©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Relationship between Eukaryotic Translation and mRNA Stability
A SHORT UPSTREAM OPEN READING FRAME STRONGLY INHIBITS TRANSLATIONAL INITIATION AND GREATLY ACCELERATES mRNA DEGRADATION IN THE YEAST SACCHAROMYCES CEREVISIAE(*)

Carla C. Oliveira (§) , John E. G. McCarthy (¶)

From the (1) Department of Gene Expression, National Biotechnology Research Center (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5`-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5`-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5`-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation.


INTRODUCTION

Given that the translational machinery and the RNA degradation apparatus interact with the same mRNA molecules, it would come as no surprise if translation and mRNA decay were to mutually influence each other. It is obvious that mRNA degradation ultimately eliminates the template of translation. Yet the issue as to the role of translation in controlling mRNA decay is far from being resolved (1, 2) . This is not for the lack of indications that there could be a link between the two. ( a) Rapid degradation of yeast MAT1 (3) and of mammalian early response genes (4) is dependent on the translation of destabilizing elements within the respective coding regions. ( b) Degradation of at least some yeast mRNAs is accelerated by nonsense codons introduced into the coding region (5, 6, 7) . Nonsense codons inserted into mammalian mRNAs seem to destabilize nuclear, rather than cytoplasmic, mRNA (8) . In yeast, the so-called nonsense-mediated decay pathway is dependent on trans-acting factors (encoded by the UPF genes (9) ), one of which (Upf1p) seems to be associated with ribosomes (10) . ( c) Two means of inhibiting translation lead to stabilization of mRNA. These involve the inhibition of elongation using cycloheximide (1, 2, 11, 12) and the use of a mutation in tRNA nucleotidyl transferase (12) . Both of these experimental strategies impose a general, rather than an mRNA-specific, block on translation. ( d) Finally, ribosomes have been found associated with certain ribonuclease activities (2, 13) .

However, other data are not easily reconciled with the above observations. Most importantly, translational initiation can be inhibited by more than 90%, using stem-loops inserted into the 5`-untranslated region (UTR),() without stabilizing the mRNA (14, 15, 16) . This clearly constitutes a challenge to any model proposing tight coupling between translation and mRNA decay.

A required approach toward understanding the relationship between translation and mRNA degradation is to restrict engineered changes in the cell to minimal alterations in the sequence of a specific mRNA. This greatly reduces the risk of misleading or artifactual effects that might be associated with general manipulations of, for example, cellular translational capacity (see point c, above). The present paper approaches this question by making use of a further important property of the eukaryotic 5`-UTR. The presence of one or more additional start codons upstream of the main reading frame inhibits cap-dependent translation (17) . This effect may be more pronounced in the yeast Saccharomyces cerevisiae, where the context of an AUG codon has a less significant modulating effect upon the efficiency of start-site selection than in higher eukaryotes (18) . An upstream AUG can be combined with a translational termination codon within the 5`-UTR, thus creating an upstream open reading frame (uORF). There are many examples of uORFs in the 5`-UTRs of vertebrate mRNAs (19) , and also a small number of them in S. cerevisiae (20, 21) . The GCN4 gene of yeast is of special interest in that it is subject to translational regulation mediated by four uORFs in its 5`-UTR (21) . The mechanism of induction of GCN4 involves phosphorylation of eIF-2, which in turn inhibits the eIF-2B-catalyzed exchange of GDP/GTP on eIF-2. Regulation of the availability of eIF-2GTP in this way is thought to influence start-site selection within the leader.

We show that the creation of a single short uORF in a synthetic 5`-UTR upstream of a reporter gene in yeast strongly inhibits translation. Moreover, given appropriate experimental design, a 5`-UTR that is otherwise free of uAUGs and of stable secondary structure can be converted to an uORF-containing leader by means of a single base change. This approach allows us to compare the translation and degradation of two mRNAs that differ minimally in terms of sequence but greatly in terms of the translational efficiency of a reporter gene (in this case the Tn9 cat gene). In comparison, strong inhibition of translation by secondary structure can only be achieved via the introduction or substitution of several nucleotides in the 5`-UTR. Further manipulations of the 5`-UTR were made in order to influence the pathway and efficiency of start-site selection on the main ORF, thus providing information about the type of mechanism responsible for the observed translation rates. We have investigated how the ribosomal loading of an mRNA is affected by the introduction of an uORF and how this relates to the decay rate. The results provide striking new evidence of a link between mRNA translation and decay.


MATERIALS AND METHODS

Yeast Strains

BWG1-7a (MATa, leu2-3, leu2-112, his4-519, ade1-100, ura3-52). Y262 (MAT, ura3-52, his4-539, rpb-1) (22) . Yeast was cultured and transformed according to standard methods (23) .

