(Received for publication, April 10, 1997, and in revised form, June 12, 1997)
From Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
tat, an essential gene of human
immunodeficiency virus, when placed under the control of the RNA
polymerase III promoter from the adenovirus VA
RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and
Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in
VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5-untranslated region of VA-Tat RNA. The results of transfection experiments showed
that highly stable secondary structure was inhibitory to Tat synthesis,
whereas structures of lower stability were not inhibitory, confirming
that uncapped mRNA is subject to scanning. Furthermore, translation
of the downstream ORF was reduced but not eliminated by mutations that
caused the upstream ORF to overlap the Tat ORF. Extending the overlap
of the two ORFs further decreased the translation of the downstream
ORF. This observation implies that ribosomes reinitiate after
termination, possibly after migrating in a 3
to 5
direction through
the overlap region of the mRNA. Similar results were obtained with
a capped polymerase II transcript, indicating that the translation
of polymerase II and polymerase III transcripts occurs through
similar mechanisms.
All known eukaryotic cellular mRNAs are capped, and most of
them are functionally monocistronic with initiation of translation taking place at the 5 proximal AUG codon. These features are consistent with the scanning model of translational initiation (1).
According to this model, cap-binding initiation factors bind to the 5
cap structure of the mRNA and facilitate assembly of the 40 S
initiation complex. The complex then migrates along the 5
-untranslated
region (5
-UTR)1 of the RNA
until it encounters the first initiation codon, AUG, present in a
favorable sequence context, whereupon it binds the 60 S ribosomal
subunit to complete the 80 S initiation complex (1). Translation then
proceeds into the elongation phase. A recent reinterpretation of the
findings proposes that an initiation factor complex, rather than the 40 S ribosomal subunit, scans the 5
-UTR, causing unwinding of the RNA,
which then facilitates ribosome binding (2). According to this
alternative model, the complex can bind randomly along the RNA but
binds to the 5
cap most efficiently (3). Exceptions to the "first
AUG" rule were initially attributed to leaky scanning, but it is now
clear that internal initiation and reinitiation can occur. Picornaviral RNAs and some cellular mRNAs contain long 5
-UTRs with several AUGs
upstream of the authentic initiator codon. These RNAs are also
furnished with an internal ribosome entry site that allows ribosomes to
bind internally (3). The internal ribosome entry site element confers
cap-independent translation upon the mRNA and plays a role
comparable to that of the cap structure in binding the 40 S ribosomal
subunit to the mRNA for translation. The translation of mRNAs
containing upstream open reading frames (ORFs) has been thoroughly
studied in the yeast GCN4 gene (4). In this case, it has
been proposed that the ribosome, after translating an upstream ORF, may
retain competence to reinitiate at the downstream ORF.
The efficiency of mRNA translation is dependent on several features
including the local sequence context surrounding the initiation codon
(5). In accordance with the scanning model, very stable secondary
structures in the 5-UTR are inhibitory to translation, presumably
because they obstruct the movement of the scanning moiety along the
5
-UTR (5). Structures of low stability are apparently readily
penetrated and do not have any effect on the translation of the
mRNA. On the other hand, a stable structure in the coding region
does not inhibit the progress of elongating 80 S ribosomes (1). The
presence of an upstream ORF in its 5
-UTR is another feature that can
also affect the translational efficiency of the main open reading frame
of an mRNA (6). In conformity with the scanning model, the upstream
ORF generally exerts a negative effect on the translation of a
downstream ORF. Reinitiation at the downstream ORF after translation of
the upstream ORF has also been observed (7, 8). In instances where the two ORFs overlap, it has been proposed that ribosomes terminate at the
end of the upstream ORF then migrate backward to the initiation codon
of the downstream ORF where they reinitiate (9-11). This 3
to 5
movement, here called "backscanning," may not be universal, since
translation of some downstream ORFs is abolished by the introduction of
an overlapping upstream ORF (12-14). An alternative explanation for
this phenomenon is that ribosomes paused at the termination codon of
the upstream ORF may slow the progress of following ribosomes that have
bypassed the upstream AUG, thereby facilitating their initiation at the
downstream AUG.
