From the
An infectious, in vitro transcript from a full-length
cDNA clone of the barley yellow dwarf virus (PAV serotype) genome
translated efficiently in a wheat germ translation extract. Deletions
in a region that we call the 3` translational enhancer, located between
bases 4,513 and 5,009 in the 5,677-base genome, reduced translation of
the 5`-proximal open reading frames from uncapped RNA by at least
30-fold. Deletions elsewhere in all but the 5` end of the genome had no
effect on translation. Presence of a m
Luteoviruses are particularly rich in unusual translational
control mechanisms
(1, 2) , e.g. ribosomal
frameshifting
(3, 4) and stop codon
suppression
(5) . The genomic RNA of luteoviruses is about 5.7
kilobases long and is not polyadenylated
(6, 7) . The
genes in the 5`-half of the genome are translated directly from genomic
RNA, whereas the 3`-half is expressed via subgenomic
mRNA(s)
(5, 8, 9) . The luteoviruses, including
the barley yellow dwarf viruses (BYDVs),
The 3`-UTRs of eukaryotic mRNAs can also regulate
initiation of translation
(22, 23) . A ubiquitous 3`-UTR
sequence that enhances translation initiation is the polyadenylate
sequence (poly(A) tail) of 30 to 200 adenosine residues at the 3`
termini of most eukaryotic mRNAs (24-27). Other sequences can
functionally substitute for a poly(A) tail. For example, the
pseudoknot-rich portion of the 3`-UTR of tobacco mosaic virus RNA,
which is not polyadenylated, enhances the translation efficiency in
vivo(28) . The 5` cap and this pseudoknot-rich domain or a
poly(A) tail stimulate translation synergistically
(27) . In the
case of satellite tobacco necrosis virus (STNV), which has an uncapped,
nonpolyadenylated, 1239-nucleotide (nt) RNA
genome
(14, 29) , the 3`-UTR facilitates cap-independent
translation in vitro of its coat protein gene or heterologous
genes in the presence of the viral 5`-UTR
(30, 31) . Here
we report that BYDV-PAV genes are translated cap-independently in wheat
germ extracts. A sequence of at most 500 bases, located between 4.5 and
5 kilobases from the 5` end of the viral genome, stimulates translation
of viral or heterologous reporter genes from uncapped RNA by 30- to
over 100-fold. This cap-independent translation may also depend on the
presence of a portion of the 5` end of the viral genome. This report
should further our understanding of interactions between mRNAs and
eukaryotic translation machinery.
Construction of a full-length clone
(pPAV6), from which infectious BYDV-PAV genomic RNA can be transcribed
in vitro, was described previously
(4) . To make pPAVM1,
pPAV6 was digested with EcoRI to remove the
EcoRI
pCIGUS was made so that
the GUS coding sequence was flanked by the 5`-terminal 169 nt of
BYDV-PAV RNA at its 5` end and bases 4513-5677 in its 3`-UTR. We
started with clone pPAVGUSRT8
(34) which contains the GUS gene
including an upstream multiple cloning site from pAGUS1
(33) inserted between base 3477 and the ScaI
pT7GUSA(+) was constructed to add a poly(A) tail
to the GUS 3`-UTR. pT7GUS1 was first digested with SmaI and
ScaI. Then the fragment containing the GUS gene was
gel-purified and ligated to SmaI-ScaI-digested
pSP64poly(A) (Promega), which has a run of 30 adenosine residues
between the SmaI and EcoRI sites in its multiple
cloning site. pGUSEA1 was made by cloning the
PstI-EcoICR I fragment of pCIGUS that spans the T7
promoter, the first 169 nt of BYDV-PAV genomic RNA, and the GUS coding
region into pSP64poly(A) cut with the same enzymes.
Translation in vitro was carried out with 8 ng/µl
capped or uncapped transcripts as mRNAs in a wheat germ extract in the
presence of 0.34 µM [
The
amount of 39-kDa translation product from all capped transcripts varied
less than 3-fold, regardless of the 3` truncation (,
Fig. 1B). Surprisingly, uncapped transcripts containing
bases up to and 3` of base 5009 (PstI site) yielded one-third
to one-half as much 39-kDa product as their capped counterparts, but
those which contained larger 3` truncations, i.e. lacked bases
3` of nucleotide 4513 (ScaI site), yielded 30- to 50-fold less
39-kDa product (; Fig. 1B, lanes 10 and 12) than either their capped forms or the larger,
uncapped transcripts. Thus, the translation of viral RNA was nearly
cap-independent when transcribed from viral cDNA digested with
SmaI, BclI, or PstI. In contrast,
translation of transcripts from pPAV6 linearized at the
ScaI
These experiments also revealed that a more distal portion of the
genome appears to be required for efficient -1 ribosomal
frameshifting. As reported previously
(4) , the 99-kDa product of
ribosomal frameshifting can be seen clearly among the translation
products of full-length (SmaI-linearized) transcript
(Fig. 1B, lanes 3 and 4). However,
transcripts truncated at the BclI
To further define the
sequence(s) necessary for cap-independent translation, transcripts
synthesized from internal deletion mutants and linearized with various
restriction enzymes were translated in wheat germ extracts
(Fig. 2). Mutants containing any deletions between the
BalI
To test the second possibility that 3`-TE(-)
transcripts were much less stable than 3`-TE(+) transcripts, their
stabilities in the translation reaction were assessed by Northern blot
hybridization. Approximately 50% of the transcripts from pPAV6
linearized with either ScaI (3`-TE(-)) or SmaI
(3`-TE(+)) remained intact after 1 h of incubation in the wheat
germ extract under our standard translation conditions (Fig. 3).
