Binary specification of nonsense codons by splicing and cytoplasmic translation

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The EMBO Journal Vol.17 No.12 pp.3484–3494, 1998 Binary specification of nonsense codons by splicing and cytoplasmic translation Different PTCs located in exons 1 and 2 direct the affected Rolf Thermann, Gabriele Neu-Yilik, mRNAs to NMD (Baserga and Benz, 1988; Enssle et al., Andrea Deters, Ute Frede, Kristina Wehr , 1,2 1993), which is associated with the lack of clinical Christian Hagemeier, Matthias W.Hentze symptoms in heterozygotes. In contrast, NMD does not and Andreas E.Kulozik occur when PTCs are located in the final exon 3. In these cases, heterozygous patients are clinically affected by an Department of Pediatrics, Charite´-Virchow Medical Center, Augustenburger Platz 1, Humboldt University, D-13353 Berlin and unusual dominant form of β-thalassemia (Thein et al., Gene Expression Programme, EMBL, Meyerhofstrasse 1, 1990; Hall and Thein, 1994). D-69117 Heidelberg, Germany In mammals, the mechanism(s) by which PTC-mutated Corresponding authors mRNAs are specifically recognized and targeted for decay is largely unknown, and both nuclear and cytoplasmic R.Thermann and G.Neu-Yilik contributed equally to this work mechanisms have been implicated to be involved in NMD. A nuclear mechanism is suggested by the following Premature translation termination codons resulting observations. (i) PTC-mutated mRNA but not pre-mRNA from nonsense or frameshift mutations are common is less abundant than wild-type mRNA in nucleus-associ- causes of genetic disorders. Complications arising from ated fractions of transfected cells, in the bone marrow of the synthesis of C-terminally truncated polypeptides patients with β-thalassemia or in fibroblasts of patients can be avoided by ‘nonsense-mediated decay’ of the with triose phosphate isomerase (TPI) deficiency (Maquat mutant mRNAs. Premature termination codons in the et al., 1981; Daar and Maquat, 1988; Belgrader et al., β-globin mRNA cause the common recessive form of 1993, 1994; Simpson and Stoltzfus, 1994; Kugler et al., β-thalassemia when the affected mRNA is degraded, 1995; Carter et al., 1996; Kessler et al., 1996). (ii) The but the more severe dominant form when the mRNA fate of PTC-mutated β-globin mRNA can be influenced escapes nonsense-mediated decay. We demonstrate that by the promoter from which it is expressed (Enssle et al., cells distinguish a premature termination codon within 1993). (iii) In some cases, PTCs have been reported to the β-globin mRNA from the physiological translation induce alternative splicing events (Naeger et al., 1992; termination codon by a two-step specification mechan- Dietz et al., 1993; Dietz and Kendzior, 1994; Lozano ism. According to the binary specification model pro- et al., 1994; Dietz, 1997). (iv) Intronic sequences have posed here, the positions of splice junctions are first been implicated to play a role in NMD of mutant TPI tagged during splicing in the nucleus, defining a stop mRNAs (Cheng et al., 1994). (v) The relative position of codon operationally as a premature termination codon a PTC with regard to the exon/intron structure of the by the presence of a 3 splicing tag. In the second step, unspliced pre-mRNA has been shown to play a crucial cytoplasmic translation is required to validate the role. mRNAs with a PTC in the last exon are usually not 3 splicing tag for decay of the mRNA. This model subject to NMD (Baumann et al., 1985; Baserga and explains nonsense-mediated decay on the basis of con- Benz, 1988; Urlaub et al., 1989; Thein et al., 1990; ventional molecular mechanisms and allows us to Mashima et al., 1992; Enssle et al., 1993; Belgrader and propose a common principle for nonsense-mediated Maquat, 1994; Cheng et al., 1994; Carter et al., 1996). (vi) decay from yeast to man. The stability of wild-type and PTC-mutated cytoplasmic Keywords: dominant β-thalassemia/mRNA stability/ mRNA was reported to be similar in transfected cells nonsense-mediated decay/translation/3-UTR splicing (Humphries et al., 1984; Baserga and Benz, 1992; Cheng and Maquat, 1993; Lozano et al., 1994). On the other hand, there are many lines of evidence that implicate a cytoplasmic mechanism in NMD. (i) In Introduction transgenic mice with a PTC-mutated human β-globin gene, Nonsense and frameshift mutations introduce premature RNA degradation products were found in the cytoplasm of translation termination codons (PTCs) into the open read- erythroid bone marrow cells (Lim and Maquat, 1992; Lim ing frames (ORFs) of the affected mRNAs and are common et al., 1992). (ii) A translational ORF starting with an causes of genetic disorders. Surprisingly, PTCs usually AUG is necessary for NMD (Naeger et al., 1992; Simpson direct the affected mRNAs to rapid degradation, a process and Stoltzfus, 1994). (iii) NMD can be suppressed by termed nonsense-mediated mRNA decay (NMD). The co-transfecting appropriate suppressor tRNAs (Takeshita physiological importance of NMD is related to the reduc- et al., 1984; Belgrader et al., 1993) and (iv) by inhibiting tion in the synthesis of C-terminally truncated proteins, global translation (Qian et al., 1993; Lozano et al., 1994; thus avoiding dominant-negative effects of non-functional Menon and Neufeld, 1994; Carter et al., 1995), or (v) by polypeptides (Maquat, 1995). alterating the structure of the 5-untranslated region PTCs in human β-globin mRNA represent a clinically (5-UTR) of a PTC-containing mRNA to inhibit translation well-documented example of this beneficial NMD effect. in cis (Belgrader et al., 1993; Kugler et al., 1995). 