Molecular Cell, Vol. 7, 1103–1109, May, 2001, Copyright 2001 by Cell Press
Base-Pairing between Untranslated
Regions Facilitates Translation
of Uncapped, Nonpolyadenylated Viral RNA
Liang Guo,2 Edwards M. Allen, and W. Allen Miller1
Interdepartmental Genetics
Plant Pathology Department
351 Bessey Hall
Iowa State University
Ames, Iowa 50011
Summary
Translationally competent mRNAs form a closed loop
via interaction of initiation factors with the 5ⴕ cap and
poly(A) tail. However, many viral mRNAs lack a cap
and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop
by direct base-pairing (kissing) of a stem loop in the
3ⴕ untranslated region (UTR) with a stem loop in the
5ⴕ UTR. This allows a sequence in the 3ⴕ UTR to confer
translation initiation at the 5ⴕ-proximal AUG. This basepairing is also required for replication. Unlike other
cap-independent translation mechanisms, the ribosome enters at the 5ⴕ end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by
which mRNA UTRs can communicate.
Introduction
The 5⬘ m7GpppN cap and poly(A) tail on eukaryotic
mRNAs stabilize the message and interact synergistically to facilitate efficient translation initiation (Gallie,
1991; Preiss and Hentze, 1998; Tarun and Sachs, 1995).
Eukaryotic initiation factor (eIF) 4E binds the 5⬘ cap,
poly(A) binding protein binds the poly(A) tail, and both
of these proteins bind eIF4G to form a closed loop mRNA
(Wells et al., 1998). This loop is required for efficient
recruitment of the 43S ribosomal subunit complex to
the 5⬘ untranslated region (UTR) of the mRNA (Hentze,
1997; Sachs, 2000). The 3⬘–5⬘ UTR interaction explains
how translation of many mRNAs is controlled by signals
in the 3⬘ UTR (Gunkel et al., 1998; Wickens et al., 2000).
The exact mechanism(s) by which these regulatory 3⬘
ends interact with the 5⬘ UTR and how closed loop
mRNA facilitates translation initiation remain to be elucidated.
Many efficiently translated viral mRNAs lack a 5⬘ cap
and/or a poly(A) tail. The uncapped RNAs of picornaviruses contain a long, structured, internal ribosome entry
site (IRES) in the 5⬘ UTR that recruits ribosomes directly
(Jackson, 2000). In most cases, eIF4E is unnecessary
for their translation. The capped, nonpolyadenylated
RNAs of rotaviruses (Piron et al., 1998) and tobamoviruses (Gallie and Walbot, 1990) harbor sequences in the
3⬘ UTRs that functionally mimic the poly(A) tail. Rotavirus
protein NSP3A binds the poly(A)-mimic sequence and
communicates with the 5⬘ end via eIF4G (Michel et al.,
1
Correspondence: wamiller@iastate.edu
Present address: Cereon Genomics, 45 Sidney Street, Cambridge,
Massachusetts 02139
2
2000). RNAs of some plant, yeast, and animal (hepatitis
C virus, Ito et al., 1998; Jackson, 2000) viruses are translated independently of both a cap and a poly(A) tail.
How they act as efficient messages, and whether they
form a closed loop, is unknown.
Members of genus Luteovirus (Wang et al., 1997) and
the large Tombusviridae family (Meulewaeter et al.,
1998; Qu and Morris, 2000; Wu and White, 1999) contain
sequences in the 3⬘ UTR that promote cap-independent
translation initiation at the 5⬘-proximal AUG on viral
RNAs. A particularly efficient sequence is the 100 nt
cap-independent translation element (TE) in the 3⬘ UTR
of Barley yellow dwarf luteovirus (BYDV) RNA (Wang et
al., 1997). The 5⬘ UTRs of either BYDV genomic RNA
(gRNA) or subgenomic RNA 1 (sgRNA1) are also necessary for the TE to function from the 3⬘ UTR, but these
5⬘ UTRs are not necessary in mRNAs that harbor the TE
in the 5⬘ UTR (Wang et al., 1999). Thus, the viral 5⬘ UTRs
are needed only for communication with the 3⬘ TE and do
not serve other essential roles in ribosome recruitment.
Here, we demonstrate that the 3⬘ and 5⬘ UTRs interact
by direct Watson-Crick base-pairing, to bring about efficient translation of uncapped RNA.