Thus, the stabilities of 3`-TE(+) and 3`-TE(-) transcripts
were indistinguishable.
The results presented here reveal that sequence(s) within a
region near the 3` end of the genome (bases 4513-5009), of
BYDV-PAV RNA confer the ability of a BYDV-PAV gene or a heterologous
gene to be translated very efficiently from uncapped mRNAs in wheat
germ extracts. The viral 5` leader (bases 1-169) may also be
required, but only one alternative leader was tested. It is possible
that other efficient (e.g. viral) leaders may substitute for
that of BYDV-PAV. Other known stimulatory sequences include the 5`
leaders of tobacco mosaic virus
(16) , alfalfa mosaic virus
RNA4
(17) , and potato virus X
(18) , but these do not
replace the need for a cap. Indeed, the 5`-UTR of BYDV-PAV alone
stimulates such cap-dependent translation relative to a vector-derived
5`-UTR (Fig. 7). The 5`-UTRs of tobacco etch virus
(19) ,
potato virus S
(44) , and picornaviruses (45) confer
cap-independent translation, but do not require the presence of a
sequence 3` of the coding region. The poly(A) tail in the 3`-UTRs of
most mRNAs stimulates translation, but has only modest effects in
vitro as observed by us (Fig. 8) and
others
(25, 46) . Thus, the observation reported here in
which the 3`-UTR functionally substitutes for a 5` cap structure
requires a revision of the traditional concepts regarding the
mechanisms of translation initiation in which mRNA recognition requires
only 5`-terminal structures and sequences
(47) .
The
stimulation of translation of uncapped mRNA by the 3`-UTR resembles the
behavior of the 3`-UTR of STNV RNA. On STNV RNA, the 5` 150 bases of
the 3`-UTR and a portion of the 5`-UTR act together to facilitate
cap-independent translation
(30, 31) in wheat germ
extracts. However, this naturally uncapped RNA
(14) differs in
several ways from BYDV-PAV RNA. (i) It is only 1239 nt
long
(29) , with a 29-base 5`-UTR, 600-base ORF that encodes coat
protein, and a 600-base 3`-UTR. (ii) The 3` stimulatory region in the
3`-UTR is adjacent to the ORF it stimulates in contrast to the 3`-TE in
BYDV-PAV RNA which is separated from the stimulated (39K) ORF by
several ORFs and kilobases. (iii) The 5`- and 3`-UTRs of STNV stimulate
translation of heterologous genes (
The
discovery of this cap-independent translation enhancer has led us to
wonder whether the 5` end of the BYDV-PAV genome is capped.
Previously
(7) , we proposed that the BYDV-PAV genome contains a
VPg because subgroup II luteoviral RNAs have
VPgs
(6, 21) . However, being a subgroup I luteovirus,
much of the genome of BYDV-PAV, including the essential replication
genes, is more closely related to dianthoviruses which have capped
mRNAs
(48) . Preliminary evidence indicates that neither of these
structures is present and that BYDV-PAV RNA is uncapped: the 5`
terminus of BYDV-PAV RNA from virions is accessible to alkaline
phosphatase and polynucleotide kinase, with or without pretreatment
with the cap-removing enzyme, tobacco acid pyrophosphatase.
Absence of a 5` cap would suggest that the 3`-TE is
necessary for virus viability. Consistent with this, all deletions in
the 3`-TE render the RNA noninfectious in protoplasts
(34) , even
though the proteins encoded by the 50K and 6.7K ORFs in which much of
the 3`-TE resides are unnecessary for replication in protoplasts. Even
if BYDV-PAV RNA were naturally capped, a role for a cap-independent
translation element would not be unprecedented. Naturally capped mRNAs
of the immunoglobulin heavy chain binding protein gene and a few other
eukaryotic cellular genes contain sequences that allow efficient
cap-independent translation in vivo(45) .