3484 © Oxford University Press Binary specification of nonsense codons Although in principle, both nuclear and cytoplasmic mechanisms could cooperate in NMD, the difficulties in reconciling this complex set of findings have recently led to contradictory models for NMD in mammals. Some of these models involve unorthodox features of gene expression such as nuclear scanning of the ORF (Urlaub et al., 1989; Dietz et al., 1993; Aoufouchi et al., 1996; Carter et al., 1996; Li et al., 1997). In this report, we have analyzed NMD of human β-globin mRNA. Exploiting specific informative manipulations of mRNAs with and without PTCs, we show that nuclear splicing and cyto- plasmic translation co-operate to enact a mechanism that distinguishes physiological from premature translation termination codons. The proposed binary specification model not only explains the findings described here, but can reconcile most experimental data on NMD in mammalian cells on the basis of molecular mechanisms that are consistent with the conventional understanding of the gene expression pathway. Results Sequence context is not sufficient for PTC versus physiological translation termination codon definition Both translation initiation and physiological translation termination (Ter) codons display typical sequence contexts that affect the efficiency of their function (Kozak, 1986; McCaughan et al., 1995). We first examined whether PTCs were distinguished from Ter by their direct sequence context or by the position of the translation termination codon relative to other cis-acting elements. The first set of constructs was designed to analyze the role of the Ter sequence context (Figure 1A). A comparison of the wild-type β-globin gene with that bearing a naturally Fig. 1. Sequence context does not exert a dominant influence to occurring nonsense mutation at position 39 (PTC 39) was distinguish operationally a premature from a physiological termination codon. (A) Human β-globin gene constructs used for transfection. The used to document NMD in our HeLa cell transfection structural modifications and the nomenclature of the constructs are system. Relative to wild-type, PTC 39 was expressed shown diagrammatically. The ORF is represented by boxes, and the typically at a 4- to 5-fold lower level (Figure 1B, lanes 1 untranslated regions and introns by lines (see Materials and methods and 2). When PTC 39 was replaced by the physiological for details). (B) Northern blot of cytoplasmic HeLa cell RNA and β-globin termination codon Ter including its direct hybridization with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after sequence context of 15 flanking nucleotides on both sides normalization for transfection efficiency. (PTC 39-Ter; Figure 1A), NMD was observed, albeit to a somewhat lesser extent (Figure 1B, compare lanes 1 and 3). Replacement of PTC 39 by the physiological stop 1), suggesting that the position of PTC/Ter within the codon and its 30 immediately flanking nucleotides is thus penultimate exon may play an important role. not sufficient to bypass NMD. When the exon 2 sequences 3 of PTC 39 were replaced by the entire exon 3 to A minimal distance of a PTC mutation from the transfer a wider Ter sequence context into the vicinity of final intron is critical for nonsense-mediated decay PTC 39 (Figure 1A, PTC 39-E2/3), mRNA expression In most cases, PTCs within the last exon fail to specify remained low and was comparable with the level for PTC NMD. A distinct positional boundary for NMD at ~50 39-Ter (Figure 1B, compare lanes 1 and 4). Therefore, nucleotides upstream of the final intron previously has exon 3 does not contain dominant sequences that are been established for PTCs in TPI mRNA. If a PTC is capable of redefining PTC 39 as a bona fide Ter, or of located upstream of that boundary, the affected mRNA is redirecting the mRNA to a non-NMD pathway. Construct degraded. If the PTC is closer to the intron, the mRNA WT-E3-E3-Ter and the control WT-E3-E3-CAA remains stable (Cheng et al., 1990, 1994; Cheng and (Figure 1A) were designed to examine the effect of Ter Maquat, 1993). In contrast, the T-cell receptor-β (TCR-β) in its physiological exonic context but in an upstream gene does not exhibit a similar boundary: PTCs as close position. The accuracy of the predicted splice events was as eight nucleotides upstream from the last intron specify confirmed by cDNA sequencing of RT–PCR products NMD (Carter et al., 1996). The second set of constructs (data not shown). Both mRNAs were expressed at high with nested mutations (Figure 2A) was therefore designed levels (Figure 1A, compare lanes 5 and 6 with lane to define whether the known positional effects of PTC 3485 R.Thermann et al. Fig. 2. The distance of the translation termination codon from the final splice junction is critical for NMD. (A) Schematic representation of human β-globin gene constructs with the position of nested PTC mutations. (B) Northern blot of cytoplasmic RNA of HeLa cells transfected with the constructs shown in (A) and hybridization with β-globin and CAT cRNA probes. (C) The column diagram summarizes the results of three independent transfection experiments. Boxes indicate the mean and bars the maximum and minimum values obtained after normalization for transfection efficiency. (D) In-frame deletion and insertion constructs shifting the position of PTC 82 towards (PTC 82Δ) and PTC 88 away from the splice junction (PTC 88). Construct WT controls for destabilizing elements within the 27 nucleotide insertion. (E) Northern blot of cytoplasmic RNA of transfected HeLa cells and hybridization with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after normalization for transfection efficiency. mutations in the human β-globin gene (Baserga and Benz, are all expressed at much higher or normal levels. PTCs 1988; Thein et al., 1990; Enssle et al., 1993) are of the in the first and in the 5 region of the