Results
Sequences Required for 5ⴕ UTR-3ⴕ TE Interaction
To understand how the 5⬘ UTR interacts with the 3⬘ TE,
we determined its secondary structure and domains
needed for cap-independent translation. It consists of
four stem loops (Figures 1A and 1B). Stem loop IV (SL-IV)
was necessary and sufficient for significant cap-independent translation of luciferase (LUC) mRNAs containing the 3⬘ TE (Figure 1C), with sequence upstream
of SL-IV possibly enhancing translation in vivo. Interestingly, a pyrimidine-rich tract was unnecessary. Such
tracts function in many IRESes (Ito et al., 1998; Jackson,
2000). Additional viral sequence downstream of the 3⬘
TE was included in constructs tested in vivo, as shown
previously to be necessary (Wang et al., 1999). This may
be necessitated by the more competitive translation
conditions that require the presence of a poly(A) tail (or
a viral substitute) on mRNAs in vivo, but not in noncompetitive in vitro conditions (Michel et al., 2000; Preiss
and Hentze, 1998). The additional viral sequence may
also contribute to RNA stability in vivo.
Previously, we identified a candidate sequence in the
TE for involvement in 3⬘–5⬘ communication. Changes to
the loop (L-III) of stem loop III in the TE (SL-III, Figure
2A) knocked out translatability when the TE was in the
3⬘ UTR, but did not affect its function in the 5⬘ UTR
context (Guo et al., 2000). Thus, L-III may be involved
primarily in communication between the 3⬘ and 5⬘ UTRs,
and not directly in recruitment of translation machinery.
The seven bases in L-III differ from those in loop (L-IV),
of SL-IV in the 5⬘ UTR, only in the middle base. This
difference results in complementarity between the central five bases in the two loops (Figure 2A). We propose
that base-pairing (kissing) between these loops forms
a translationally competent closed loop mRNA.
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Figure 1. 5⬘ UTR Element Required for 3⬘ TE-Mediated Translation
(A) Alignment and structure probing of the BYDV genomic 5⬘ UTR. 5⬘ UTRs of known BYDV isolates (Genbank accession numbers: PAV-Vic,
X07653; PAV-ILL, AF235167; PAV-JPN, D85783; PAV-P, D11032; PAV-129, AF218798; MAV-PS1, D11028) were aligned using CLUSTAL W
(Thompson et al., 1994). Arrows and shading indicate predicted base-paired regions. Covariations that support base-pairing are underlined.
Dashed box: pyrimidine-rich domain (PRD). The substrate was a 5⬘ end-labeled transcript consisting of the 143 nt 5⬘ UTR of isolate PAV-ILL
followed by the first 47 nt of the LUC ORF. T1 nuclease sequencing ladder and 0.4 M imidazole cleavage reactions (cuts most single-stranded
nucleotides) are shown on the horizontal gel (top of gel at right) under the aligned sequences.
(B) The MFOLD-predicted (http://bioinfo.math.rpi.edu/mfold/rna/form1.cgi) secondary structure of the 5⬘ UTR superimposed with imidazole
cleavage sites.
(C) Translation of 3⬘ TE-containing reporter transcripts with viral 5⬘ UTR truncations or substitutions indicated at left. Thick lines: portions of
5⬘ UTR with positions indicated by base numbers. Thin line: vector sequence. In the map of the reporter construct (top), 3⬘ TE represents 101
nt 3⬘ TE (nts 4818–4918) for in vitro constructs and 869 nt viral 3⬘ end (nts 4809–5677) for in vivo constructs. Luciferase activity is in relative
light units (RLU), normalized to the construct with the complete BYDV 5⬘ UTR (1–143). Each RNA was translated at least in triplicate (Experimental
Procedures).
Potential Base-Pairing between UTRs
Is Phylogenetically Conserved
The potential kissing stem loop interaction between the
3⬘ and 5⬘ UTRs is conserved in all mRNAs known or
suspected to harbor a 3⬘ TE. This includes all genomic
and subgenomic mRNAs of all sequenced BYDV isolates
(Figure 1A and [Guo et al., 2000]), the related Soybean
dwarf virus (SDV), and the unrelated Tobacco necrosis
virus (TNV, Tombusviridae), which also has uncapped,
nonpolyadenylated RNA (Figure 2A). The predicted kissing stem loop in the 5⬘ UTR of BYDV sgRNA1 is in a
region necessary for 3⬘ TE-mediated cap-independent
translation of the RNA (Wang et al., 1999). The putative
3⬘ TEs of SDV and TNV were identified by a conserved,
essential (Guo et al., 2000) 17 nt tract followed by a
stable stem loop resembling SL-III. In TNV, the sequences of the 5⬘ UTR loop and putative L-III differ from
those in BYDV and SDV, supporting the notion that basepairing between the loops, rather than their sequences,
is important for function (Figure 2A).