How can a
3` sequence behave like a cap? Gallie and Tanguay
(46) showed
that a poly(A) tail is bound by the same factors (eIF-4F and eIF-4B)
that bind the m
Mechanisms by which the 3`-TE may act by enhancing translation
initiation at the 5` end can be envisioned by comparison with STNV RNA.
Danthinne et al.(30) and Timmer et al.(31) proposed that direct base pairing occurs between small
stretches of the 3` translation enhancing domain of STNV RNA and the
5`-UTR to facilitate return of ribosomes that have completed
translation to the 5` end of the genome. In BYDV-PAV RNA, there were no
obvious, phylogenetically conserved regions in the 5`-UTR to which
portions of the 3`-TE might base pair, nor were any striking
similarities between STNV and BYDV-PAV primary or secondary structures
detected. Danthinne et al.(30) identified a potential
18 S ribosomal RNA binding sequence in the 3`-UTR of STNV RNA. Upon
sequence comparison of three complete subgroup I luteoviral
genomes
(52, 53) and partial sequences of nine
additional BYDV-PAV isolates
(54) , we found an intriguing
17-base sequence, beginning at the BamHI
A possibility remains that the 3`-TE acts by preventing
degradation of uncapped mRNA. Because stability of total added mRNA in
wheat germ was unaffected by the presence of the 3`-TE (Fig. 3),
differential stability could be explained only if just a few percent of
the mRNA molecules were actually associated with polysomes and being
translated. If this polysomal fraction alone were subject to
instability in the absence of 3`-TE, the degradation might not be
detected in our Northern blots. In this case, either the 3`-TE or a 5`
cap must prevent instability.
Although the mechanism by which a 3`
sequence can substitute for a 5`-m
We thank S. P. Dinesh-Kumar and David Higgs for
assistance with plasmid construction and technical advice and Di Rong
for early observations that led to this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
G(5`)ppp(5`)G cap on
the 5` end fully restored translational efficiency of transcripts
lacking the 3` translational enhancer. The translation enhancer reduced
inhibition of translation by free cap analog, did not affect RNA
stability, and did not function in reticulocyte lysates. When placed in
the 3`-untranslated region of uncapped mRNA encoding the
-glucuronidase gene, the translation enhancer stimulated
translation more than 80-fold, in the presence of the viral, but not a
plasmid-derived, 5` leader. A polyadenylate tail could not substitute
for the 3` translation enhancer. These observations provide an extreme
example, in terms of distance from the 5` end and level of stimulation,
of an mRNA in which a sequence near the 3` end stimulates translation.
(
)
have
been classified into subgroups I and II, according to genome
organization and other biological
properties
(1, 2, 10, 11) . The 5`-halves
of the genomes of the two subgroups are essentially unrelated, whereas
the 3`-halves of all luteoviruses have significant homology, with the
exception of the approximately 800 3`-terminal bases of subgroup I
genomes which are absent in subgroup II luteoviruses. The 5`-half of
the genome of BYDV-PAV, a member of subgroup I, encodes a 39-kDa
protein (39K ORF) and an overlapping open reading frame (60K ORF) which
has the conserved amino acid motifs shared by RNA-dependent RNA
polymerases
(6) . This 60K ORF is expressed via a -1
ribosomal frameshift at the end of the 39K ORF to produce a 99-kDa
protein (Fig. 1A)
(3, 4) .
Figure 1:
Effect of 3` truncation on translation
of transcripts of the BYDV-PAV genome. A, genome organization
of BYDV-PAV RNA showing locations of ribosomal frameshift site and
subgenomic RNAs (sgRNAs 1, 2, and 3) detected in infected cells but not
virions (5, 9). Selected restriction sites are numbered according to
their position in the genome (7). Abbreviations: Kp,
KpnI; Sc, ScaI; Ps, PstI;
Bc, BclI; Sm, SmaI. B,
wheat germ translation products of transcripts of BYDV-PAV cDNA clone
pPAV6 transcribed in the presence (C, lanes 3,
5, 7, 9, and 11) or absence
(U, lanes 4, 6, 8, 10, and
12) of mGpppG. Lane 1, products of BMV
RNA with molecular masses in kilodaltons shown at left;
lane 2, no added template; lanes 3-12,
transcripts of pPAV6 linearized with the following restriction
endonucleases; lanes 3 and 4, SmaI
(full-length, infectious transcripts); lanes 5 and 6,
BclI; lanes 7 and 8, PstI;
lanes 9 and 10, ScaI; lanes 11 and
12, KpnI. Expected sizes, in kilodaltons, of
translation products from BYDV-PAV transcripts are shown on the
right. All reactions were performed with 0.2 µg of added
mRNA in a volume of 25 µl at 25 °C for 1 h in the wheat germ
extract (Promega), containing 0.34 µM
[
S]methionine (1,071
Ci/mmol).