Plasmid Constructions

The cloning of short upstream reading frames into the leader region of the cat mRNA was performed by introducing synthetic oligodeoxyribonucleotides into the YCpCATEX1 plasmid (24) (see ). The oligonucleotide B1A, bearing an ORF of six codons, was inserted into the BamHI site at the 5`-end of the cat leader. A control construct was prepared using the synthetic B1O fragment, which differs from B1A in only one nucleotide (where B1A has AUG, B1O has AAG). The resulting leader does not contain an uORF. In order to increase the leader length upstream or downstream of the uORF, additional spacer fragments were cloned between the EcoRV and BamHI sites, respectively, creating USP10 and USP30. The leader length between the uORF and the cat coding region was increased by inserting a synthetic fragment between the XhoI and NdeI sites of B1A (DSP30). Further extension of the spacer region was achieved by inserting the 50-nucleotide fragment SP50 (24) into the AflII site of DSP30, giving DSP80. OL136 was generated by destroying the termination codon of the uORF by means of digestion of YCpCATEX1-B1A with AflII, followed by treatment with mung bean nuclease and religation, yielding an extended uORF that overlaps with the cat gene, terminating 136 nucleotides downstream of the A of the cat start codon. uATG has three extra in-frame AUG codons upstream of the original B1A uORF start codon. B1ASS bears a secondary structure with a stability of -22 kcal/mol located inside the uORF, extending the uORF length from 6 to 21 codons. B1SS is a control for B1ASS, bearing the same secondary structure, but no uORF. B1X3 has been described elsewhere (24) and was used as a control for B1AX3, which bears the uORF B1A and a secondary structure (X3) with a predicted stability of -17 kcal/mol, five nucleotides upstream of the cat start codon. The GCN4 leader, which bears four uORFs, was cloned upstream of the cat gene (after mutagenesis by PCR of the plasmid p180 (25) in order to create a BamHI and an NdeI site at the 5`- and 3`-ends of the leader, respectively).

RNA Isolation and Analysis

Total yeast RNA was isolated by the hot phenol method (26) from 150 ml of culture in YNB medium (0.67% bacto-yeast nitrogen base without amino acids, 2% dextrose) and analyzed using Northern blots after glyoxylation of 10-µg samples of total RNA, as described elsewhere (24, 27) . Primer extension analysis was performed as described previously (24) . mRNA half-life analysis (adapted from Ref. 28) was performed using yeast transformants grown in 100 ml of YNB-lactate medium at 24 °C for 18 h. Cells from these cultures were inoculated in the same volume of YNB-galactose and incubated at 24 °C for 3 h to A= 0.8. Cells were then harvested by centrifugation and resuspended in the same volume of prewarmed (36 °C) YNB-glucose medium. A 20-ml sample was collected at this time (time 0). The cell cultures were further incubated at 36 °C in a shaking water bath, and samples were collected at various time points thereafter. Total RNA isolated from the different samples was used for Northern blots. The resulting labeled bands were excised from the blotting membrane and used for scintillation counting (24) .

CAT Assay

Fresh cultures of the yeast transformants were allowed to grow in YNB-galactose to A= 0.8-1.0. Cells from 10 ml of culture were used for analysis of CAT activity (adapted from Ref. 29). After autoradiography of separated acetylated chloramphenicol forms, spots were counted in a scanning machine (linear analyzer, Chroma 2D, Berthold). Protein quantitation was performed by the BCA method (bicinchoninic acid protein assay (30) ).

Simulation of Amino Acid Starvation Conditions

Yeast cells were grown in YNB-Gal medium to A= 0.3 (as described in Ref. 31). 3-Aminotriazole (3-AT) was added to a final concentration of 10 m M, and incubation at 30 °C was continued for another 6 h. Cells were harvested by centrifugation and washed with water, and the pellet was frozen and stored at -20 °C until used for CAT assays and RNA analysis.

Polyribosome Analysis

In a procedure adapted from Sagliocco et al. (15) , yeast cell extracts were prepared from cultures in 100 ml of YNB-Gal medium ( A= 0.8) and loaded on a 12-ml diethyl pyrocarbonate 15-45% sucrose gradient. Total RNA was extracted from 600-µl fractions and resuspended in 10 µl of treated water. 5 µl of RNA suspension was glyoxylated and separated by electrophoresis in a 1.3% agarose gel. After blotting to a nylon membrane, Northern blot analysis was performed using P-labeled DNA fragments. Quantitation of the relative amounts of mRNA was performed as above.


RESULTS

Effects of uORFs on Translation of the cat Gene

Short open reading frames (uORFs) were inserted into the leader region of the cat mRNA in the plasmid YCpCATEX1 (24) using a series of oligodeoxyribonucleotides ( Fig. 1 and Table I). The presence of an uORF strongly inhibited translation of the cat gene, as seen, for example, by comparing the CAT levels of yeast cells bearing the constructs B1O and B1A (B1A supports 6.5% of the CAT activity supported by the control B1O; Fig. 2 A). These two constructs differ by only one nucleotide in the 5`-UTR.