Base-Pairing between UTRs Is Necessary
for Cap-Independent Translation
To test the role of base-pairing between the loops, the
potential base-pairing was disrupted by altering the central base in either L-IV or L-III, and restored in the double
mutant with both base changes. As predicted, each
point mutation (SL-IVA→U or 3⬘TEU→A, Figure 2A) reduced LUC expression to levels similar to that obtained
with vector-derived 5⬘ UTR (Figure 2B). Compared to
the single mutants, LUC expression from the double
mutant (SL-IVA→U/3⬘TEU→A) was doubled in vitro and
increased at least 10-fold in vivo (Figure 2B).
To determine the effects of mutations on mRNA stability, the functional half-lives of the mRNAs were estimated by monitoring the rate of LUC accumulation
(Meulewaeter et al., 1998). The half-lives of the mutants
were not reduced enough to account for the drastic
drop in LUC synthesis, nor did they correlate with total
LUC expression (Figure 2C). Thus, LUC synthesis reflects translation efficiency.
It is possible that the double mutant did not restore
translation fully because the wild-type 3⬘ L-III sequence
may play an additional role in recruiting ribosomes beyond the 3⬘–5⬘ communication function, or the mutation
may have altered the TE structure in an unpredicted
way. To avoid these possibilities while testing the role
of 3⬘–5⬘ UTR base-pairing, additional mRNAs with the
wild-type 3⬘ TE sequence but altered 5⬘ stem loop were
constructed. In the 5⬘ UTR, SL-IV was replaced with a
copy of SL-III from the 3⬘ TE. Thus, this mRNA harbored
one copy of SL-III in the 5⬘ UTR and one in the 3⬘ TE.
Kissing UTRs Promote Cap-Independent Translation
1105
Figure 2. Base-Pairing between the 3⬘ TE and 5⬘ UTR
(A) Secondary structures of portions of the viral 5⬘ UTRs, and the 3⬘ TE. Structures for SDV and TNV are the most stable predicted using
MFOLD (Zuker et al., 1999). Bold italics: invariant 17 nt tract. Dashed lines: potential base-pairing. Shaded boxes: predicted stem loops at
5⬘ ends of sgRNAs (Meulewaeter et al., 1992; Wang et al., 1999), with bold bases complementary to L-III. Genomic positions of bases are in
italic numbers; their positions in sgRNAs are in parentheses. Circled bases: mutated in panel B.
(B) Relative luciferase activity produced by translation of LUC mRNAs containing the indicated UTRs (with longer 3⬘ UTRs in protoplasts as
in Figure 1C). RLU were normalized against those obtained with wild-type SL-IV and 3⬘ TE flanking the LUC gene (top row). 3⬘TEBF contains
a four base duplication (GAUC) in BamHI4837 site that eliminates cap-independent translation (Wang et al., 1997). U→A or A→U indicates the
base change in one or both of the circled bases in panel A.
(C) Time course of LUC synthesis (RLU) in protoplasts electroporated with mRNAs containing some of the same UTR mutations as in panel
B. Approximate half-lives were calculated as in Meulewaeter et al. (1998).
(D) Effect of mutations on the structure of the 5⬘ UTR. LUC mRNAs with the indicated 5⬘UTRs were digested partially with nuclease T1 under
translation ionic conditions, and then used as templates for primer extension (Experimental Procedures). Sequencing ladder is in the four
lanes at right. Dots at left indicate reverse transcriptase termination sites present in the absence of T1 treatment. G105 is the guanosine in the
SL-IV loop that is predicted to base-pair to SL-III in wild-type and double mutant structures. The ratio of radioactivity in each G105 band to
total radioactivity in its lane was normalized to the ratio obtained with wild-type UTRs (WT) and indicated below each lane.
