Post-termination ribosome interactions with the 5'UTR modulate yeast mRNA stability
1999; Springer Nature; Volume: 18; Issue: 11 Linguagem: Inglês
10.1093/emboj/18.11.3139
ISSN1460-2075
Autores Tópico(s)RNA modifications and cancer
ResumoArticle1 June 1999free access Post-termination ribosome interactions with the 5′UTR modulate yeast mRNA stability Cristina Vilela Cristina Vilela Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Carmen Velasco Ramirez Carmen Velasco Ramirez Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Bodo Linz Bodo Linz Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Claudina Rodrigues-Pousada Claudina Rodrigues-Pousada Instituto Gulbenkian de Ciência, Lab. de Genética Molecular, 2780 Oeiras, Portugal Search for more papers by this author John E.G. McCarthy Corresponding Author John E.G. McCarthy Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Cristina Vilela Cristina Vilela Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Carmen Velasco Ramirez Carmen Velasco Ramirez Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Bodo Linz Bodo Linz Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Claudina Rodrigues-Pousada Claudina Rodrigues-Pousada Instituto Gulbenkian de Ciência, Lab. de Genética Molecular, 2780 Oeiras, Portugal Search for more papers by this author John E.G. McCarthy Corresponding Author John E.G. McCarthy Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK Search for more papers by this author Author Information Cristina Vilela1, Carmen Velasco Ramirez1, Bodo Linz1, Claudina Rodrigues-Pousada2 and John E.G. McCarthy 1 1Post-transcriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester, M60 1QD UK 2Instituto Gulbenkian de Ciência, Lab. de Genética Molecular, 2780 Oeiras, Portugal *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:3139-3152https://doi.org/10.1093/emboj/18.11.3139 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A novel form of post-transcriptional control is described. The 5′ untranslated region (5′UTR) of the Saccharomyces cerevisiae gene encoding the AP1-like transcription factor Yap2 contains two upstream open reading frames (uORF1 and uORF2). The YAP2-type of uORF functions as a cis-acting element that attenuates gene expression at the level of mRNA turnover via termination-dependent decay. Release of post-termination ribosomes from the YAP2 5′UTR causes accelerated decay which is largely independent of the termination modulator gene UPF1. Both of the YAP2 uORFs contribute to the destabilization effect. A G/C-rich stop codon context, which seems to promote ribosome release, allows an uORF to act as a transferable 5′UTR-destabilizing element. Moreover, termination-dependent destabilization is potentiated by stable secondary structure 3′ of the uORF stop codon. The potentiation of uORF-mediated destabilization is eliminated if the secondary structure is located further downstream of the uORF, and is also influenced by a modulatory mechanism involving eIF2. Destabilization is therefore linked to the kinetics of acquisition of reinitiation-competence by post-termination ribosomes in the 5′UTR. Our data explain the destabilizing properties of YAP2-type uORFs and also support a more general model for the mode of action of other known uORFs, such as those in the GCN4 mRNA. Introduction The 5′ untranslated region (5′UTR) of eukaryotic mRNA plays a key role in the post-transcriptional regulation of gene expression. Until very recently, attention was focused exclusively on the role of the 5′UTR in controlling translational initiation. Translational initiation exerts strong rate control on gene expression, thereby determining the specific rate of protein synthesis from a given mRNA. Small upstream open reading frames (uORFs) are a feature of at least a few percent of the mRNAs in yeast, plants and mammals (Kozak, 1991; Vilela et al., 1998), and can be important players in translational control. The best characterized example is the regulation of GCN4 translation in Saccharomyces cerevisiae mediated via four uORFs in the 591 nucleotide long leader of this gene (Hinnebusch, 1984, 1996, 1997; Thireos, 1984). In the case of the very short GCN4 uORFs, it is not the encoded product, but rather the nature of the interactions between the mRNA sequence and the translational apparatus, that is relevant for regulation. In contrast, in two other examples of uORF-mediated translational regulation of fungal genes: CPA1 in S.cerevisiae (Werner et al., 1987) and ARG1 in Neurospora crassa (Luo and Sachs, 1996), the uORF-encoded peptides are thought to be involved in the regulatory mechanism. Both classes of uORF function are also identifiable in plant and mammalian systems (Geballe, 1996). A number of