Figure 1: The various constructs described in this paper were derived from the expression plasmid YCpCATEX1 (24). The diagrams indicate the overall composition of the leader/gene combinations inserted into this plasmid. The cat gene ( open box) was preceded by 5`-UTRs either free of uAUGs ( unbroken lines) or containing uORFs ( hatched boxes). The total lengths of the respective 5`-UTRs are given at the very left of each diagram above the lines. The lengths of the uORFs and of the sequences between the 5`-end and the uORF, as well as between the uORF and the cat start codon are given (as nucleotide ( nt) values) above and below the 5`-UTRs, respectively. In the case of OL136, the uORF is extended into the cat coding region. The positions of stem-loop structures either within an uORF (B1SS and B1ASS) or in noncoding regions (B1X3 and B1AX3) are also indicated. In the cases of two pairs of constructs (B1O/B1A and B1SS/B1ASS), the 5`-UTRs differ by the identity of only one nucleotide. In the 5`-UTR of uATG there are four AUGs within the uORF. The details of construction of the various plasmids and the exact sequences of the various leaders (Table I) are given under ``Materials and Methods.''




Figure 2: The relative CAT activities directed by the constructs illustrated in Fig. 1. Panel A shows the activities of all of the constructs relative to the activity of B10, which was normalized to the value of 1.00. Overall, the values demonstrate the strong inhibition of translation caused by the presence of an uORF in the 5`-UTR of the cat gene. The GCN4 construct bears the complete 5`-UTR from this yeast gene inserted upstream of the cat gene. Panel B compares the CAT activities of those constructs with an uORF in the 5`-UTR (except B1AX3). The data are grouped into two sections. The changes in encoded CAT activity relative to B10 in group 1 are most readily explained in terms of reductions in the amount of ``leaky scanning'' of preinitiation complexes past the uAUGs in the 5`-UTRs. At least part of the remaining cat translational activity is probably attributable to reinitiation of ribosomes subsequent to termination at the uORF stop codons. The changes in encoded CAT activity relative to B1A depicted in group 2 are more easily explained in terms of modulation of the efficiency of reinitiation at the cat start codon following termination on the uORF. The relative values schematically represented here (averages of measurements performed with at least three different sets of cell extracts) were as follows (±S.D. values): B1O, 1,00; B1A, 0.065 ± 0.010; USP10, 0.039 ± 0.007; USP30, 0.041 ± 0.009; DSP30, 0.067 ± 0.008; DSP80, 0.118 ± 0.025; uATG, 0.031 ± 0.006; OL136, 0.052 ± 0.013; B1SS, 0.020 ± 0.005; B1ASS, 0.007 ± 0.002; B1X3, 0.035 ± 0.008; B1AX3, 0.004 ± 0.001; GCN4, 0.011 ± 0.001.



Other constructs were designed with the intention of defining the possible processes responsible for translation of the cat gene (Fig. 2 B). The AUG codon of the uORF in B1A is located 13 nucleotides downstream of the 5`-end of the mRNA, which is a position thought not to allow efficient recognition of this AUG by the ribosome (17, 18) . Increasing the leader length upstream of the B1A uORF (constructs USP10 and USP30; Fig. 1) resulted in further attenuation of cat expression. These results can be interpreted in terms of the scanning model of translation initiation (17) . This would predict that translation of the cat gene in the B1A construct is due primarily to leaky scanning. It has been demonstrated that eukaryotic ribosomes need at least 14 nucleotides upstream of the AUG codon for efficient translation initiation to occur (32) . Since USP10 and USP30 provide 23 and 43 nucleotides, respectively, upstream of the AUG of the uORF, the ribosomes can more efficiently recognize this AUG, initiating translation at the uORF and thus inhibiting cat translation more effectively (Fig. 2). By analogy, uATG, with its four in-frame AUGs (Fig. 1), should show decreased leaky scanning past the uORF and thus direct poorer translation of the cat reading frame. The observed reduction in cat translation relative to comparable constructs is consistent with leaky scanning providing many of the ribosomes for initiation on the cat ORF. Analysis of the above data alone does not rule out any contribution of reinitiation to the overall translation of cat. The described mutations in the leaders may not have eliminated leaky scanning completely.