This construct gave background levels of LUC activity
presumably because SL-III cannot base pair with itself
(Figure 2B). The L-IIIU→A mutation was introduced only
in the 5⬘ copy of SL-III, to allow base-pairing between
it and the wild-type L-III in the 3⬘ TE. In this case, nearly
wild-type levels of LUC were obtained (Figure 2B). Thus,
base-pairing is necessary and sufficient for communication between the 3⬘ TE and the 5⬘ UTR. Moreover, because the stem of SL-III is different from that of SL-IV,
the stem structure in the 5⬘ UTR is not important for
3⬘–5⬘ communication or ribosome recruitment. This is
supported by the very different sequence of the SL-IV
stem in the 5⬘ UTR of BYDV isolate PAV-129 (Figure 1A).
To detect the long-distance base-pairing directly, we
observed the effect of disrupting and restoring L-IV:LIII base-pairing on nuclease accessibility of L-IV. mRNAs
with viral 5⬘ UTR and 3⬘ TE were probed with nuclease
T1, which cleaves only single-stranded guanosine nucleotides, and cleavage sites were mapped by primer
extension. Although several nonspecific bands that did
not correspond to G residues appeared, presumably
due to premature termination by reverse transcriptase,
the G banding pattern was consistent with the predicted
kissing stem loop structure (Figure 2D). Disruption of
predicted base-pairing between UTRs resulted in 3- to
5-fold more T1 cleavage at the G in L-IV (G105) compared
to wild-type RNA or the double mutant in which basepairing would be restored (Figure 2D). Thus, a point
mutation over 1.6 kb downstream clearly affects
T1 nuclease sensitivity of a base (G105) in the 5⬘ UTR
(3⬘TEU→A mutants, Figure 2D), supporting the predicted
interaction.
L-III:L-IV Base-Pairing Is Necessary for Replication
To determine the biological relevance of the above mutations in their natural context, they were introduced
into full-length viral RNA, and the effects on translation
in vitro and on virus replication (which requires translation) in vivo were observed. The effects of single and
compensatory mutations in L-III and L-IV on translation
of genomic RNA (Figure 3B) were similar to their effects
on translation of the reporter gene (Figure 2B), but in the
double mutants viral RNA translation was fully restored.
Replication was assessed by quantification of subgenomic RNAs that accumulated in transfected oat protoplasts (Figure 3C). Unlike genomic RNA inoculum,
which can linger in the absence of replication, subgeno-
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Figure 4. Effect of Stable Stem-Loop at Different Sites in the 5⬘ UTR
(A) Map of 5⬘ UTRs containing stem loop, K-SL: GCGCGCACGGCCCA
AGCUGGGCCGUGCGCGC (base-paired regions underlined, ⌬G ⫽
⫺34.4 kcal/mol) at the 5⬘ end (K-SL-5⬘UTR56–143) or 3⬘ end (5⬘UTR56–143K-SL) of the 5⬘ UTR (truncated 5⬘ of nt 56). Nonviral sequence is
gray.
(B) LUC activity upon translation of transcripts. LUC activity from
uncapped mRNA with 5⬘UTR56–143 and 3⬘ TE is defined as 100%.
Figure 3. Base-Pairing between UTRs Is Necessary for Viral Translation and Replication
(A) BYDV genome organization. Black box: genomic 5⬘ UTR. Gray
box: 100 nt 3⬘ TE. Hatched box: additional 3⬘ UTR in mRNAs assayed
in protoplasts.
(B) In vitro translation products of full-length wild-type (PAV6) and
mutant BYDV RNAs. PAV6BF contains a GAUC insertion in the TE
that knocks out cap-independent translation and replication (Allen
et al., 1999). Mobilities of products of ORF 1 (39 kDa) and the fusion
of ORFs 1 ⫹ 2 generated by ribosomal frameshifting (99 kDa) are
indicated. Relative amount of 39 kDa product produced is shown
below gel.
(C) Northern blot of total RNA 24 hr after inoculation of protoplasts
with the same RNAs used in panel B. Equal loading of each lane
was verified by ethidium bromide staining. Relative accumulation of
sgRNAs is indicated below each lane. Residual full-length inoculum
RNA is in all lanes, even in the absence of replication (Allen et al.,
1999).
mic RNAs arise only if replication takes place (Allen et
al., 1999). Replication of the single mutants was reduced
to very low levels (undetectable in L-IIIU→A), and most
importantly, the compensatory mutations restored replication to a level roughly proportional to that restored in
translation in vivo (compare Figures 2B and 3C). Thus,
the base-pairing is necessary for translation of replicating viral RNA. These results also indicate that the viral
replication machinery tolerates variations in the sequences of L-III and L-IV.