studies of heterologous or modified mRNAs in yeast have indicated that uORFs can influence more than translational efficiency (Oliveira and McCarthy, 1995; Ruiz-Echevarria et al., 1996, 1998a; Linz et al., 1997). Moreover, we have estimated that there could be up to a few hundred natural yeast mRNAs containing uORFs (McCarthy, 1998; Vilela et al., 1998). Recent work has shown that uORFs can act as naturally occurring modulators of the stability of such mRNAs (Vilela et al., 1998). In an initial study of the post-transcriptional control of the uORF-containing mRNAs of YAP1 and YAP2, it was determined that the YAP2-type uORFs destabilize the mRNA by a factor of five, which constitutes a major suppressive effect on gene expression. YAP1 and YAP2 encode proteins showing strong homology to AP1-like factors in higher eukaryotes and to Gcn4p in S.cerevisiae (Harshman et al., 1988; Moye-Rowley et al., 1989; Bossier et al., 1993; Wu et al., 1993). YAP1 and YAP2 are also regulatory genes involved in the mechanisms used by the yeast cell to protect itself in situations of stress. For example, overexpression of the two related YAP1 and YAP2 genes confers general stress resistance to a variety of unrelated compounds, including metal ions and various inhibitors and drugs (Hertle et al., 1991; Schnell and Entian, 1991; Haase et al., 1992; Bossier et al., 1993; Wu et al., 1993; Hirata et al., 1994; Lesuisse and Labbe, 1995; Turton et al., 1997). The YAP2 leader has one 6-codon uORF (uORF1) and an overlapping short reading frame (uORF2) of 23 codons (Vilela et al., 1998), while the YAP1 5′UTR has one 7-codon uORF (Moye-Rowley et al., 1989). Previous results (Vilela et al., 1998) indicated the existence of two types of functional influence exerted by the respective YAP uORFs. The YAP2 uORFs act to block ribosomal scanning and also to accelerate mRNA decay, whereas the YAP1 uORF has only a negligibly small inhibitory influence on downstream translation and is not destabilizing. Strikingly, the accelerated decay imposed by the YAP2 uORFs was found to be largely upf1-independent, thus contrasting with the upf-dependent decay seen in aberrant mRNAs containing premature nonsense codons (Jacobson and Peltz, 1996). Here we investigate the mechanistic principles underlying mRNA destabilization by the respective YAP2 uORFs and examine why these uORFs are functionally so different to the YAP1 type of (non-destabilizing) uORF. The results uncover a causal link between the ribosome–mRNA interactions in the 5′UTR and the novel form of accelerated (largely UPF-independent) decay manifested by YAP2 mRNA. We also demonstrate a new role for a eukaryotic initiation factor: eIF2 modulates the destabilizing influence of YAP2 uORF-dependent termination. Moreover, additional experiments with the GCN4 mRNA suggest a unifying working model which can explain the apparent discrepancies between results obtained with different uORF-containing mRNAs. Termination-dependent mRNA destabilization mediated via the 5′UTR is thus shown to constitute a novel principle of post-transcriptional control acting on non-aberrant mRNAs. This in turn means that translation termination on non-aberrant mRNAs has an additional significance beyond that of generating complete polypeptide chains, namely as a site for modulation of gene expression via the mRNA decay rate. Results Two uORFs contribute to the destabilization of YAP2 Earlier work showed that the YAP2 leader imposes both translational inhibition and reduced stability on the YAP2 and LUC mRNAs (Vilela et al., 1998). The first step towards understanding the basis for the destabilization effect is to characterize the roles of the respective uORFs in this 5′UTR. We therefore constructed derivatives of YAP2 in which each of the uORF start codons was mutated to AAG (Figure 1). Analysis of the decay rates of the mRNAs encoded by these constructs revealed that both uORFs contribute to the overall destabilization effect of the YAP2 leader. The total effect of the natural 5′UTR therefore constitutes the combination of the destabilizing influence of the two uORFs, whereby uORF2 acts as a slightly more potent destabilizing element. Figure 1.Both YAP2 uORFs contribute to mRNA destabilization. Northern blots show the results of hybridization using RNA preparations from strains SWP154 (+)(UPF1+) and SWP154 (−)(upf1−) taken during half-life determination experiments. The upper part of the figure shows the decay of the YAP2 mRNA containing either the wild-type 5′UTR or three other derivative leaders. The wild-type endogenous PGK1 mRNA was used as an internal control. The estimated half-life values represent averages of measurements performed using at least three independent sets of RNA