Further experimental results provided a stronger case for the occurrence of leaky scanning on the cat 5`-UTRs. Translating 80 S ribosomes are considered less sensitive to secondary structure than 40 S ribosomal subunits (33) and thus should be relatively insensitive to the presence of secondary structure within the uORF in B1ASS, providing that this structure is sufficiently distant from the uORF start codon to rule out any interference with initiation on the uORF. Thus if reinitiation was responsible for cat translation in B1A, translation of this gene in B1ASS would not be expected to be very different from that of B1A. However, the results show that translation of the cat gene is very strongly inhibited in B1ASS, indicating that the translation of the cat gene in B1A is probably due primarily to leaky scanning and not reinitiation (Fig. 2). Another potential effect of the inserted stem-loop structure should be considered. A stable secondary structure (-22 kcal/mol) positioned downstream of the uAUG, might even allow better recognition of this AUG (probably because it slows ribosome scanning in the vicinity of the start codon, compare Ref. 34), increasing levels of translation of the uORF. Thus in the case of a reinitiation mechanism, cat translation would have been expected to be at least as efficient in B1ASS as in B1A. Interestingly, the levels of cat expression in cells bearing B1ASS are more similar to those observed with B1SS, which contains the same secondary structure, but no uORF in the leader.

Despite the observations discussed above, other evidence seems to indicate that reinitiation also occurs in B1A, albeit to a limited extent, and that its efficiency can be increased (Fig. 2 B). In constructs DSP30 and DSP80, the distance between the uORF and the cat gene was increased by 30 and 80 nucleotides, respectively. As argued by others (31, 35) , increasing the length between two ORFs may increase the probability that scanning ribosomes reinitiate translation at the downstream AUG. In this case, DSP30 and DSP80 should support higher levels of cat translation than B1A. This is indeed the result observed, although the difference between B1A and DSP30 is small (Fig. 2). The construct in which the uORF (+1 relative to the cat reading frame) overlaps the cat coding region (OL136) would be expected to support levels of cat expression not significantly different from that of B1A if leaky scanning was the dominant mechanism. However, if reinitiation was responsible for cat translation in B1A, the latter should decrease dramatically in OL136 (relative to B1A), since the termination codon of the uORF is inside the cat gene, 134 nucleotides downstream of the AUG. The translation of cat in OL136 was found to be reduced relative to B1A, although only by about 20% (Fig. 2). However, correction of the translation values for differences in mRNA abundance (see Fig. 5) would yield a greater inhibitory effect than this (approximately 35%).


Figure 5: The steady-state levels of the cat mRNAs encoded by the yeast transformants carrying the various cat constructs. A typical Northern blot ( panel A) indicates the different amounts (and lengths) of the respective cat mRNAs. The blot was hybridized simultaneously with radioactive probes specific for cat and PGK. The individual bands were excised from the blotting membrane after hybridization and subjected to scintillation counting. The results of cat mRNA quantitation are expressed as ratios to the PGK mRNA cpm obtained using each total cell extract ( panel B). The tabulated data represent averages of measurements performed using at least three independent RNA preparations.



The B1AX3 construct (Fig. 1) does not allow differentiation between leaky scanning and reinitiation, but the results obtained with it indicate that the effects of the elements introduced into the leader act in series, as would be expected of a cap-dependent screening mechanism. In this construct, the presence of a secondary structure of predicted stability of -17 kcal/mol five nucleotides upstream of the cat initiation codon strongly inhibits translation (Fig. 2; compare Refs. 14 and 24). This degree of inhibition is approximately equivalent to the inhibitory effect of the uORFs present in most of the constructs.

Fig. 2B summarizes the results obtained from the various CAT assays and indicates the simplest theoretical explanations of the respective effects observed. Given that the differences between the various CAT values are in some cases quite small, we do not wish to overinterpret the individual results described above, but would rather focus attention on the sum of these data. Overall, this indicates that translation of cat in B1A is largely due to leaky scanning. The remaining translation of the cat gene in B1ASS may be at least partially attributable to reinitiation, since the secondary structure is expected to strongly inhibit leaky scanning. The rate of reinitiation can apparently be increased by lengthening the spacer between the uORF and the cat start codon.

For comparative purposes, we investigated the influence of the GCN4 leader on cat translation. It was found to direct a very low level of cat expression. Indeed, after correction for relative mRNA abundance (see Fig. 5 B), the translation rate of the GCN4 construct was lower than any of the other constructs tested. The long leader of this gene, containing four uORFs, evidently inhibits translation of the main ORF very effectively. Since the uORFs in the GCN4 leader are known to mediate up-regulation of this gene in response to amino acid starvation, we investigated how the single uORF leaders used in the present work respond under the same conditions. This situation can be simulated in the cell by adding 3-aminotriazole to the medium (31) . The GCN4 leader directed a 2.5-fold increase of cat expression upon addition of 3-aminotriazole. This result indicates that the starvation-induced reinitiation in the GCN4 leader continues to function when the cat gene is substituted for the main GCN4 ORF. In contrast, translation of the cat constructs bearing single uORFs was not influenced by the addition of 3-aminotriazole (data not shown). Clearly, the single uORF leaders do not fulfill the structural requirements of a system intended to respond to changes in eIF-2GTP availability.