Recently, we reported the translation in vivo of an RNA
polymerase III (pol III) product that is an uncapped and
nonpolyadenylated mRNA (15). To generate this RNA, we placed the
human immunodeficiency virus-1 (HIV-1) Tat protein ORF under the
control of the adenovirus VA RNA1 gene promoter,
a strong pol III promoter. The resultant construct, pVA-Tat,
synthesizes VA-Tat RNA that is transported into the cytoplasm and
recruited for translation, resulting in the production of functional
Tat protein in transfected cells. The translation of the pol III
transcript is inefficient compared with that of a Tat transcript
synthesized by pol II, which is capped and presumably polyadenylated.
These findings indicate that neither the cap structure at the 5 end
nor the poly(A) tail at the 3
end of an mRNA is essential for
translation, although these features may be stimulatory.
To gain insight into the mechanism of translation of an uncapped
mRNA, we examined the effects of alterations in the structure of
VA-Tat RNA. VA-Tat RNA contains a short upstream ORF that is out-of-frame with the downstream Tat ORF. Incapacitation of the upstream ORF resulted in a small increase in translation of the downstream Tat ORF, raising the possibility that the RNA is scanned (15). As a critical test of this inference, we inserted secondary structures into the 5-UTR of VA-Tat RNA and also made point mutations that caused the two ORFs to overlap. The results of transient expression assays confirm that the RNA is scanned for translational initiation despite being uncapped. Furthermore, the data are suggestive of reinitiation at the downstream AUG after termination of translation of the upstream ORF, possibly implying that ribosomes can backscan over
the short distance of the overlap sequence.
A
BamHI restriction enzyme site was created at +72 of pVA-Tat
by site-directed mutagenesis to produce pVT.Bam. For the construction of pVT.sl.60 and pVT.60, two oligonucleotides were synthesized, one
identical to and another complementary to the sequence between nt +76
and +112 of pVA-Tat (AGCTTCGACATAGCAGAATAGGCGTTACTCGACAGAG). Both
oligonucleotides carried four extra nucleotides at the 5 end (GATC)
such that they formed cohesive ends compatible with the
BamHI restriction site. The two oligonucleotides were
annealed and ligated with pVT.Bam linearized with BamHI,
giving plasmids pVT.sl.60 and pVT.60, which have the insert in the
antisense or sense orientation, respectively, with regard to the
sequence in pVA-Tat. pVT.sl.30 and pVT.30 were similarly constructed
using oligonucleotides that correspond to nt +76 to +97 of pVA-Tat. The
upstream ORF was incapacitated in all these constructs by mutating the
upstream AUG (nt +44 to +46) to GCG.
pVA-Tat
contains termination codons in-frame with the upstream AUG (Fig. 2,
S1-S6). By site-directed mutagenesis (16), these were
mutated in plasmids pVT.S1, pVT.S1-3, and pVT.S1-5. A control plasmid
transcribed by pol II, pCMV-VT, was constructed by inserting the
BglII-AflIII fragment from pVA-Tat.A (15) between
the HindIII and AflIII sites of pCMV-Tat (17)
using conventional recombinant DNA techniques (18). It contains nt +15
to +75 of the adenovirus VA RNA gene sequence inserted
upstream of the Tat sequence in pCMV-Tat. The termination codon
mutations described above were also introduced into pCMV-VT by
site-directed mutagenesis (16).
Other Methods
Transactivation of the HIV-1 LTR by Tat was
assayed as described previously (15). Briefly, HeLa cells were
transfected with pHIV-CAT alone or together with Tat constructs
(pVA-Tat, pCMV-Tat, pCMV-VA-Tat, or mutants thereof) using the calcium
phosphate transfection protocol. CAT activity was measured in lysates
prepared at 48 h post-transfection. Northern blot analysis was
used to monitor VA-Tat RNA levels and was performed as described
previously (15) using a probe that is complementary to the 5 end
sequence of VA RNA. HeLa cell nuclear extract was prepared
according to the Dignam protocol (19) and used to transcribe linearized
plasmids as described by Laspia et al. (20).
