From the
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.
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 MAT
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),
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
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.
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%).
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-2
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-2
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
upf1
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (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) .
(
)
without stabilizing the mRNA
(14, 15, 16) . This clearly constitutes a
challenge to any model proposing tight coupling between translation and
mRNA decay.
, which in turn inhibits the eIF-2B-catalyzed exchange of
GDP/GTP on eIF-2. Regulation of the availability of eIF-2
GTP in
this way is thought to influence start-site selection within the
leader.
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.
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.
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.
GTP 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.
GTP, 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.
gene 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 upf1
mutant
(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.
Table: Sequences of the 5`-UTRs
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.