second exon thus TPI or the TCR-β type. direct mRNAs to the NMD pathway, whereas PTCs β-Globin mRNAs with a PTC at codon 26 (in exon 1) towards the 3 end of the second exon and in the third and at codons 39, 75 and 82 within the 5 two-thirds of exon are associated with high level mRNA expression. exon 2 result in NMD, and are expressed at low levels This polarity exhibits a clear boundary between 48 and (Figure 2B and C, compare lane 1 with lanes 2–5). In 66 nucleotides upstream of intron 2 (Figure 2), suggesting contrast, mRNAs bearing PTCs towards the 3 end of that the distance between the PTC and the exon 2–intron 2 exon 2 at codons 88, 91, 95, 98 and 103 (Figure 2B and splice junction may be important. This was tested directly C, lanes 6–10) and those with PTCs in exon 3 at codons with a set of deletion and insertion constructs (Figure 2D) 106, 107, 114 and 121 (Figure 2B and C, lanes 11–14) shifting the PTC towards or away from the boundary. 3486 Binary specification of nonsense codons Construct PTC 82Δ differs from PTC 82 by a 27 nucleotide in-frame deletion, and PTC 88 differs from PTC 88 by a 27 nucleotide in-frame insertion. In PTC 82Δ, the distance between PTC 82 and the splice junction was thus reduced from 66 to 39 nucleotides, which results in a switch from low to high mRNA expression (Figure 2E, compare lanes 3 and 4). In PTC 88, the distance between PTC 88 and the splice junction was increased from 48 to 75 nucleotides, which redirects this mRNA to the NMD pathway (Figure 2E, compare lanes 5 and 6). The wild- type control with the same insertion (WT) is expressed at normal levels (Figure 2E, lane 7), demonstrating that the 27 nucleotide insert per se does not harbor destabilizing elements. The distance between the PTC and the final intron thus represents the reference point for a distinct positional polarity of the TPI type. Splicing in the 3-UTR subverts a wild-type mRNA to nonsense-mediated decay The data described above and previous findings (Carter et al., 1996) indicate the importance of splicing 3 of premature translation termination codons for the NMD pathway. They also suggest the provocative possibility that a wild-type mRNA could be subverted to the NMD pathway by the introduction of a spliceable intron at sufficient distance downstream from Ter within the 3-UTR. The constructs shown in Figure 3A were designed to test this hypothesis directly. Construct WT-SP-Ter contains a heterologous intron plus 15 nucleotides of flanking sequences at both sides 62 nucleotides down- stream of Ter. Importantly, and in contrast to previously reported experimental designs (Carter et al., 1996; Li et al., 1997), the ORF was not altered by this manipulation, and specific cis-acting intronic effects such as those reported for TPI RNA (Cheng et al., 1994) were excluded here. The controls contain either just the two 15 nucleotides flanking sequences (WT-spf and PTC 39-spf) or the Fig. 3. Splicing in the 3-UTR directs a wild-type mRNA to the NMD identical intron without splice donor and acceptor signals pathway. (A) Human β-globin gene constructs with modifications of the 3-UTR. The presence or absence of GU...AG motifs indicates (WT-sp). Analysis of these constructs (Figure 3B) reveals whether or not the intron was furnished with a splice donor and that: (i) controls WT-spf and PTC 39-spf are comparable acceptor site (see Materials and methods for details). (B) The Northern with wild-type and PTC 39, respectively (Figure 3B, blot of cytoplasmic RNA of transfected HeLa cells was hybridized to compare lanes 1 and 2); (ii) the presence of an unspliceable β-globin and CAT cRNA probes. The percentage values refer to the intron in construct WT-sp does not influence the level of mean of three independent experiments after normalization for transfection efficiency. wild-type mRNA expression (Figure 3B, compare lanes 1 and 6; note the slower migration of WT-sp mRNA); and (iii) the spliceable intron of construct WT-SP-Ter is intron, i.e. 3 of the positional boundary defined in Figure 2 removed from the mature mRNA (Figure 3B, compare (Figure 3A). Neither mRNA is subject to NMD (Figure 3B, lanes 3 and 6), resulting in NMD of the WT-SP-Ter compare lane 1 with lanes 4 and 5), demonstrating that: mRNA, i.e. the physiological stop codon is operationally (i) the positional boundary established for the natural final redefined as a PTC by the presence of the downstream intron of the β-globin gene (Figure 2) can be translocated intron, and an mRNA with a wild-type ORF is directed when the exon–intron structure of the gene is altered; (ii) to the NMD pathway. Note that WT-spf and WT-SP-Ter the same positional requirements appear to apply to 3- encode identical mature mRNAs, with the latter but UTR introns; and (iii) a 3-UTR intron must be spliceable not the former being subject to NMD. The functional to subvert a wild-type mRNA to NMD. In conclusion, relationship between the position of Ter and the 3 splice NMD depends in cis on the position of the premature or event was examined further: (i) with construct WT- physiological translation termination codon relative to the SP-CAA that differs from WT-SP-Ter by a Ter→Gln most 3 spliceable intron. These findings also unequivoc- (TAA→CAA) mutation, thus removing the translational ally identify a nuclear component of the NMD pathway. termination signal 5 of the intron inserted into the 3-UTR; and (ii) with construct WT-SP-CAA-Ter that Nonsense-mediated decay of mutant mRNAs differs from WT-SP-CAA by a novel Ter 18 nucleotides requires their cytoplasmic translation downstream of the CAA, thus repositioning the translation Findings from several laboratories have implicated a role termination codon to only 44 nucleotides 5 of the novel for translation or ‘translation-like’ mechanisms in NMD 3487 R.Thermann et al. (see above). In studies employing general translation of human β-globin genes (constructs shown in Figure 4C) inhibitors (Qian et al., 1993; Lozano et al., 1994; Menon thus allows the role of cytoplasmic translation in NMD and Neufeld, 1994; Carter et al., 1995) or suppressor to be probed specifically. A wild-type β-globin mRNA tRNAs (Takeshita et al., 1984; Belgrader et al., 1993), bearing an IRE within its 5-UTR (WT-IRE) is stable possible pleiotropic effects of these approaches complicate regardless of the availability of iron (Figure 4D, lanes 1 the interpretation of the data. Furthermore, these and other and 3). In the presence of iron, translation is readily studies using specific translational inhibitory alterations detectable by immunoblotting (Figure 4D, lane 5), whereas of the 5-UTRs of PTC mRNAs (Belgrader et al., 1993; hardly any protein can be identified in iron-deficient cells Kugler et al., 1995) could not distinguish unambiguously (Figure 4D, lane 2). The iron-dependent regulation of between conventional cytoplasmic translation and the less translation is lost when a point-mutated non-functional orthodox nuclear translation-like mechanisms that are a IRE is introduced into the β-globin 5-UTR: the mRNA critical feature of the nuclear scanning model (Carter is translated constitutively (Figure 4C, construct WT- et al., 1996; Li et al., 1997). IREΔC, Figure 4E lanes 4 and 7) and also is stable The interaction between iron-responsive elements (Figure 4D, lanes 5 and 7). In contrast, PTC 39 mRNA (IREs) and iron-regulatory proteins (IRPs) enables the bearing a functional IRE (Figure 4C, construct PTC 39- specific translational regulation of mRNAs bearing IREs IRE) is subject to NMD when translation is enabled, but in their 5-UTRs (Klausner et al., 1993; Hentze and Kuhn, escapes NMD in iron-deficient cells where IRP binding 1996). In iron-deficient cells, IRP binding to an IRE inhibits the cytoplasmic translation of the mutated mRNA specifically inhibits the formation of a 48S pre-initiation (Figure 4). The NMD escape is due to translational complex at the 5 end of the mRNA, thus blocking an inhibition rather than to iron deficiency per se, because a early step of translation (Gray and Hentze, 1994). Elevation PTC 39 mRNA bearing a non-functional IRE (Figure 4C, of intracellular iron levels inactivates IRP binding and constructs WT-IREΔC and PTC 39-IREΔC) is subject to thus permits the translation of the mRNA. IRP-1 (98 kDa) NMD in both iron-replete and iron-deficient cells and IRP-2 (105 kDa) are expected to be expressed exclus- (Figure 4D, compare lanes 3 with 4, 5 with 6 and 7 with 8). ively in the cytoplasm, because they are too large to The β-globin antibodies fail to detect any polypeptide traverse the nuclear membrane passively and they do not expressed from the PTC 39 mRNAs, hence it was not contain a known nuclear localization signal for active possible formally to demonstrate translational control of nuclear import. This spatial restriction was assessed dir- these mRNAs by immunoblotting (Figure 4E, lanes 1, 3, ectly by immunofluorescence studies of cells with an IRP-1- 6 and 8). We therefore analyzed the polysome association specific antibody (Pantopoulos et al., 1995) that show a (Figure 5) of the mRNAs expressed from the constructs cytoplasmic signal excluding nuclei (Figure 4A). Although shown in Figure 4C. This analysis confirms that: (i) wild- IRP-1 is the predominant IRP in mammalian cells, this type β-globin mRNAs are associated preferentially with analysis does not exclude the presence of IRP-2 in the heavier, and PTC 39 mRNAs with lighter polysomes when nucleus. We therefore performed gel retardation experi- translation is enabled, reflecting the differences in length ments with nuclear and serial dilutions of cytoplasmic of the respective ORFs (Figure 5A, C and D); and (ii) the extracts from HeLa cells with an IRE probe (Pantopoulos IRE–IRP interaction results in an accumulation of the et al., 1995) that forms electrophoretically co-migrating wild-type and the PTC 39 mRNAs at the top of the gradient, complexes with IRP-1 and IRP-2 with equal affinity showing that they are not associated with ribosomes (Hentze and Ku¨hn, 1996). Binding activity was readily (Figure 5B). We conclude that specific inhibition of detectable with as little as 0.07 μg of cytoplasmic protein ribosome association and cytoplasmic translation in cis (Figure 4B, lanes 1–8). In contrast, no signal was detectable prevents the degradation of PTC 39 mRNA. Thus, cyto- in the nuclear fraction, even when 25 μg of nuclear protein plasmic translation of the mutated mRNA is necessary (i.e. a 350-fold higher amount than 0.07 μg) was for NMD. used (Figure 4B, lanes 12–14). To exclude non-specific inhibitory effects on complex formation exerted by com- Discussion ponents in the nuclear extract, we compared complex formation between the IRE probe and recombinant human Cells have evolved mechanisms to distinguish wild-type IRP-1 in assays with or without 25 μg nuclear protein from mutated mRNAs, and to degrade the latter. These (Figure 4B, lanes 15 and 16). Taken together, these data mechanisms can operate when PTCs, but not missense show that IRP-1 and IRP-2 can be identified readily in mutations, disrupt the ORF. Nonsense-mediated decay is the cytoplasm and that neither appears to be present in an ill-understood, yet biologically and medically import- the nucleus. The introduction of an IRE into the 5-UTR ant, mechanism as demonstrated by mutations in the smg Fig. 4. Cytoplasmic translation is necessary to direct a PTC-mutated mRNA to the NMD pathway. (A) Immunofluorescence of B6 cells with pre- immune serum (left) and IRP-1-specific antibody (anti-IRP; right). (B) Gel