Polysomal Gradient Analysis of cat Translation

It could be argued that the uORF in B1A is poorly translated and that leaky scanning is the sole mechanism responsible for cat translation. This would mean that the complete leader as such would allow very restricted initiation (at whatever AUG codon). In order to investigate this more directly, we performed polysomal gradient analysis on cell extracts from cells carrying the constructs B1O, B1A, and OL136 (Figs. 3 and 4). The results demonstrate that the mRNA B1O is mainly associated with polysomes, corroborating the data from the CAT assays showing that this mRNA is efficiently translated (Fig. 3 A). In contrast, the B1A mRNA, which directs only low rates of CAT synthesis, was associated primarily with monosomes and disomes, showing only poor representation in the larger polysomal fractions. The data are compared directly with those of B1O in Fig. 4A. Despite the relatively low steady-state amount of B1A mRNA in the cell (see Fig. 5 B), it is evident that the cat Northern blot signal of this construct in the monosome/disome region is comparable with that of B1O. This result is consistent with relatively efficient initiation on the uORF of B1A, but not on the main cat ORF. Finally, the OL136 mRNA was found associated with larger polysomes than B1A (Figs. 3 B and 4 B). Again, this mRNA is relatively poorly represented in the cell (Fig. 5 B), yet the Northern blot signals (Fig. 4 B) in the monosomal and early polysomal fractions of the gradient are relatively strong. Thus the uORF in this construct was probably also translated considerably more efficiently than the cat coding region, but being considerably longer than the other uORFs, it can accommodate a larger number of translating ribosomes at any one time.


Figure 3: Spectrophotometric and Northern blot analysis of polysomal gradients made using extracts derived from cells bearing cat expression constructs. The results obtained with B10 ( panel A) and OL136 ( panel B) are compared. The Northern blot analysis shows that a large proportion of the B10 cat mRNA is associated with polysomes, whereas there is a shift toward monosomes and smaller polysomes in the case of OL136.




Figure 4: Quantification of the polysome gradient analysis. The plot in panel A serves to illustrate the effects of the single base change that converts B10 to B1A. The counts/min values of the Northern blot bands corresponding to the cat mRNA are expressed in each case as a ratio to the equivalent cpm values corresponding to the PGK mRNA found in the same fractions of the gradient (see ``Materials and Methods'' and compare Fig. 5). Creation of the uORF causes a major shift from polysomes to monosomes. Panel B compares the counts/min corresponding to the cat and PGK mRNAs in the gradient fractions isolated using an extract from cells containing the construct OL136. In the latter, cat mRNA is associated with larger polysomes than in the case of B1A. The data shown represent the results of one out of a total of two sets of measurements made with independent cell extracts.



Quantitative and Qualitative Analysis of the uORF-containing mRNAs

The mRNA levels of these constructs were controlled by means of Northern blotting. Total RNA was extracted from the same cell cultures tested for CAT activity and hybridized with DNA probes specific for cat and PGK genes. Quantitative analysis of the results (Fig. 5 B) revealed that the abundance of the uORF-bearing mRNAs is relatively low, with the exception of GCN4-cat mRNA, which is present in significantly greater amounts. Particularly striking is the observation that mRNAs bearing only one base change (B1O and B1A; B1SS and B1ASS) are present in greatly differing amounts. It should be noted that the relative CAT activities given in Fig. 2have not been corrected for the reduced abundance of the uORF-bearing mRNAs. This correction would result in increased estimated translational rates relative to B1O. However, since all of the uORF constructs have similarly reduced mRNA levels, the internal comparisons within this group remain valid. Primer extension analysis revealed that all of the cat mRNAs synthesized in vivo possessed the expected leader sequences, including the uORFs (Fig. 6). Using the same amount of total RNA in these experiments, we also observed different intensities of the extension bands that correlated with the differences in the steady-state amounts of cat mRNA in the cells (compare Fig. 5).


Figure 6: Primer extensions performed using RNA extracts from the yeast transformants. These experiments confirmed the expected 5`-ends of the respective mRNAs. The positions of the 5` termini with respect to the cat start codon are indicated by arrowheads. In some cases, there were additional bands (marked by an asterisk) corresponding to particularly strong premature terminations of the reverse transcriptase reaction at the sites where stem-loop structures were present in the 5`-UTR (compare Ref. 24). Where two neighboring lanes have bands running at the same height, only one arrowhead is used to indicate the positions of both (see e.g. B1SS/B1ASS). The length of the complete extension along the GCN4 mRNA leader cannot be deduced from this autoradiograph. The cause of the additional, shorter bands is also likely to be the presence of structure in the sequence. As in Fig. 5, the variations in signal strength reflect differences in the amounts of cat mRNA in the 10-µg samples used in the primer extension experiments. The sequencing reaction products obtained using B10 served as a standard for the calculation of extension lengths.