preparations (± SD). The lower part of the figure shows the influence of the YAP2 leader on the decay of the ‘mini’-PGK1 mRNA (compared with a control construct lacking the uORFs). The light grey boxes preceding the YAP2 and mini-PGK1 reading frames represent the YAP2 uORFs. The inverted ‘v’, bridging the two dark grey boxes, indicates the region of PGK1 deleted in the mini-PGK1 reading frame. The X symbols indicate where AUG start codons have been mutated to AAGs. Download figure Download PowerPoint We next examined whether the uORF-dependent destabilization effect can be transferred to a further, more stable, yeast mRNA. In other studies, PGK1 has frequently been used as a model of relationships between translation and mRNA stability. One particular deletion derivative, the so-called ‘mini-PGK1’ gene, has been a favoured tool in investigations of the phenomenon of nonsense-dependent decay (Peltz et al., 1993a). The mini-PGK1 sequence is believed not to contain any of the ‘downstream elements’ (Peltz et al., 1993a) that have been proposed to mediate the acceleration of mRNA degradation observed upon the introduction of premature stop codons into the first two-thirds of the PGK1 reading frame. We found that, as with the YAP2 ORF itself, the YAP2 leader acted to destabilize the mini-PGK1 mRNA (Figure 1). We explored a further aspect of the destabilization mediated by the YAP2 ORFs, namely its dependence on Upf1. Since UPF1 dependence is typical of a number of nonsense-destabilized mRNAs (Peltz et al., 1993a,b), the observation that the YAP2 uORFs act on YAP2 to a large extent independently of this gene (Figure 1) indicated that they do not force the mRNA to follow the nonsense-dependent decay pathway described in previous investigations of aberrant mRNAs (Jacobson and Peltz, 1996). Interestingly, the YAP2 leader::mini-PGK1 mRNA was also destabilized in a UPF1-independent fashion (data not shown). In control experiments (data not shown), we found that the upf1− strain used in this study did show stabilization of an mRNA (BIAcat) shown previously to respond to inactivation of the UPF1 gene (Linz et al., 1997). This confirmed that the strains used here were capable of supporting upf1-dependent accelerated decay. Specific uORF properties contribute additively to destabilization If specific sequence elements individually or collectively determine translation and mRNA turnover rates, it should be possible to convert one type of uORF into another type by modifying its sequence environment. We therefore investigated what modifications are needed to convert the YAP1 uORF into an inhibitory, destabilizing type of uORF (Figure 2A). Since we wished to establish the generality of the relationship between strong translational inhibition by an uORF and its ability to destabilize, we used components of GCN4 uORF4 and its flanking sequences to modify the YAP1 uORF in its natural leader (see Table I for details). The use of the cat (chloramphenicol acetyl transferase) gene enabled us to monitor both the translation and the stability of the mRNA. Like the YAP1 and YAP2 mRNAs, this reporter mRNA is one of the more rapidly degraded transcripts in S.cerevisiae. The initial change was the substitution of the downstream sequence of GCN4 uORF4 (puY1du4G4). This was followed by the penultimate codon of GCN4 uORF4 combined with mutation of the U at −3 to A (pAmuY1du4G4), and finally by substitution of the complete GCN4 uORF4 sequence (pu4G4). U is a less favoured nucleotide that lowers the efficiency of AUG recognition by the 40S ribosomal subunit (Cigan and Donahue, 1987; Cavener and Ray, 1991; Yun et al., 1996). Its substitution by A therefore increases the efficiency of start-codon recognition. A control construct in which the start codon of the destabilizing uORF was mutated to AAG (pΔAmuY1du4G4) served to confirm that the destabilizing effect was specifically associated with translation of the uORF. In conclusion, the experiments in Figure 2A demonstrate how a non-destabilizing type of uORF can be progressively converted to a destabilizing type in a series of small (additive) steps. In further experiments, another reporter mRNA (LUC, encoding firefly luciferase) was also found to be subject to the same stepwise translational inhibition and destabilization as cat (data not shown), thus confirming the relationship observed. Figure 2.Progressive conversion of a YAP1-type uORF to a YAP2-type destabilizing element. Starting from the YAP1 leader preceding the cat gene, the uORF was progressively converted into a destabilizing uORF by introducing internal and flanking elements from the GCN4 uORF4 (A). The final stage is the complete substitution by the GCN4 uORF4 plus its downstream 10-nucleotide