We showed previously that an RNA polymerase III product that is
uncapped and nonpolyadenylated can serve as an effective mRNA in
mammalian cells (15). The HIV-1 Tat protein ORF was placed under the
control of the strong pol III promoter from the adenovirus VA
RNA1 gene to construct pVA-Tat. In transfected HeLa
cells, pVA-Tat is transcribed to produce VA-Tat RNA, which is
transported into the cytoplasm and recruited for translation by
ribosomes, resulting in the production of functional Tat protein. This
indicated that neither the cap structure at the 5 end nor the poly(A)
tail at the 3
end of an mRNA is essential for translation.
The VA-Tat transcript is 368 nt long and contains two out-of-frame
ORFs: the first ORF is 45 nt long and lies between nucleotides 44 and
88, whereas the second, which encodes Tat, lies between nucleotides 124 and 339 and is 216 nt long. The two ORFs are separated by 37 nt, and
both have start codons in good sequence contexts for translational
initiation (AUCAUGGCG and GAAAUGGAG, respectively). Mutation of the upstream AUG to GCG (in construct pVA-Tat.I), effectively eliminating the upstream ORF, increases synthesis of Tat from the downstream ORF by about 2-fold (15). This
inhibitory effect of the upstream AUG raised the possibility that the
RNA is scanned. To examine this possibility, we investigated the effect
of introducing secondary structure into the 5-UTR of VA-Tat.I RNA.
Stable secondary structure in the 5
-UTR is envisaged to obstruct the
movement of ribosomes or other moieties that are scanning the RNA from
the 5
end, preventing their access to the start codon. Therefore, the
inhibitory effect of secondary structure in the 5
-UTR on the
translation of an mRNA provides evidence for scanning (1). To
create secondary structure in the 5
-UTR of VA-Tat.I RNA, a stretch of
sequence from the 5
-UTR was duplicated in an inverted orientation such
that the pairing of the two sequences forms a stem structure topped by
a 4-nt loop (Fig. 1A). The
plasmid pVT.sl.30 contained 22 nt (nt +76 to +97 with respect to the
transcription start site) inserted at +76, and pVT.sl.60 contained 37 nt (+76 to +112) inserted at the same position. The predicted stability of the stem and loop structures in VT.sl.30 RNA and VT.sl.60 RNA was
31 and
60.5 kcals, respectively. Two control plasmids, pVT.30 and
pVT.60, were also constructed that contained the equivalent sequences
inserted in the sense orientation; transcripts of these plasmids were
not predicted to form stable secondary structures in the 5
-UTR.
The translational activities of these transcripts were tested in an
HIV-LTR transactivation assay, which takes advantage of the fact that
HIV-1 Tat protein stimulates transcription from the HIV-LTR (21).
pVA-Tat or derivatives thereof were transfected with the reporter
construct pHIV-CAT, which contains the HIV-LTR driving the CAT reporter
gene. The expression of Tat protein was monitored by measuring the
increase in CAT activity in cells cotransfected with Tat-producing
plasmids as compared with basal CAT activity in cells transfected with
pHIV-CAT alone. Fig. 1B shows the results of such an assay
performed using the stem and loop insertion mutants of pVA-Tat. Cells
transfected with parental pVA-Tat.I produced 20-60-fold higher levels
of CAT enzyme activity than cells transfected with pHIV-CAT only. For
purposes of comparison, the level of transactivation given by the
parental Tat plasmid was assigned a value of 100. Cells transfected
with control constructs (pVT.30 and pVT.60) that were not predicted to
contain secondary structure in their 5-UTR produced levels of CAT
activity about 20% lower than that of pVA-Tat.I. The increased length
of the 5
-UTR probably accounts for this small decrease in activity.
Cells transfected with pVT.sl.30 exhibited similar CAT enzyme activity,
whereas transactivation by pVT.sl.60, which encodes a structure of
higher stability (
G =
60.5 kcal/mol), was 5-fold lower than
that given by the parental plasmid. Northern blot analysis using a
probe complementary to the 5
end of VA RNA verified that
similar levels of RNA were produced by pVA-Tat.I, pVT30, and pVT60, but
pVTsl30 and pVTsl60 transcripts were not detected (data not shown),
presumably because their secondary structures interfere. It is unlikely
that these transcripts are degraded, however, because secondary
structure generally increases RNA stability, and pVTsl30 expresses
nearly as much transactivation activity as the parent plasmid,
pVA-Tat.I.