cat mRNA Half-life Measurements

One interpretation of the above results is that the presence of an uORF destabilizes the cat mRNA. To test this hypothesis, the half-lives of some of the cat mRNAs were analyzed (Fig. 7). The estimated half-lives varied from 4-5 min (B1O, B1SS) to 1.5 min (B1A, OL136, and B1ASS). Thus the differences in measured cat mRNA levels among the constructs are primarily attributable to differences in mRNA stability rather than to variations in transcription rates. It is therefore evident that the introduction of an uORF into the 5`-UTR of the cat gene accelerates degradation of the encoded mRNA. It is noteworthy that the estimated half-lives fall into two groups, whereby the decisive difference is the presence or absence of an uORF in the 5`-UTR. A similar degree of destabilization is observed irrespective of whether the uORF terminates upstream or within the cat reading frame. Comparison of the results obtained with B1SS and B1ASS reveals that the introduction of a stem-loop into the uORF has no measurable effect on the mRNA half-life.


Figure 7: The effects of the introduction of an uORF into the 5`-UTR on cat mRNA stability. Northern blots ( panel A) show the results of hybridization performed using RNA preparations from cells (transformants of Y262) taken at various time-points during a half-life determination experiment. Transformants of the rpb-1 strain were subjected simultaneously to both a temperature shift and a medium shift (galactose/glucose) in order to inactivate polymerase II-dependent transcription (of the cat gene). The cat mRNA counts/min values are expressed as a ratio to the corresponding actin mRNA values and plotted as logarithms ( y axis) versus time ( panel B). The estimated mRNA half-lives (in minutes) calculated from these data were (±S.D. values): B1O, 5.0 ± 0.2; B1A, 1.2 ± 0.1; OL136, 1.2 ± 0.1; B1SS, 3.0 ± 0.1; B1ASS, 1.0 ± 0.1. A further single set of data obtained using the strain BWG1-7a yielded estimated half-lives of B1O = 4.0, B1A = 1.5, OL136 = 1.5, B1SS = 5.0, and B1ASS = 1.8.




DISCUSSION

The results presented in this paper show that the introduction of an uORF into the 5`-UTR of an mRNA in yeast both inhibits translation and leads to accelerated degradation. In the constructs described here, the degree of translational inhibition varies between 60% and greater than 90%, after correction for the relative steady-state mRNA abundance in the cell. These levels of inhibition are greater than the inhibitory effect exerted by the first GCN4 uORF (50%) when this is the sole uORF present in the GCN4 leader (37) . Variation of the spacer lengths between the 5`-end and the uORF, and between the uORF and the cat gene leads to changes in translational efficiency. These changes can be best explained if it is assumed that leaky scanning is primarily responsible for initiation at the cat start codon. Reinitiation is evidently very inefficient on these leaders. Moreover, we also observed that cat translation could not be significantly altered by conditions under which the translation of GCN4 is positively regulated. Thus the GCN4 induction mechanism, which effectively involves changes in the availability of eIF-2GTP, does not influence translation directed by the single uORF leaders we have described. This may be at least partly due to the fact that reinitiation contributes poorly to the overall level of initiation on the single uORF leaders. Reinitiation is thought to be the process primarily regulated on the GCN4 leader. It should also be emphasized that in the GCN4 5`-UTR, regulation is thought to involve kinetic control of initiation at the fourth uORF (uORF4) and the main reading frame. The ribosomes that reach the uORF4 have probably terminated previously on uORF1, so that translation of both uORF4 and GCN4 is performed by reinitiating ribosomes (36) . In our case, the single uORF is the first reading frame encountered by the scanning ribosome.

The combination of an uORF and secondary structure, or the presence of very stable secondary structure alone, more effectively inhibited translation than a single uORF. Despite this, we have shown that secondary structure does not destabilize the cat mRNA (compare Refs. 14-16). Thus it is not the degree of translational inhibition per se that determines whether mRNA is destabilized but rather the mechanism by which recognition of the main ORF's start codon is rendered less efficient. Given that a drastic reduction in cat mRNA stability can be achieved by means of the substitution of only one nucleotide in the leader of B1O or B1SS, the described results argue against there being any major role of mRNA structural changes in the observed destabilization. It seems most likely therefore, that the presence of a translatable uORF is the decisive factor that triggers events responsible for rapid decay of the mRNA.