The 5`
termini of eukaryotic cellular mRNAs have a cap structure
(mG(5`)ppp(5`)N)
(12, 13) , while the 5`
termini of viral RNAs can be either capped, uncapped
(14) , or
covalently linked to a viral protein (VPg)
(15) . Genomes of
viruses with any of these structures can have sequences near the 5` end
that enhance translation. For example, the 5`-untranslated regions
(UTRs) of naturally capped RNAs of tobacco mosaic virus
(16) ,
alfalfa mosaic virus
(17) , and potato virus X
(18) and of
VPg-linked viral RNAs such as tobacco etch virus
(19) and the
picornaviruses
(20) all stimulate translation by a variety of
mechanisms. Although a VPg is attached to the 5` termini of subgroup II
luteoviral genomes
(6, 21) , the chemical nature of the
5` terminus of BYDV-PAV and other subgroup I luteoviruses has not been
reported.
Construction of Internal Deletion Mutants
DNA
manipulations were performed essentially as described by Sambrook
et al. (32). All plasmids were cloned in Escherichia coli (strain DH5f`) except when BclI was used to digest
the DNA, in which case plasmids were prepared in
dam
strain GM33. Restriction sites discussed
below are numbered by the base position in the BYDV-PAV
genome
(7) . pAGUS1
(33) was a gift from J. Skuzeski, now
at Oregon State University.
-EcoRI
fragment
and religated. The same strategy was used to make pPAVM2, in which the
BamHI
-BamHI
fragment
was deleted from pPAV6. To construct pSPM, pPAV6 was first digested
with ScaI which cuts at base 4513 in the BYDV-PAV cDNA genome
and in the ampicillin resistance gene in the vector. The resulting
5.5-kilobase fragment was purified by 1% low melting point agarose gel
electrophoresis and digested with PmlI, and this
ScaI
-PmlI
fragment was ligated with ScaI
-ScaI
fragment to give rise to
pSPM in which the PmlI
-ScaI
fragment was deleted from the BYDV-PAV genome. To delete the
BalI
to ScaI
region of
the genome, pPAV6 was digested with these enzymes and religated. After
gel purification, the
BalI
-ScaI
fragment was
discarded, and the remaining two fragments were ligated to give rise to
pBS6.
Construction of BYDV-PAV/GUS Chimeric RNA
The
E. coli uidA gene which encodes -glucuronidase (GUS) was
cloned from NcoI-EcoICR I-digested pPAGUS1
(33) into similarly cut pGEM5Zf(+) (Promega) to give rise
to pT7GUS1. To construct pT7GUS3`-d2, the ScaI
-ScaI
fragment of pPAV6 was cloned
into EcoICR I-linearized pT7GUS1.
site in pPAV6. pPAVGUSRT8 was digested with Eco47
III
and Bsp120 I (codon 5 of the GUS ORF), made
blunt-ended with T4 polymerase, and religated to produce pCIGUS. In
pCIGUS, the entire 5`-UTR and the first nine codons of the GUS coding
sequence were derived from the first 169 nt of BYDV-PAV RNA. The
ScaI site was destroyed in the process, so pCIGUS was
linearized at a unique EcoICR I site in the GUS-derived
portion of the 3`-UTR to generate transcripts lacking viral sequences
in the 3`-UTR.
In Vitro Transcription and Translation
Capped and
uncapped RNAs were prepared by in vitro transcription with T7
DNA-dependent RNA polymerase of the linearized plasmids using the
Ambion Inc. mMessage mMachine and MegaScript
kits, respectively, according to the manufacturer's
instructions. An 8:1 ratio of m
G(5`)ppp(5`)G:GTP was used
in reactions to produce capped transcripts. The integrity of all the
transcripts was ascertained by 1% agarose gel electrophoresis, and the
concentration of the RNA was determined spectrophotometrically.
S]methionine
(1,175 Ci/mmol) in 25-µl reactions for 1 h as described by the
manufacturer (Promega) or in a rabbit reticulocyte lysate (Promega) as
described previously
(5) , except where indicated.
S-Labeled polypeptides were resolved by loading one-fifth
of the in vitro translation reaction mixture on a 5% stacking
and 10% resolving polyacrylamide gel and electrophoresis using a
discontinuous buffer system
(35) . After the gel was fixed in a
25% ethanol, 10% acetic acid solution overnight, it was prepared for
fluorography by shaking in 20 volumes of dimethyl sulfoxide, twice,
each for 30 min. Then the gel was treated by gentle shaking in a 20%
(w/v) solution of 2,5-diphenyloxazole in dimethyl sulfoxide. After the
gel was soaked in water with shaking for 30 min and dried,
radioactivity was visualized and digitized by exposure to a
PhosphorImager 400E (Molecular Dynamics). Radioactivity was quantified
using Imagequant 3.22 software (Molecular Dynamics). Thus, the
``relative units'' of radioactivity used in this manuscript
are arbitrary.