retardation analyses with an IRE probe and serial dilutions of cytoplasmic (lanes 1–11) and nuclear (lanes 12–14) protein from fractionated HeLa cells and recombinant human IRP-1 together with (lane 15) or without (lane 16) nuclear protein. The IRE probe forms co-migrating complexes (arrow) with IRP-1 and IRP-2. (C) Wild-type and PTC 39-mutated human β-globin gene constructs with functional (IRE, bold stem loop) and non-functional (IREΔC, thin stem loop) IREs in the 5-UTR (see Materials and methods for details). (D) Northern blot of cytoplasmic RNA of transfected HeLa cells that were grown in medium either supplemented (iron)or not (iron–) with heme arginate as an iron source. Translation of the constructs containing a functional IRE could thus be regulated specifically (translation– or translation). Hybridization was performed with β-globin and CAT cRNA probes. The percentage values refer to the mean of three independent experiments after normalization for transfection efficiency. (E) Immunoblot of total protein of HeLa cells transfected with the constructs shown in (C). The medium was either supplemented (iron) or not (iron–) with heme-arginate. Hybridization was performed with human β-globin- and vigilin-specific antibodies. Vigilin is a protein that is expressed independently of the availability of iron and served as a control for equal loading of the lanes. 3488 Binary specification of nonsense codons 3489 R.Thermann et al. Fig. 5. Translation-competent wild-type and PTC 39 β-globin mRNAs are associated with polysomes. The top panel shows representative gradient profiles of HeLa cells transiently transfected with the constructs described in Figure 4C. The number of the fractions that were used for Northern blotting are indicated below the gradient profiles. The individual panels show Northern blots of the different fractions after hybridization with β-globin- or β-actin-specific probes. The left side represents cells that were transfected with wild-type and the right side those transfectd with a PTC 39-mutated construct. The table indicates the modification of the 5-UTR, the iron status of the cells and the specific translational competence of the transfected β-globin gene. (a) Functional IRE with iron supplementation resulting in translational competence. (b) Functional IRE without iron supplementation resulting in translational incompetence. (c) Non-functional IRE with iron supplementation. (d) Non-functional IRE without iron supplementation. (e) Representative blots hybridized with a β-actin-specific probe controlling for the quality of the gradient and general translational competence of the transfected cells. genes that are required for NMD in Caenorhabditis PTC-mutated mRNA degradation is independent of the elegans, and display synthetic lethality with nonsense recognition of translational sense within the nucleus but mutations in the unc-54 myosin heavy chain B gene (Pulak occurs while the mRNA is still physically associated with and Anderson, 1993), or by forms of β-thalassemia with the nucleus. Those PTC-mutated mRNA molecules that symptomatic heterozygotes and a dominant mode of escape the nucleus-associated degradation mechanism are inheritance (Thein et al., 1990; Hall and Thein, 1994). stable once they have reached the cytoplasm (Maquat, NMD involves different phases: discrimination of a 1995). In contrast, different variations of ‘nuclear scan- PTC from the physiological stop codon, commitment of ning’ models postulate the existence of a (ribosome- the mutated mRNA to NMD, and nucleolytic degradation like) structure that scans nuclear (pre-)mRNAs for their of the mRNA. Much work has focused on the early phases translational sense and induces either altered splicing to of NMD. A complex array of findings obtained from avoid the PTC or the degradation of PTC-mutated mRNAs various mammalian systems has led to the formulation of within the nucleus (Urlaub et al., 1989; Dietz et al., 1993; several models. In spite of significant progress to distin- Dietz and Kendzior, 1994; Aoufouchi et al., 1996; Carter guish between these models, at least two mutually exclus- et al., 1996; Li et al., 1997). ive models remain (Urlaub et al., 1989; Maquat, 1995; Our strategy based on PTC mutations of the β-globin Carter et al., 1996). Neither of the two models can fully gene aimed to unambiguously identify necessary and account for the available data (see below). One central sufficient components during the discrimination and com- issue revolves around the question of whether NMD is a mitment phase of NMD. The first set of experiments nuclear, a nucleus-associated or a cytoplasmic mechanism. addresses the role of splicing as a nuclear component of The analyses of the TPI gene suggested that PTC recogni- the NMD pathway. In the TPI gene, a sequence element tion takes place after splicing and that intronic sequences in the final intron was implicated in marking the mRNAs provide a mark that translating ribosomes must approxi- in cis. NMD depends on the distance between this sequence mate or traverse in order to confer mRNA stability element and the PTC mutation, but not on splicing per se (Maquat, 1995). However, the inhibition of translation (Cheng et al., 1994). In contrast, an analysis of the TCR-β was shown not to destabilize the mRNA (Belgrader et al., gene implicated a functional role for splicing but no distinct 1993). According to the co-translational export model, positional effect (Carter et al., 1996). The interpretation of 3490 Binary specification of nonsense codons the TCR-β data is complicated by two aspects; the tion is blocked, the tags are not encountered and the duplication or deletion of exonic and intronic sequences mRNA remains stable (Figure 6, pathway b). If the tags of that