These results resemble those reported for a preproinsulin mRNA that
is transcribed by pol II (1), indicating that VA-Tat RNA is indeed
scanned during translation despite its uncapped status. The scanning
mechanism seems able to unwind secondary structures of low stability
(G =
30 kcal/mol) but is blocked by more stable structures
(
G =
60 kcal/mol) in an uncapped mRNA as in capped
mRNA (1). Furthermore, the decrease in activity accompanying the
increased length of the 5
-UTR in VT.30 and VT.60 and VT.sl.30 is
similar to that observed for capped mRNAs and is consistent with
ribosome scanning. Our results indicate that the scanning process does
not require the 5
cap structure, but they do not necessarily exclude a
role for the cap-binding initiation factor, eIF4F, and associated
factors. Conceivably, such factors are recruited by uncapped mRNA,
albeit inefficiently, by a cap-independent mechanism.
Given that VA-Tat RNA is scanned, it is not surprising that mutation of
the termination codon of the upstream ORF of VA-Tat RNA, causing the
two ORFs to overlap by 10 nt, leads to a decrease in transactivation
activity (Ref. 15; Fig. 2B).
The magnitude of the decrease (about 2-fold) is relatively slight,
however, prompting us to consider possible explanations for the small
effect. First, the start codon of upstream ORF may be leaky,
i.e. not recognized by ribosomes despite its good context,
so the ribosomes proceed to scan to the Tat AUG. Second, as VA-Tat RNA
lacks a cap structure at its 5 end, ribosomes might bind to VA-Tat RNA randomly and then commence scanning; those ribosomes binding between the two AUGs should not be affected by the overlap of the two ORFs.
Third, ribosomes might reinitiate after translating the upstream ORF,
an exercise that would seem to require 3
to 5
ribosome movement
(backscanning) across the overlap region. Backscanning has been
observed over a span of 80-90 nt (10), but an overlap of 92 nt was
resistant to backscanning (9). To distinguish among the three
possibilities with regard to VA-Tat RNA translation, we studied the
effect of increasing the extent of the overlap of the two ORFs to more
than 100 nt. If either of the first two explanations pertains,
increasing the overlap would not be expected to affect the translation
of the Tat ORF. On the other hand, if the Tat ORF is translated by
reinitiation after backscanning, an increase in the length of the
overlap would be expected to decrease Tat ORF translation, since
backscanning is observed only over short distances.
We exploited the six termination codons present in-frame with the upstream AUG of VA-Tat RNA (Fig. 2A, S1-S6) to extend the overlap. By site-directed mutagenesis, the in-frame stop codons were eliminated sequentially without altering the amino acids encoded by the Tat ORF. The resultant plasmids, pVA-Tat.S1, S1-3, and S1-5, contained mutations in the first stop codon, in the first three stop codons, and in the first five stop codons, respectively, yielding overlaps between the two ORFs of 10, 115, and 220 nt (Fig. 2A). The mutants were then tested in the HIV-CAT transactivation assay, giving results shown in Fig. 2B. Northern blot analysis indicated that similar levels of RNA were generated by the mutant and parental plasmids (data not shown). All constructs with overlapping ORFs produced decreased CAT activity compared with the wild type pVA-Tat construct, and extending the overlap from 10 to 115, or 220 nt, caused a decrease in translation of the downstream ORF. pVT.S1, which contains an overlap of 10 nt, was about 50% as active as wild type pVA-Tat, whereas pVT.S1-3 and pVT.S1-5, which have overlaps of 115 and 220 nt, respectively, both produced only about 20% of wild type activity. These results imply that Tat activity from RNA containing the S1 mutation only (10-nt overlap) may be partially accounted for by reinitiation requiring backscanning. Since increasing the overlap from 115 to 220 nt gave no further decrease in Tat activity, the Tat activity produced by these constructs may be due to leaky scanning. In other words, it seems that only 20% of the ribosomes scanning VA-Tat RNA failed to recognize the first AUG, and of those that do initiate translation at the upstream AUG, about 40% are able to backscan after translating the upstream ORF and reinitiate at the Tat ORF.