RNA Stability Assay
1-2 pmol of uncapped
transcripts from SmaI- or
ScaI
-linearized pPAV6 were incubated in the
wheat germ extract in the translation conditions (above). At indicated
time points, 5-µl aliquots were removed. RNA was extracted with
phenol using an aurin tricarboxylate buffer followed by lithium
chloride precipitation as described by Wadsworth et al.(36) and modified by Seeley et al.(37) . The
final ethanol-precipitated RNA pellet was resuspended in 10 µl of
diethyl pyrocarbonate-treated water. 5 µl of this RNA was subjected
to electrophoresis in a 0.8% agarose gel for Northern blot
hybridization. The conditions used for probe labeling and Northern
hybridization were described previously
(5) . The probe was
P-labeled RNA, complementary to BYDV-PAV bases
2737-2985, derived by in vitro transcription of
EcoICR I-linearized pSP9
(5) .
Identification of a Sequence That Allows BYDV-PAV RNA
to Be Translated Efficiently in the Absence of a
5`-m
To ensure that
translation conditions were sensitive to changes in the translation
efficiency of the RNA template
(38, 39) , a subsaturating
mRNA concentration of 8 ng/µl (4 nM for full-length
BYDV-PAV RNA) was used in the translation reactions
(4) . RNAs
containing a guanosine 5`-triphosphate or a
mG(5`)ppp(5`)G Cap Structure
G(5`)ppp(5`)G-cap were transcribed from pPAV6 templates
linearized with SmaI
,
BclI
, PstI
,
ScaI
, or KpnI
and
translated in the wheat germ extracts under previously optimized
conditions
(4) (Fig. 1B). Despite its anomalously
slow mobility and occasional appearance as a doublet, the prominent
band was shown previously to be the product of the 39K ORF (4, 40).
Because the vast majority of incorporated
[
S]methionine was in the 39-kDa polypeptide, the
translation efficiency was defined as the amount of radioactivity
incorporated in this protein as measured with a PhosphorImager.
or upstream sites was highly
cap-dependent. Thus, a sequence between the ScaI
site and the PstI
site is at least in
part responsible for efficient translation in the absence of a 5` cap.
site gave
less frameshift product, and those with larger deletions yielded no
frameshift products at all (Fig. 1B, lanes
7-12). Thus, sequences near the 3` end of the genome are
required for efficient frameshifting at bases 1152-1158. This
remarkable phenomenon was unaffected by capping and it will be
discussed in a separate publication.
and ScaI
sites were
still translated cap-independently, if the transcripts were synthesized
from PstI- or SmaI-linearized templates
(Fig. 2). Cap-independent translation was abolished in
transcripts linearized at BamHI
(Fig. 2).
Transcripts synthesized from SmaI- and
PstI-linearized pPAVM2, in which the sequence immediately 5`
of BamHI
was deleted, also failed to permit
cap-independent translation. We conclude that (i) no sequences from the
BalI
to ScaI
sites and
from PstI
to the 3` end of the genome
(SmaI
) are required for cap-independent
translation, (ii) the regions from ScaI
to
BamHI
or BamHI
to
PstI
alone were insufficient, and (iii)
reducing the spacing between the ScaI-PstI region and
the 5` end of the genome did not reduce cap-independent translation.
Thus, a region of the RNA between bases 4513 and 5009 is required for
BYDV-PAV RNA to be translated independently of the presence of a
5`-m
G(5`)ppp(5`)G cap. We define the required
ScaI
to PstI
region as
a 3` translation enhancer (3`-TE). In the following experiments, the
transcripts having intact 3`-TE are called 3`-TE(+), whereas
transcripts having no, or an incomplete 3`-TE, are 3`-TE(-).
Figure 2:
Effect of deletions in BYDV-PAV
transcripts on cap-independent translation. Schematic diagram of
BYDV-PAV cDNA clones with internal deletions and corresponding
transcripts from these clones after linearization with various
restriction enzymes. Bold lines below genome organization
represent transcribed RNAs containing deletions indicated by the
dotted lines. Deletion and 3` truncation sites correspond to
the restriction endonuclease sites indicated below genome organization.
The dependence of each transcript on a 5` cap for efficient translation
is indicated at the right. ``+'' indicates that the
translation efficiency (yield of product from 39K ORF or its deleted
forms) of uncapped transcripts is no more than 3-fold lower than that
of their capped counterparts, and 30-50-fold higher than that of
uncapped transcripts indicated by ``-.''
``-'' also indicates that the translation efficiency of
uncapped transcripts is 30-50-fold lower than that of their
capped counterparts. The 3`-TE is
shaded.
Analysis of Conditions for Cap-independent
Translation
The reduced amount of 39-kDa product from uncapped,
3`-TE(-) transcripts could result from (i) a sequence-specific
difference in optimal ionic conditions for in vitro translation, (ii) sharply decreased stability of the transcript,
(iii) inactivation of critical factor(s) involved in translation in the
wheat germ extract due to unknown reasons, or (iv) a decreased level of
translatability of the transcript. These possibilities were tested.