What could be the mechanism of destabilization via an uORF? Previous work has shown that nonsense mutations within the initial two-thirds of the PGK1-coding region accelerate the decay rate of the PGK1 mRNA up to 12-fold (7) . It was proposed that destabilization requires an element downstream of the nonsense codon that contains potential sites of translational reinitiation. One experimental result found consistent with this proposal was that the introduction of a stem-loop structure immediately downstream of a nonsense codon stabilizes an otherwise unstable PGK1 transcript. In most of our constructs, the termination codon of uORF is followed by the start codon of the cat gene (Fig. 1). Yet the introduction of a stem-loop structure between the uORF and the cat ORF (B1AX3) does not result in a stabilized mRNA (Fig. 5). Moreover, the OL136 mRNA is also destabilized, despite the fact that its uORF terminates 136 nucleotides downstream of the cat start codon (Fig. 7). Thus our results do not provide evidence for reinitiation playing an important role in the destabilization of the cat mRNA. In any case, for reasons already outlined in this paper, it is likely that reinitiation is generally very limited on the single uORF leaders. In conclusion, none of our data provide support for a model in which translational reinitiation is required for mRNA destabilization by an uORF. If reinitiation is involved, we would have to argue that the destabilization mechanism is not only triggered by very low rates of reinitiation, but is also insensitive to large reductions in reinitiation efficiency. The discrepancy here to the results obtained with the PGK1 gene may be attributable to the involvement of a different mechanism in destabilization induced by an uORF as opposed to a nonsense codon.

Only one other case of an unstable mRNA bearing an uORF has been analyzed in yeast. The PPR1 mRNA has a half-life of 1 min, which can be extended by exchanging the 5`-UTR for that of the URA3 mRNA (38) . The PPR1 leader contains a six-codon uORF that overlaps in the +1 reading frame with the PPR1 coding region. However, elimination of this uORF by modification of the start codons, or by shifting its reading frame to that of the PPR1 coding region, had no effect on mRNA stability. The authors concluded that this uORF1 has no significant effect on PPR1 mRNA degradation. Nevertheless, these results do not prove that the PRP1 uORF cannot (at least potentially) influence mRNA decay. It has not been ruled out that the rate-controlling step in the degradation of the highly unstable PPR1 mRNA is associated with another part or property of this transcript so that the potential effects of the uORF on stability are effectively obscured. The influence of the much more complex GCN4 leader on mRNA stability have hardly been explored. Certainly, the presence of the four uORFs does not result in very rapid degradation of the mRNA (see Ref. 2 and Fig. 5). This therefore reinforces the idea that an uORF-bearing leader can, but need not, contribute to mRNA destabilization. That such leaders may behave differently in this respect is reflected in the differential effects of inactivation of the upf1gene upon the degradation rates of these mRNAs. The protein encoded by this gene seems to be a trans-acting factor involved in nonsense-mediated RNA decay (9, 10) . Its inactivation stabilizes PGK mRNAs containing early nonsense codons. Surprisingly, the PPR1 mRNA is also more stable in an upf1mutant (2) , despite the fact that the uORF is thought not to be relevant to the degradation of this transcript (38) . On the other hand, inactivation of UPF1 does not stabilize the GCN4 mRNA. Overall, the data obtained with PPR1 and GCN4 provide us with no clear picture of the functional significance of uORFs in terms of mRNA decay. It will undoubtedly be important to determine the rate-controlling steps of degradation in these different cases in order to be able to explain the apparently contradictory effects observed.

In conclusion, the well defined modular system we have described here has been used to demonstrate a link between translation and mRNA degradation at the level of a specific transcript. A large decrease in stability could be induced by a single nucleotide substitution in the 5`-UTR of the cat mRNA, clearly showing that destabilization is not exclusively coupled to premature termination within a gene's coding region. It is unlikely that the specific signals suspected to be involved in nonsense-mediated decay of the PGK1 mRNA are responsible for destabilization of the cat mRNA. The mechanism of destabilization may therefore differ from that of the nonsense-mediated decay pathway proposed elsewhere (2, 7) . The ``minimal'' uORF system we have described should prove useful as a basis for further studies of the relationship between translation and mRNA decay.

  
Table: Sequences of the 5`-UTRs



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a stipend from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) of Brasil.

To whom correspondence should be addressed. Tel.: 49 531 6181 430; Fax: 49 531 6181 458.

The abbreviations used are: UTR, untranslated region; ORF, open reading frame; uORF, upstream ORF; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank Dr. Alistair Brown (Aberdeen University, United Kingdom) for sending us the yeast strain Y262 and Dr. Alan Hinnebusch (National Institutes of Health, Bethesda, MD) for p180.