To test the effect of the overlaps on the translation of RNA generated
by pol II, the same mutations were introduced into pCMV-VT that
contained the CMV immediate early promoter, driving transcription of
the VA-Tat sequence (Fig. 3A).
The construct contains a polyadenylation signal downstream of the Tat
sequence to ensure proper termination and processing of the pol II
transcript, and the first 11 nt of VA-Tat sequence were deleted so as
to remove the A box of the VA RNA gene promoter and impair
pol III transcription. To verify that transcription of this plasmid was
indeed dependent on pol II, its sensitivity toward -amanitin was
tested in a transcription assay in vitro (Fig.
3B). The plasmid was cut at +189 nt (with respect to the CMV
transcription start site) with MfeI, and run-off transcription was performed in HeLa cell nuclear extract in the presence and absence of
-amanitin. Fig. 3B shows that
transcription from pCMV-VT, like that from pCMV-Tat which also contains
a pol II promoter (17), was eliminated by 5 µg/ml
-amanitin,
whereas transcription from pVA-Tat containing pol III promoter was not. Thus transcription of pCMV-VT is dependent on the pol II promoter as a
result of the deletion in its VA RNA gene promoter.
In the transactivation assay (Fig. 3C), overlap of the ORFs by 10 nt (S1) caused a decrease in translation of the downstream Tat ORF of CMV-Tat by about 40%, and increasing the length of the overlap (S1-3 and S1-5) caused a further decrease in expression. The additional decrease was small, however, possibly indicating that backscanning can occur over longer distances in the case of capped pol II transcripts than in the uncapped pol III transcripts. Mutation of the upstream AUG, which eliminates the upstream ORF, abolished the inhibition of Tat ORF translation in these stop codon mutants as expected (Fig. 3D), verifying that the effect of the stop codon mutations on Tat ORF translation is dependent on the integrity of the upstream ORF.
We also tested the effects of overlapping ORFs in a wheat germ cell-free translation system using both capped and uncapped mRNAs synthesized in vitro. As observed in vivo, the overlap of the two ORFs was inhibitory to translation of the downstream Tat ORF. In RNAs that contained long overlaps of the two ORFs, the translation product from the downstream ORF was reduced significantly (data not shown). Similar results were obtained with both capped and uncapped RNA, confirming that capped RNA and uncapped RNA are translated by similar mechanisms that allow backscanning.
The 7-methyl guanosine cap structure that characterizes the 5 end of
eukaryotic mRNAs has a role in their stability, processing, and
transport into the cytoplasm. It also enhances the efficiency with
which mRNA binds initiation factor eIF4F (22). This factor being
limiting in most eukaryotic cells, capped mRNA has a clear advantage for translation initiation (22-24). In this paper we assessed the significance of cap for the mechanism of translational initiation. We found that capped and uncapped mRNA are translated by similar mechanisms although with very different
efficiencies.2 Both were
scanned from the 5
end of the RNA, indicating that the process of
scanning does not require the cap. In addition, both allowed
translation to initiate at a downstream ORF after the translation
of an upstream ORF. When the two ORFs overlap, reinitiation appears to
involve movement of the ribosomes in a backward direction along both
capped and uncapped mRNA. We therefore concluded that the
mRNA cap is not an essential feature of the translation initiation
mechanism, although it is possibly required for efficient translation
by virtue of recruiting eIF4F.
Eukaryotic cellular mRNA is believed to be synthesized exclusively
by RNA polymerase II, but the mammalian cell is capable of utilizing an
RNA pol III transcript as an mRNA to produce functional protein
(15). Although pol III genes are transcribed with great efficiency, the
translational efficiency of the resultant RNAs is very low due to the
absence of the 5 cap. Therefore it seems likely that one of the
reasons why pol III genes have not been shown to code for proteins is
the lack of the cap on their transcripts. Mechanisms such as
trans-splicing and "cap-snatching" are used by trypanosomes (25)
and influenza virus (26) to cap mRNAs that are transcribed by
polymerases other than pol II (pol I and viral polymerase,
respectively). It remains to be seen whether a comparable mechanism for
capping of uncapped cellular RNA exists and allows the efficient
translation of pol III transcripts.
We thank Patricia Wendel for technical help.