Because different mRNAs can have different optimal potassium ion
concentrations
(38, 41) , these concentrations were
varied. At all potassium ion concentrations, uncapped, 3`-TE(-)
transcripts gave extremely low amounts of 39-kDa product which was
about 25- to 50-fold less than that from uncapped 3`-TE(+)
transcripts (data not shown). Thus, the extreme cap dependence of the
3`-TE(-) transcripts could not be compensated for by changes in
ionic conditions.
Figure 3:
The 3`-TE does not affect transcript
stability in wheat germ extracts. pPAV6 was linearized with
SmaI or ScaI (Fig. 1A) prior to
transcription. Transcripts were added to a standard wheat germ
translation system as in Fig. 1, with aliquots being removed at
indicated time points. Total RNA was extracted from the translation
mixture and analyzed by Northern blot hybridization (see
``Materials and Methods''). A, uncapped transcripts
from SmaI-cut pPAV6. B, uncapped transcripts from
ScaI-cut pPAV6. C, quantitation of transcripts as in
A and B. Data represent averages of two and three
experiments for SmaI-linearized (filled circles) and
ScaI-linearized (open circles) transcripts,
respectively.
To test the third possibility, that the
absence of cap-independent translation in the uncapped, 3`-TE(-)
transcripts was due to some kind of selective inactivation of the wheat
germ translation system, translation of naturally capped brome mosaic
virus (BMV) RNAs was observed in the presence of various BYDV-PAV
transcripts. BMV RNA was translated with the same efficiency in the
presence of capped or uncapped, 3`-TE(-) or 3`-TE(+)
BYDV-PAV transcripts (Fig. 4, lanes 1 and
6-9). Conversely, the presence of BMV RNA did not affect
the translation of capped or uncapped, 3`-TE(-) or 3`-TE(+)
transcripts (Fig. 4, compare lanes 2 and 6,
3 and 7, 4 and 8, and 5 and 9). Thus, there was no inhibition of the wheat germ
translation machinery by the uncapped, 3`-TE(-) BYDV-PAV
transcript. The lack of competition between BMV and BYDV-PAV RNAs
verified that the mRNA levels were subsaturating and thus
rate-limiting. All the above results taken together support the
hypothesis that the 3`-TE acts to increase efficiency with which
ribosomes and translation factors initiate translation of the uncapped
viral transcript.
Figure 4:
Effect of capped and uncapped,
3`-TE(+) and 3`-TE(-) pPAV6 transcripts on translation of
BMV RNA. The reactions are the same as in Fig. 1B, except
that, in lanes 6-9, 0.1 µg of BMV RNA was added to
the translation mixture. Even-numbered lanes contain products
of capped transcripts (C); lanes 3, 5,
7, and 9 contain products of uncapped transcripts
(U). Lane 1, BMV RNA only; lanes 2,
3, 6, and 7, transcripts from
SmaI-linearized pPAV6; lanes 4, 5,
8, and 9, transcripts from ScaI-linearized
pPAV6. Mobilities (in kilodaltons) of BMV translation products and
expected major translation product from pPAV6 transcripts
(arrow) are at left.
The 3`-TE appears to functionally substitute for a
cap structure. Thus, we tested whether addition of free cap analog
inhibited translation of 3`-TE(+) transcripts differently from
3`-TE(-) transcripts. As shown in Fig. 5, twice as much
mG(5`)ppp(5`)G (27 µM) was required to achieve
50% inhibition of translation of uncapped, 3`-TE(+) transcript as
was required for 50% inhibition of capped, 3`-TE(-) transcript
(13 µM). Consistent with this, inhibition of translation
of the transcript containing both a cap and 3`-TE falls in between.
Although translation of capped transcripts was higher than uncapped
3`-TE(+) transcripts in these conditions (), their
sensitivity to inhibition by free cap analog was greater.
Figure 5:
Inhibition of translation by free cap
analog. Capped and uncapped transcripts (4 nM) of pPAV6
linearized with SmaI (3`-TE(+)) or ScaI
(3`-TE(-)) were translated in wheat germ extracts containing the
indicated amounts of cap analog (mG(5`)ppp(5`)G). The 100%
relative amount of 39-kDa product is defined for each transcript as the
relative radioactivity incorporated in the 39-kDa product (measured
using Imagequant 3.22) in the absence of added cap analog. Data
represent the averages of two separate
experiments.