region. Elimination of the start codon of one of the destabilizing uORF constructs by mutation to AAG (pΔAmuY1du4G4) reverts the leader to its non-destabilizing status. Sequences derived from the destabilizing type of uORF (YAP2 uORF1 and GCN4 uORF4) also induce destabilization of the YAP1 mRNA (B). These changes in uORF structure diminished yeast resistance to H2O2 (C). The CAT activities are corrected for variations in the cat mRNA levels of the respective strains and given to two significant figures. The boxes preceding the cat main ORF represent uORFs (grey = YAP1 uORF; black = GCN4 uORF4). Download figure Download PowerPoint Table 1. puY1 series of constructsa puY1 UGCAUGAACACGAGCCAUUUUUAGUUUGUUUAAG puY1du4G4 UGCAUGAACACGAGCCAUUUUUAGCGGUUACCUU pAmuY1du4G4 AGCAUGAACACGAGCCAUCCGUAGCGGUUACCUU pu4G4 AAGAUGUUUCCGUAACGGUUACCUU pΔAmuY1du4G4 AGCAAGAACACGAGCCAUCCGUAGCGGUUACCUU aThese sequences correspond to the segment of the YAP1 5′UTR which is modified in Figure 2. The table shows nucleotides 79–112 of the wild-type YAP1 leader (puY1) followed by the sequences that are substituted for it in the other constructs. The derivative leaders all contain different portions of the GCN4 uORF4 and its flanking sequences. The uORF in each vector is shown underlined and the GCN4 uORF4 sequences are given in bold. Taking four of the series of leader sequences shown in Figure 2A, we next investigated whether sequences derived from the GCN4 leader could be used to destabilize the YAP1 mRNA, which is not normally destabilized by its own leader (Vilela et al., 1998). Progressive increases in the destabilizing potential of the uORF were indeed reflected in reductions in the half-life of YAP1 mRNA (Figure 2B). As with the cat gene, the individual changes associated with the respective steps were relatively small, but added up to a maximum overall destabilization of ∼3-fold, thus showing again how small changes in mRNA sequences can be used to achieve progressive modulation of mRNA function. This also means that uORF-mediated destabilization is not an all-or-nothing effect. It should be pointed out that previous studies have confirmed that small changes in mRNA half-life in this range can be reproducibly measured in S.cerevisiae and are significant in terms of cellular decay kinetics (Herrick et al., 1990; Cui et al., 1995; Hatfield et al., 1996; Hennigan and Jacobson, 1996). The YAP mRNAs we are studying here belong to the more unstable end of the scale of mRNA stabilities, but we have observed major changes in the decay rate of both YAP mRNAs in response to alterations in uORF structure and function. Comparison of the effects of the pAmuY1du4G4 leader on the stability of cat (Figure 2A) and YAP1 (Figure 2B) mRNAs revealed that the degree of destabilization imposed is very similar. Up to this stage therefore, we had shown that five different mRNAs (YAP1, YAP2, cat, LUC and ‘mini-PGK1’) were subject to uORF-dependent destabilization via a largely upf-independent pathway. Manipulation of uORF structure in the YAP leaders modulates the cellular stress response We examined how uORF structure can influence the physiological function of one of the YAP mRNAs, investigating how manipulation of the normal YAP1 uORF changes the tolerance of S.cerevisiae to oxidative stress. Strikingly, the relatively moderate change (compared with, for example, pu4G4) in stability and expression caused by the leader pAmuY1du4G4 was already sufficient to drastically decrease tolerance to H2O2 in the plate assay (Figure 2C). This result illustrates how sensitively the stress response can be modulated by uORF-mediated post-transcriptional control of this YAP gene. Overall, the experiments shown in Figure 2 demonstrate the principle that a wide range of post-transcriptional control can be imposed generally on yeast mRNAs via alterations in the structure and immediate environment of short uORFs. The effects can be subjected to fine or coarse control, depending on the combination and number of individual small changes in mRNA structure, and can clearly be of physiological significance. The post-termination behaviour of ribosomal subunits is linked to mRNA destabilization We next proceeded to investigate the principles underlying uORF-dependent destabilization. In order to be able to focus on the properties of the individual uORFs, we inserted them into a synthetic leader which supports a translation efficiency that is comparable to the average efficiency of natural yeast mRNAs (Oliveira et al., 1993b; Table II; Figure 3). The system chosen for these more detailed studies contained the cat gene transcribed from the inducible PGPF promoter (Oliveira et al., 1993b; Oliveira and McCarthy, 1995). Our initial results