particular gene significantly alters the ORFs of the are encountered by elongating ribosomes within the ORF different TCR-β-related mRNAs, and the use of a TCR- (as would happen with tags representing all former introns β intron rather than a heterologous one. It is difficult, upstream of termination codons), the mRNA is not valid- therefore, to exclude a possible effect of specific cis-acting ated for the NMD pathway. In this case, the tags may intronic or exonic sequences that have been implicated in remain ‘neutral’, could be ‘approved’ or removed, and affecting mRNA stability in other mammalian systems the mRNA is not degraded (Figure 6, pathway a). If (Cheng et al., 1994; Liu and Mertz, 1996). mRNA translation occurs and a tag 3 of the translation In the experiments shown in Figure 3, a spliceable stop codon is identified (as would happen with PTCs in intron was inserted into the 3-UTR of the human all but the final exon and with 3-UTR intron tags), the β-globin mRNA without changing the structure of the tag is validated for NMD and the mRNA targeted for mature mRNA (compare WT-spf with WT-SP-Ter). More- degradation (Figure 6, pathway c). If PTC mutations are over, the intron used is unrelated to the β-globin gene, located at an insufficient distance from the final tag avoiding the inadvertant inclusion of confounding homo- (Figure 2) (Baumann et al., 1985; Urlaub et al., 1989; logous cis-acting elements. The results demonstrate that Thein et al., 1990; Mashima et al., 1992; Enssle et al., a structurally wild-type mRNA without any changes in its 1993; Belgrader and Maquat, 1994; Cheng et al., 1994), ORF is directed to the NMD pathway by a splicing event this may be (falsely) ‘approved’ or removed by the leading in the 3-UTR (Figure 3). Downstream splicing can edge of the elongating ribosome, which thus plays an therefore serve as a sufficient means for redefining the mRNA protective role. The factor or the complex that physiological translation stop codon Ter as a PTC. The validates downstream tags for NMD remains to be identi- operational specification of a PTC includes the requirement fied, but could well represent (components of) a ribosomal for a minimal distance between the termination codon and post-termination complex (Ruiz-Echevarria et al., 1998). the 3-splicing event (Figure 2). This position dependence It is possible that such a complex could contain homologs displays a clear boundary similar to that described for the of the UPF proteins (Applequist et al., 1997) which are TPI gene (Cheng et al., 1994). The potential mechanistic required for NMD in yeast and, interestingly, have been basis of the boundary effect is discussed below. shown to be ribosome associated (Ruiz-Echevarria The other issue that we directly addressed is the et al., 1996). specific role of translation in the cytoplasm. Whereas the This binary specification model can explain or account introduction of hairpins into the 5-UTR or the use of for many previous, seemingly contradictory findings that suppressor tRNAs (Takeshita et al., 1984; Belgrader et al., were decribed above. Our model can also explain a recent 1993) may interfere with the function of the notional report demonstrating that translation re-initiation after a nuclear ribosome-like structure that represents a critical premature stop codon results in the escape of TPI mRNA feature of the nuclear scanning model (Carter et al., 1996; from the NMD pathway (Zhang and Maquat, 1997). One Li et al., 1997), the IRE/IRP system specifically regulates of the most puzzling aspects of NMD has been the nuclear mRNA translation in the cytoplasm (Figure 4A and B; or cytoplasmic site of PTC identification and the frequently Klausner et al., 1993; Hentze and Kuhn, 1996). The design nucleus-associated site of mRNA degradation. Our find- of the experiments shown in Figures 4 and 5 also excludes ings functionally assign the relevant translational activity possible pleiotropic effects of general inhibitors of protein to the cytoplasm, and thus do not support some of the synthesis. Moreover, our strategy exploits the functional unconventional aspects of the nuclear scanning model. (IRE/IRP) rather than preparative (subcellular fractiona- The nuclear association of PTC mRNA degradation in tion) definition of the cytoplasmic compartment. This some (but not all) cases of NMD is consistent with the allows us to distinguish whether the influence of transla- binary specification model. We suggest that some mRNPs tional features on the NMD pathway is conferred by the undergoing nuclear export have an inherent transient conventional cytoplasmic translation apparatus or by a affinity for the preparatively defined ‘nuclear fraction’ notional translation-like nuclear mechanism (Carter et al., while being accessible to the cytoplasmic translation 1996; Li et al., 1997) and also avoids uncertainties apparatus on the cytoplasmic side of the nuclear envelope. about the purity of nuclear preparations. We show that The residual levels of PTC-mutated mRNAs of com- conventional cytoplasmic translation is a necessary com- monly 10–30% of the wild-type levels may be explained ponent to validate a PTC-mutated β-globin mRNA for the by a 100% efficiency of tagging and/or by tag dissoci- NMD pathway (Figures 4 and 5). ation. The normal cytoplasmic stability of the remaining Our results suggest a binary specification model for the 10–30% PTC mRNAs that bear no tag is predicted by the discrimination and commitment phase of NMD (Figure 6). binary specification model. An observation that can be In the first step, splicing induces the tagging of the position explained by neither the binary specification nor by any of (former) introns. It is conceivable that such a tag could of the other models is the