The 3`-TE
did not stimulate translation of uncapped transcripts in reticulocyte
lysates. Uncapped 3`-TE(+) or 3`-TE(-) RNAs both gave about
one-eighth as much 39-kDa product as capped 3`-TE(+) and
3`-TE(-) transcripts (Fig. 6). Because reticulocyte lysates
were less discriminatory against the uncapped transcripts than were
wheat germ extracts, any enhancement by the 3`-TE would have been less
extreme than in wheat germ. This relatively less cap dependence in
reticulocyte lysates has been observed previously
(42) .
Figure 6:
Comparison of 3`-TE function in wheat germ
and reticulocyte lysates. Yield of 39-kDa product produced in wheat
germ (A) or reticulocyte lysates (B) containing 4
nM transcripts from SmaI (3`-TE(+))- or
ScaI (3`-TE(-))-linearized pPAV6 is plotted with the
amount of product from capped, 3`-TE(+) transcript defined as
100%. Experiments were repeated three times. Error bars represent 1 S.D. Conditions for translation in rabbit reticulocyte
lysates (Promega) were as described previously
(5).
BYDV-PAV Sequences Confer Cap-independent Translation on
a Heterologous Gene
To determine the role of the portion of the
39K ORF that was in all the deletion mutants, this ORF was replaced
with a nonviral gene. Construct pCIGUS has the 5`-terminal 169 nt of
BYDV-PAV, including all 141 bases of the BYDV-PAV 5`-UTR and the first
nine codons of the 39K ORF fused in-frame with the E. coli uidA (GUS) gene lacking its own start codon (Fig. 7). In
this plasmid, the 3`-UTR of the GUS gene contains the 3` end of the
BYDV-PAV genome from the ScaI to the SmaI sites,
including the 3`-TE. Because the ScaI site was destroyed in
construction of pCIGUS, linearization with EcoICR I in the GUS
gene-derived portion of the 3`-UTR was used to create transcripts
lacking all viral sequence in the 3`-UTR. The presence of both the
viral 5` sequence and 3`-TE flanking the GUS gene enhanced translation
of uncapped transcript by more than 80-fold (Fig. 7, compare
lanes 7 and 9 to lane 5). Unlike in the
viral genomic context, capping of these transcripts did not increase
their ability to be translated. These capped transcripts gave about 4
times as much product as the capped transcript lacking the 3`-TE
(Fig. 7, compare lanes 6 and 8 to lane
4). To test the role of the 5` leader sequence, pT7GUS3`-d2 was
constructed. This plasmid contains a 5` leader derived from
pGEM5Z(f+) in place of the BYDV-PAV 5` sequence. All transcripts
of this plasmid translated poorly in the absence of a 5` cap, whether
or not they contained the 3`-TE (Fig. 7). The translation
efficiency of transcripts from EcoICR I-linearized pT7GUS1,
which lacks all viral sequences, was similar to those corresponding
transcripts synthesized from pT7GUS3`-d2 (Fig. 7, compare
lanes 2 and 3 with lanes 10-15). Thus,
the 3`-TE does not function in the presence of the vector-derived 5`
leader sequence. The 5` viral leader sequence itself can enhance the
translation efficiency by about 4-fold relative to the leader derived
from the multiple cloning site of pGEM5Z(f+) (Fig. 7,
compare lane 4 with 2 and lane 5 with
3). This stimulation is reminiscent of other efficient viral
leader sequences such as the tobacco mosaic virus
sequence
(16) .
Figure 7:
Translation of GUS from transcripts
containing viral sequences in the UTRs. Schematic diagrams of chimeric
GUS constructs (not to scale) are shown at the top. The
open box represents the GUS coding region. Gray shaded
boxes indicate sequences derived from BYDV-PAV, including the
5`-UTR and first 9 codons of 39K ORF (left of GUS ORF) fused in-frame
with the GUS gene, and the 3`-terminal 1164 nt of the BYDV-PAV genome
(right of GUS ORF). Sequence from EcoICR I to PstI
represents the 3`-TE (ScaI-PstI fragment from pPAV6).
The black box represents multiple cloning site sequence (bases
1-37) from pGEM5Zf(+) as the 5`-UTR in pT7GUS1 and
pT7GUS3`-d2. The restriction enzymes used for linearizing the plasmids
for transcription are EcoICR I (Eco), BamHI
(Bam), PstI (Pst), or SmaI
(Sma). Solid and dotted lines connecting
maps to the lanes indicate 5` and 3` ends, respectively, of the
transcripts used to generate the products shown in the lanes below.
Phosphorimage shows translation products of BMV RNA (lane 1),
transcripts from pT7GUS1 (lanes 2 and 3), pCIGUS
(lanes 4-9), and pT7GUS3`-d2 (lanes
10-15). Restriction endonuclease with which each plasmid was
linearized prior to transcription is shown above each lane, as is the
presence (C) or absence (U) of a cap structure.
Translation efficiency, indicated below each lane, is the relative
amount (%) of GUS protein (68-kDa product) detected, with the products
of the capped transcript of SmaI-cut pCIGUS transcript
(lane 8) defined as 100.