revealed that the destabilization effects of uORFs are independent of the type of promoter used to transcribe the constructs under study. While the absolute half-lives measured using the PGPF promoter (Figure 3A) are somewhat shorter than with the constitutive PTEF1 promoter (compare Linz et al., 1997), the degree of destabilization measured for a given construct was, within the limits of experimental error, identical in both cases. Since the repressible PGPF promoter offered enhanced accuracy at the fastest degradation rates, this promoter was chosen for the remaining analysis. The changes in turnover rate observed clearly confirm that the functional influence of an uORF can be shifted progressively between the identified two states via changes in small, defined regions within, or flanking, the uORF. Figure 3.Post-termination events modulate the destabilization potential of uORFs. Four different uORFs (YAP1 uORF, YAP2 uORF1, GCN4 uORF1 and GCN4 uORF4) together with their respective downstream sequences were inserted upstream of the cat gene in pcat (A). A stable hairpin loop capable of strongly inhibiting translation (−28.8 kcal/mol) was inserted 5 nucleotides upstream of the cat mRNA in each construct (B). The presence of the stem–loop decreased, in all cases, both translation and the mRNA half-life. This effect was eliminated when the start codon of the uORF was mutated to AAG (pΔu4G4. cat). Download figure Download PowerPoint Table 2. Sequences used in the reconstructed 5′UTRs of the puORFcat series of constructsa pcat AAGGATCCAATTATCTACTTAAGAACACAAACTCGAGAACATATG puORFcat AAGGATCCAAAAAAAGATCT….uORF + 10 nt…CTCGAGTAAACATAGAAACTTAAGACAAAGTATAGATACACTACGTAAACTACATATG S: stem–loop CTCGAGTAAACATAGAAACTTAAGCTCAAGTATAGATACAC (−28.8 kcal/mol) CAGCTTACGCCCGCCAAACAGGCGGGCGTAAGCTGCATATG s: stem–loop CTCGAGAATTATCTACATAAGAACACAAAA (−8.6 kcal/mol) CTCGAAGATACAAAAAAGTATCCTCGAAAACATATG 60 nt spacer CTCGATATTTATAAAAACAATTACCACAAACAACAATACTTTCTTAAAGATCTTAACCTCGAG 30 nt spacer CTCGATATTTATAAAAACAATTACCACAAACAACCTCGAG aThese are the partially synthetic leader sequences shown schematically in Figures 3 and 4. Each uORF (YAP1uORF, YAP2uORF1, GCN4uORF1 and GCN4uORF4) and respective downstream sequence (uORF + 10 nt) was inserted in the form of a BglII/XhoI oligodeoxyribonucleotide pair. The two stem–loop sequences were inserted as XhoI/NdeI oligodeoxyribonucleotide pairs into puORFcat, creating the S and s derivatives of this vector. The restriction sites used in the cloning of the different sequences are underlined. The first set of experiments showed that the cat mRNA is subject to destabilization by the YAP2 type of uORF in the synthetic leader context, but not by the YAP1-type (Figure 3A). The uORFs were inserted into the leader together with their respective (10 nucleotide) downstream sequences. Neither the YAP1 uORF (puY1cat) nor the GCN4 uORF1 (pu1G4cat) changed cat mRNA stability. In contrast, destabilization was caused by YAP2 uORF1 (pu1Y2cat) and was even stronger in the case of GCN4 uORF4 (pu4G4cat). Only partial upf1-dependence was evident. All of the evidence accumulated so far suggested that post-termination ribosomes play a decisive role in the destabilizing mechanism. We therefore decided to introduce a structural ‘hurdle’ into the mRNA downstream of the uORFs (Figure 3B). By inserting a stem–loop structure of sufficient stability to block the progress of scanning 40S subunits (Kozak, 1986; Oliveira et al., 1993b; Vega Laso et al., 1993; see Figure 3B), we could expect to achieve at least one of two objectives: first, to prevent reinitiation downstream of the uORFs; second, to induce an enhanced rate of ribosomal release from the mRNA subsequent to termination on the uORFs. The result of this manipulation was striking: all of the uORF-containing mRNAs were destabilized, irrespective of which type of uORF was present (Figure 3B). The insertion of the same stem–loop structure at the equivalent position into a leader that was identical except for the absence of an uORF had no effect on mRNA stability (pScat). Moreover, the requirement for recognition of the uORF by ribosomes in order for destabilization to occur was confirmed by a control in which the start codon of GCN4 uORF4 was mutated to AAG (pΔu4G4. cat). Post-termination ribosomes lose the ability to destabilize mRNA during scanning As indicated above, one possible explanation of the potentiation effect of a stem–loop structure placed downstream of an uORF is that the RNA structure promotes release of terminating ribosomes. This may even occur downstream of an uORF that allows post-termination scanning, since the ribosomal subunits proceeding beyond the stop codon may be devoid of one or more factors required