effect of PTC mutations on be a spliceosome-associated protein that remains bound splice-site choice (Dietz et al., 1993; Dietz and Kendzior, to, and escorts the mRNA into the cytoplasm (Visa et al., 1994; Dietz, 1997) or the splicing process per se (Lozano 1996). In the exceptional cases where additional specific et al., 1994; Aoufouchi et al., 1996), which may be cis-acting sequences (Cheng et al., 1994) can substitute induced by another PTC-dependent mechanism. for the presence of a spliceable downstream intron (such In yeast, NMD has been perceived to be enacted by a as the TPI mRNA), the tag (protein) may be recruited by mechanism which is profoundly different from that used these specific sequences. During or after nuclear export, in multicellular eukaryotes. NMD in yeast is splicing the second step requires cytoplasmic translation. If transla- independent, cytoplasmic and apparently dependent on a 3491 R.Thermann et al. Fig. 6. The binary specification model of NMD. The details of the model are described in the text. at the 3 end. In construct WT-E3-E3-Ter, the entire exon 2 is replaced specific ‘downstream sequence element’ (DSE) (Ruiz- in-frame by a PCR fragment containing the entire coding sequence of Echevarria et al., 1996). Interestingly, the DSE is thought exon 3, termed exon 3*. Construct WT-E3-E3-CAA differs from WT- to require recognition by a ribosomal post-termination E3-E3-Ter by a TAA→CAA mutation of the translation termination complex (Ruiz-Echevarria et al., 1998). The binary speci- codon of exon 3*. Constructs PTC 26, PTC 75, PTC 82, PTC 91, PTC 95, PTC 98, PTC fication model provides a conceptional bridge between 103, PTC 106, PTC 107, PTC 114 and PTC 121 were created by NMD in yeast and mammals. Hypothetically, the mammal- site-directed mutagenesis (Hagemeier, 1996). Construct PTC 88 was ian splicing tag could be substituted by the DSE (or a generated by replacing the sequences in the wild-type construct by a DSE-binding protein) in Saccharomyces cerevisiae, where PCR fragment derived from a proband with heterozygous β-thalassemia introns are rare. All subsequent steps of NMD in yeast carrying a ΔG frameshift mutation at codon 82/83, which results in a TGA PTC at codon 88 (Vetter et al., 1997). and man could then conceivably be alike. Further charac- Constructs PTC 82Δ and PTC 88 were generated by site-directed terization of the splicing tag and of the function of the mutagenesis (Hagemeier, 1996). Construct PTC 82Δ differs from con- DSE represent experimental tests of this hypothetical struct PTC 82 by a 27 bp deletion of codons 91–99. In construct PTC prediction. 88, a 27 bp fragment was inserted into construct PTC 88 in-frame at codon 99. Construct WT differs from PTC 88 by the absence of the In conclusion, we present specific evidence that the PTC 88 mutation. discrimination and commitment to degrade PTC-mutated In the constructs shown in Figure 3A, the ApaI and XhoI sites of the human β-globin mRNAs involves the co-operation of Bluescript polylinker were deleted. New ApaI and XhoI restriction sites conventional nuclear and cytoplasmic molecular mechan- were then inserted by site-directed mutagenesis into the 3-UTR at a isms. Our findings and their interpretation raise the pos- position 41 bp downstream of the physiological stop codon. Construct WT-SP-Ter was generated by introducing the MINX-intron (Zillmann sibility of a common principle for NMD from yeast to et al., 1988) with 30 bp flanking sequences including its splice donor man. Further implications of our findings relate to the and acceptor sites into the ApaI and XhoI sites in the 3-UTR. Construct common usage of introns in the 3-UTRs of expression WT-SP-CAA differs from WT-SP-Ter by a TAA→CAA mutation of Ter. vectors in molecular biology and gene therapy, which may The novel in-frame Ter of this construct is located 41 bp downstream of the inserted intron. Construct WT-SP-CAA-Ter differs from WT-SP- warrant re-evaluation. CAA by a new TAA that was created by site-directed mutagenesis at an in-frame position 18 bp downstream of the former Ter. Construct WT-sp differs from construct WT-SP-Ter by the absence of the intron flanking Materials and methods sequences including the splice donor and acceptor sites. Construct WT- Plasmid constructs spf differs from WT-SP-Ter by the absence of the intron, but retains the The constructs shown in Figures 1A–3A contain a 4.4 kb human intron flanking sequences in the 3-UTR. In construct PTC 39-spf, the β-globin gene with a linked SV40 enhancer inserted as a NotI fragment WT CAG at position 39 of construct WT-spf was replaced by a TAG PTC. into pBlueskriptSK II (Stratagene) (Enssle et al., 1993). The wild-type The constructs shown in Figure 4A contain the human β-globin gene and the PTC 39 constructs are derived from a healthy proband and from fragment as described above in a pGEM-5Zf () expression vector a patient with homozygous β-thalassemia, respectively. Construct PTC (Promega). In constructs WT-IRE and PTC 39-IRE, a 35 bp fragment 39 was modified by overlap extension PCR (Pogulis et al., 1996) to containing a functional IRE (Hentze et al., 1987) was inserted by site- generate construct PTC 39-Ter, in which the stop codon at position 39 directed mutagenesis (Hagemeier, 1996) into the 5-UTR 37 nucleotides (TAG) and the 15 bp flanking both sides were replaced by the physiologi- downstream of the cap structure of the wild-type and the PTC 39 cal stop codon (TAA) including 30 bp of its immediate flanking constructs (Figure 1A) (Goossen and Hentze, 1992). Constructs WT- sequences. In construct PTC 39-E2/3, exon 2 sequences 3 of PTC 39 IREΔC and PTC 39-IREΔC differ from the IRE constructs by a deletion were replaced in-frame by a PCR fragment containing exon 3 including of a single cytosine residue, which functionally inactivates the IRE the physiological translation termination codon and a novel splice donor (Hentze et al., 1988). 