A Poly(A) Tail Does Not Substitute for the BYDV-PAV 3`
Translational Enhancer
Because a poly(A) tail confers stability
on eukaryotic mRNAs and also enhances translation initiation in
vivo(24, 25, 26, 27) , we compared
the effect of a poly(A) tail on translation of GUS from capped and
uncapped transcripts. The GUS gene was subcloned from pT7GUS1 into
vector pSP64poly(A) which contains a run of 30 adenosine residues in
the multiple cloning site, to create plasmids pT7GUS(A+) and
pGUSEA1 (Fig. 8). Transcripts from these plasmids differ only in
their 5`-UTRs. The 5`-UTR of the pT7GUS(A+) transcript is derived
from the vector, whereas that in pGUSEA1 contains the same 169-base
5`-UTR and first 9 codons as pCIGUS. Transcription of either of these
plasmids when linearized with EcoRI gives a transcript ending
in ACCGAAUU. Linearization with EcoICR I gives an
otherwise identical transcript lacking this 3`-terminal sequence. All
uncapped transcripts translated very poorly whether or not they
contained a poly(A) tail or a viral 5` leader (Fig. 8). In all
cases, capped transcripts yielded 20 to 50 times as much GUS as
uncapped transcripts, although the amount of GUS protein made from
uncapped transcripts was so low that these -fold increases are very
approximate. Any increase in translation owing to polyadenylation was
2-fold or less, regardless of whether the transcripts were capped.
Thus, the poly(A) tail does not substitute for the 3`-TE, nor does it
act synergistically
(43) with a 5` cap to stimulate translation
in the wheat germ extract translation system.
Figure 8:
Effects of a poly(A) tail on GUS
translation. Schematic diagrams of chimeric GUS constructs (not to
scale) are shaded and aligned with lanes as in Fig. 7.
A indicates location of the 30-base polyadenylate
sequence. A, translation products of transcripts from
pT7GUS(A+) which contains the same vector-derived 5` leader as
pT7GUS1 (Fig. 7) after linearization with EcoRI (lanes 2 and 3) or EcoICR I (lanes 4 and
5). Lanes 2 and 4, capped transcripts
(C); lanes 3 and 5, uncapped transcripts
(U). B, translation products of transcripts from
pGUSEA1 which contains the same BYDV-PAV-derived 5` leader as pCIGUS
(Fig. 7) and the same 3`-UTR as pT7GUS(A+) (panel A).
pGUSEA1 was linearized with EcoICR I (lanes 3 and
4) or EcoRI (lanes 5 and 6).
Lane 2, no added RNA. Lane 1 (both panels),
BMV RNA.
-globin or GUS) less than the
native coat protein gene
(30, 31) , whereas the BYDV-PAV
sequences provided greater stimulation and more complete
cap-independent translation of GUS than of viral genes.
(
)
In support of this, uncapped, full-length in vitro transcripts of BYDV-PAV RNA (49) are far more infectious than
capped transcripts
(34) . Furthermore, another group has found
that BYDV-PAV RNA seems to lack a VPg.
(
)
G cap
(50) , but with lower affinity.
If these or other factors bound the 3`-TE with higher affinity, it
could perhaps function like a cap. The fact that more free cap analog
is required to inhibit translation of 3`-TE-containing RNA than capped
RNA suggests that the 3`-TE would have a higher binding affinity to
initiation factors than cap analog. Furthermore, translation of
uncapped RNAs containing a picornaviral internal ribosome entry site
requires the presence of cap-binding protein eIF-4F (51). Thus, the
concept that the same factors can recognize both a sequence and a
modified nucleotide (m
G) provides an explanation for how a
sequence like the 3`-TE can substitute for a cap structure.
site,
GGAUCCUGGGAAACAGG, that is absolutely conserved. Consistent with the
model of Danthinne et al.
(30) , the underlined
hexanucleotide has the potential to base pair near the 3` end of wheat
18 S rRNA
(55) . No conserved, potential 18 S ribosomal RNA
binding sequence was identified in the 5`-UTRs. Obviously, further
structural comparisons await narrowing down the 3`-TE to its mini-mal
functional size and determining the specific role(s) of sequence(s) in
the 5`-UTR.
GpppN cap structure is
still unclear, the occurrence of 3` sequence-mediated cap-independent
translation in BYDV-PAV RNA and the unrelated STNV RNA which have
little sequence similarity suggests that these two viruses arrived by
convergent evolution at a similar strategy. This provides yet another
example of novel mechanisms by which viruses interact with host
translational machinery to control their gene expression.
Table:
Effect of 3` truncations on the cap dependence
and yield of 39-kDa product from BYDV-PAV RNA transcripts
-glucuronidase; TE, translation enhancer; BMV, brome mosaic virus.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.