REFERENCES
  1. Sachs, A. B. (1993) Cell 74, 413-421 [Medline] [Order article via Infotrieve]
  2. Belasco, J., and Brawerman, G. (eds) (1993) Control of Messenger RNA Stability, Academic Press, San Diego
  3. Caponigro, G., Muhlrad, D., and Parker, R. (1993) Mol. Cell. Biol. 13, 5141-5148 [Abstract]
  4. Wellington, C., Greenberg, M. E., and Belasco J. G. (1993) Mol. Cell. Biol. 13, 5034-5042 [Abstract]
  5. Losson, R., and Lacroute, F. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5134-5137 [Abstract]
  6. Pelsey, F., and Lacroute, F. (1984) Curr. Genet. 8, 277-282
  7. Peltz, S. W., Brown, A., and Jacobson, A. (1993) Genes & Dev. 7, 1737-1754
  8. Cheng, J., and Maquat, L. (1993) Mol. Cell. Biol. 13, 1892-1902 [Abstract]
  9. Leeds, P., Wood, J. M., Lee, B.-S., and Culbertson, M. R. (1992) Mol. Cell. Biol. 12, 2165-2177 [Abstract]
  10. Peltz, S. W., Trotta, C., Feng, H., Brown, A., Donahue, J., Welch, E., and Jacobson, A. (1993) in Protein Synthesis and Targeting in Yeast (Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G., eds) pp. 1-10, Springer-Verlag, Berlin, Germany
  11. Herrick, D., Parker, R., and Jacobson, A. (1990) Mol. Cell. Biol. 10, 2269-2284 [Medline] [Order article via Infotrieve]
  12. Peltz, S. W., Donahue, J. L., and Jacobson, A. (1992) Mol. Cell. Biol. 12, 5778-5784 [Abstract]
  13. Stevens, A. (1980) J. Biol. Chem. 255,3080-3085 [Abstract/Free Full Text]
  14. Vega Laso, M. R., Zhu, D., Sagliocco, F., Brown, A. J. P., Tuite, M. F., and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462 [Abstract/Free Full Text]
  15. Sagliocco, F. A., Zhu, D., Vega Laso, M. R., McCarthy, J. E. G., Tuite, M. F., and Brown, A. J. P. (1994) J. Biol. Chem. 269, 18630-18637 [Abstract/Free Full Text]
  16. Beelman, C. A., and Parker, R. (1994) J. Biol. Chem. 269, 9687-9692 [Abstract/Free Full Text]
  17. Kozak, M. (1991) J. Biol. Chem. 266, 19867-19870 [Free Full Text]
  18. Donahue, T. F., and Cigan, M. (1990) Methods Enzymol. 193, 366-372
  19. Kozak, M. (1991) J. Cell Biol. 115, 887-903 [Abstract]
  20. Bossier, P., Fernandes, L., Rocha, D., and Rodrigues-Pousada, C. (1993) J. Biol. Chem. 268, 23640-23645 [Abstract/Free Full Text]
  21. Hinnebusch, A. G. (1994) Trends Biochem. Sci. 19, 409-414 [CrossRef][Medline] [Order article via Infotrieve]
  22. Nonet, M., Scafe, C., Sexton, J., and Young, R. (1987) Mol. Cell. Biol. 7, 1602-1611 [Medline] [Order article via Infotrieve]
  23. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics, pp. 117-122, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Oliveira, C. C., van den Heuvel, J., and McCarthy, J. E. G. (1993) Mol. Microbiol. 9, 521-532 [Medline] [Order article via Infotrieve]
  25. Hinnebusch, A. G. (1985) Mol. Cell. Biol. 5, 2349-2360 [Medline] [Order article via Infotrieve]
  26. Köhrer, K., and Dorndey, H. (1991) Methods Enzymol. 194, 398-405 [Medline] [Order article via Infotrieve]
  27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  28. Parker, R., Herrick, D., Peltz, S. W., and Jacobson, A. (1991) Methods Enzymol. 194, 415-423 [Medline] [Order article via Infotrieve]
  29. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051 [Medline] [Order article via Infotrieve]
  30. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85 [Medline] [Order article via Infotrieve]
  31. Abastado, J. P., Miller, P. F., Jackson, B. M., and Hinnebusch, A. G. (1991) Mol. Cell. Biol. 11, 486-496 [Medline] [Order article via Infotrieve]
  32. Sedman, S. A., Gelembiuk, G. W., and Mertz, J. E. (1990) J. Virol. 64, 453-457 [Medline] [Order article via Infotrieve]
  33. Liebhaber, S. A., Cash, F., and Eshleman, S. S. (1992) J. Mol. Biol. 226, 609-621 [Medline] [Order article via Infotrieve]
  34. Kozak, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8301-8305 [Abstract]
  35. Kozak, M. (1987) Mol. Cell. Biol. 7, 3438-3445 [Medline] [Order article via Infotrieve]
  36. Grant, C. M., Miller, P. F., and Hinnebusch, A. G. (1994) Mol. Cell. Biol. 14, 2616-2628 [Abstract]
  37. Mueller, P. P., and Hinnebusch, A. G. (1986) Cell 45, 201-207 [Medline] [Order article via Infotrieve]
  38. Pierrat, B., Lacroute, F., and Losson, R. (1993) Gene ( Amst.) 131, 43-51 [CrossRef][Medline] [Order article via Infotrieve]

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