3492 Binary specification of nonsense codons The structure of all constructs was confirmed by DNA sequence was added to each fraction to a final concentration of 0.5%. Subsequently, analysis of the insert including the promotor and the entire transcribed fractions were subjected to phenol extraction. The extracted RNA was region from the cap site to the polyadenylation signal. analyzed by Northern blotting as described above. Transfection efficiency was determined by assaying for CAT activity. Cell culture and transfections HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) Immunoblotting under standard conditions. Cells were transiently transfected by calcium Cytoplasmic protein was extracted after lysis in cold lysis buffer plus phosphate precipitation (Ausubel et al., 1994) with 20 μgofthe 10 μg/ml PMSF as described for polysome gradients. Equal amounts of test constructs and 15 μgofan Escherichia coli chloramphenicol cells were analyzed on 5–15% SDS–polyacrylamide gradient gels. acetyltransferase (CAT) gene cloned into the multiple cloning site of the The proteins were electroblotted onto PVDF membranes (Immobilon, plasmid pSG5 (Green et al., 1988), which served as a control for Schleicher and Schuell) using a semi-dry apparatus. Membranes were transfection efficiency. Cells were washed after 20 h and harvested stained with Ponceau S to ensure that the transfer was complete and 24 h later. uniform, and then blocked with 5% skimmed milk in Tris-buffered saline For the experiments with the IRE-containing constructs, culture media (TBS) plus 0.05% Tween-20. The primary β-globin antibody was raised were supplemented with 100 μM of the iron source heme arginate in rabbits against the N-terminal 13 amino acids of human β-globin (Leiras, Turku, Finland) 8 h after washing. For iron starvation, it was (Eurogentec, Seraing, Belgium). Immunoblotting was performed using sufficient to grow the cells in unmodified medium that contained only 2 standard conditions and a 1:2000 dilution of the antibody. The vigilin 0.25 μmol of Fe . At 16 h after addition of heme arginate, i.e. 24 h antibody was a rabbit antiserum against the 15 C-terminal amino acids after transfection, the cells were harvested. of human vigilin and was diluted 1:6000. The secondary antibody (anti- rabbit IgG coupled to horseradish peroxidase; Dianova) was diluted Isolation and analysis of RNA 1:3000. Specific staining was detected using a bioluminescence kit Total cytoplasmic RNA was purifed from the supernatant of homogenized (Boehringer Mannheim) according to the manufacturer’s instructions. cells with TRIzol reagent (Gibco-BRL). Northern blot analysis was performed as previously described (Enssle et al., 1993) with 1–2 μgof total cytoplasmic RNA or, when fractions of polysome gradients were analyzed, with half of each fraction. Blots were hybridized using in vitro Acknowledgements transcribed P-labeled antisense cRNA β-globin and CAT probes. The template for the β-actin probe was a 383 bp human β-actin cDNA Heme arginate was generously provided by Leiras, Turku, Finland. We PCR fragment, which was subjected to single strand PCR in the presence thank Dr Kostas Pantopoulos (EMBL, Heidelberg) for performing the of [ P]dCTP (Konat et al., 1994). Hybridization was carried out at experiment shown in Figure 4A. This study was supported by the 65°C overnight. Deutsche Forschungsgemeinschaft (DFG). The signals were quantified by imaging in a GS-250 Molecular Imager (Bio-Rad). β-Globin mRNA levels are expressed as a percentage of wild-type after normalization for transfection efficiency. References Indirect immunofluorescence Aoufouchi,S., Yelamos,J. and Milstein,C. (1996) Nonsense mutations B6 cells were grown on coverslips in DMEM. Coverslips were washed inhibit RNA splicing in a cell-free system: recognition of mutant twice with phosphate-buffered saline (PBS), and cells were permeabilized codon is independent of protein synthesis. Cell, 85, 415–422. with 0.5% Triton X-100 in CKS buffer (100 mM NaCl, 300 mM sucrose, Applequist,S.E., Selg,M., Raman,C. and Jack,H.M. (1997) Cloning and 10 mM PIPES pH 6.8, 3 mM MgCl , 1 mM EGTA) containing 0.1 mM characterization of HUPF1, a human homolog of the Saccharomyces phenylmethylsulfonyl fluoride (PMSF) for 2 min on ice. Subsequently, cerevisiae nonsense mRNA-reducing UPF1 protein. 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Baserga,S.J. and Benz,E.J.,Jr (1992) Beta-globin nonsense mutation: Gel retardation experiments deficient accumulation of mRNA occurs despite normal cytoplasmic These were performed exactly as previously described (Pantopoulos and stability. Proc. Natl Acad. Sci. USA, 89, 2935–2939. Hentze, 1995). Baumann,B., Potash,M.J. and Kohler,G. (1985) Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the Cell fractionation mouse. EMBO J., 4, 351–359. Cytoplasmic and nuclear fractions from HeLa cells used for IRE/IRP Belgrader,P. and Maquat,L.E. (1994) Nonsense but not missense bandshift analyses were prepared essentially as described (Kugler et al., mutations can decrease the abundance of nuclear mRNA for the 1995), with the following modifications: the lysis buffer contained 0.5% mouse major urinary protein, while both types of mutations can Triton X-100, 0.2% Na deoxycholate instead of 0.5% NP-40 and was facilitate exon skipping. Mol. Cell. 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Journal

The EMBO Journal – Springer Journals

Published: Jun 15, 1998

Keywords: dominant β‐thalassemia; mRNA stability; nonsense‐mediated decay; translation; 3′‐UTR splicing

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