for stable association with the mRNA during the scanning process. Hinnebusch and colleagues have proposed previously that ribosomal subunits which resume scanning subsequent to GCN4 uORF1 rebind eIF2–Met–tRNAi (thus becoming re-initiation competent) at a rate that is slow compared with termination (Abastado et al., 1991). Accordingly, we suspected that such ribosomal subunits may be particularly sensitive to the presence of a stem–loop structure because their association with the mRNA in the post-termination phase is relatively unstable. We set out to test this hypothesis (Figure 4A). First of all, we decreased the stability of the stem–loop in order to bring it down to a level which is known to cause only partial inhibition of cat translation (Vega Laso et al., 1993). This showed a slightly reduced destabilization effect (compare pu1Y2. cat, Figure 3B, with pu1Y2. cat, Figure 4A), thus confirming that the size of the thermodynamic barrier presented to the post-termination ribosomal subunits on the mRNA controls the degree of disruption of their normal scanning behaviour. Secondly, we increased the distance of the stem–loop from the uORF in order to allow post-termination ribosomes more time to become initiation-competent before reaching the obstruction (compare puY160Scat, Figure 4A, and puY1. cat, Figure 3B). The result was abrogation of the destabilization effect, indicating that the time/distance between termination and negotiation of the stem–loop enabled the ribosomal subunits to regain their more resistant status. That this was a progressive effect dependent on distance was confirmed by a construct with a spacer of intermediate length between the uORF and the stem–loop (puY130Scat, Figure 4A). Figure 4.Re-acquisition of re-initiation competence prevents destabilization. Replacement of the −28.8 kcal/mol secondary structure in pu1Y2. cat by a less stable one (−8.6 kcal/mol) in pu1Y2. cat resulted in higher stability (A). Complete abrogation of the destabilization effect was achieved by increasing the distance of the stem–loop from the uORF (puY160Scat). Partial destabilization was measured with an intermediate construct, puY130Scat. In a gcd2− strain, spacers between the uORF and the stem–loop were no longer sufficient to prevent destabilization (B). Both cat mRNA and endogenous PGK1 mRNA were labelled by hybridization in the Northern blots. Download figure Download PowerPoint In the GCN4 system, the influence of eIF2 activity on the behaviour of ribosomes that have terminated on uORF1 plays an important role in controlling downstream reinitiation events (Hinnebusch, 1997). It was therefore a logical step to investigate whether decreasing the level of active eIF2 affected the decay of the stem–loop-containing mRNAs. We compared the stability of the puY160Scat mRNA in a gcd2− strain and in an isogenic GCD2+ strain (Figure 4B). The gcd2− strain is defective in the δ subunit of eIF2B, and therefore maintains a reduced level of active eIF2. The decay rate was higher in the former strain, suggesting that the activity of eIF2B, and thus of eIF2, plays a role in uORF-dependent decay. No such effect was seen with a control construct in which the AUG of the uORF had been converted to AAG (puΔY160Scat; Figure 4B). Re-initiation prevents destabilization caused by post-termination ribosomes While we report here evidence that translational termination on a natural 5′UTR promotes destabilization, previous work on nonsense-dependent accelerated decay in aberrant mRNAs carrying premature stop codons has stressed the role of reinitiation following termination in promoting the destabilization process (Peltz et al., 1993b). The experiments in Figure 3B already indicated that blocking reinitiation did not prevent destabilization. However, we also examined the effect of reinitiation on the destabilizing influence of the YAP2 uORF1 by inserting the YAP1 uORF upstream of it (Figure 5A; puY1u1Y2). Since the YAP1 uORF allows efficient resumption of scanning (Vilela et al., 1998), the YAP2 uORF downstream of it is translated by a mixture of ribosomes that have ‘overlooked’ the YAP1 uORF and a number of reinitiating ribosomes. Termination on YAP2 uORF1 in this construct is accordingly at a level comparable to that seen with YAP2 uORF1 alone (data not shown), yet the mRNA is no longer destabilized (Figure 6). Re-initiation therefore suppresses the destabilization mechanism. Figure 5.Combinations of the YAP1 and YAP2 uORFs. YAP1-type (dark grey) and YAP2-type (light grey) uORFs were combined preceding the LUC reporter gene. Removal of one nucleotide from the inter-uORF region (puY1fu1Y2) had no effect on expression (A). Modific
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