Ribosome Entry at the 5ⴕ End
Finally, we explored whether the ribosome must enter
the mRNA from the 5⬘ end. Translation initiates only at
the 5⬘-proximal AUG in BYDV gRNA (Wang et al., 1997).
Thus, a 5⬘ scanning mechanism seems likely. Alternatively, the 3⬘ TE may facilitate ribosome entry directly
at L-IV. To distinguish between these mechanisms, we
introduced a stable stem loop (K-SL) that blocks ribo-
some entry on capped mRNAs when located ⱕ12 nt
from the 5⬘ end, but allows ribosome binding, scanning,
and translation when located more 5⬘-distally in the 5⬘
UTR (Kozak, 1989). Presumably the 43S ribosome complex requires a single-stranded 5⬘ terminus to bind the
mRNA, but then can melt out distal secondary structures
as it scans toward the first AUG. In the 5⬘-proximal construct (K-SL-5⬘UTR56–143), BYDV bases 1–55 were replaced by K-SL (Figure 4A). In the 5⬘-distal construct
(5⬘UTR56–143-K-SL), K-SL was inserted between SL-IV and
the start codon. As shown in Figure 4B, the 5⬘-proximal
stem loop blocked translation, whereas the 5⬘-distal
stem loop had little effect. This effect was observed with
3⬘ TE-mediated translation of uncapped mRNAs, and, in
agreement with Kozak (1989), on capped mRNAs (Figure
4B). Thus, the ribosome must scan from the very 5⬘
end for translation of uncapped mRNAs in a manner
indistinguishable from scanning on capped, polyadenylated mRNAs, and markedly different from IRES-containing (Hentze, 1997) or other uncapped, polyadenylated mRNAs (Preiss and Hentze, 1998).
Discussion
The fact that BYDV has evolved to require base-pairing
between UTRs suggests that there is strong selective
pressure for formation of closed loop mRNA, and that
a closed loop may be necessary in all mRNAs, regardless of the terminal structures. In support of this conclusion, uncapped, polyadenylated mRNAs (Bergamini et
al., 2000), and capped, nonpolyadenylated mRNAs (Michel et al., 2000) are also likely to form closed loops.
The formation of the closed loop by long-distance
pairing of just five bases raises questions regarding stability and specificity of this interaction. First, it is possible
that host proteins strengthen the long-distance interaction. Second, the base-pairing between the 3⬘ TE the 5⬘
UTR is probably more stable than predicted by Watson-
Kissing UTRs Promote Cap-Independent Translation
1107
Crick interactions alone, because kissing stem loops are
kinetically and thermodynamically favored compared to
equivalent base-pairing of linear RNAs (Zeiler and Simons, 1997). However, internal sequences within the
coding regions of the mRNA might be expected to compete with L-IV of the 5⬘ UTR for base-pairing to L-III of
the TE. In fact, BYDV genomic RNA contains an internal
loop capable of base-pairing to L-III. This loop is in the
5⬘ UTR of subgenomic RNA 1 (Figure 2A). In that context,
it probably does base-pair to L-III during translation,
whereas it does not inhibit translation from its internal
location in the full-length genomic RNA context. Thus,
the proximity to the 5⬘ end may be necessary for stable
base-pairing. This is supported by the requirement for
ribosomal scanning from the 5⬘ end in Figure 4.
The TE-mediated translation mechanism differs from
those of other mRNAs by requiring the 3⬘–5⬘ interaction
even in the poly(A) tail-insensitive wheat germ extract.
This interaction, via the kissing stem loop, is required
only when the TE is in the 3⬘ UTR (Guo et al., 2000).
Thus, we propose that the closed loop is necessary to
deliver either ribosomes or initiation factors to the 5⬘
UTR, and that mRNA can then enter the ribosome for
scanning and translation only via its 5⬘ terminus. This
model is supported by preliminary data indicating that
the TE specifically binds canonical initiation factors (E.
Allen, our unpublished data). The mechanism is complicated by the requirement for additional viral sequence,
mostly downstream of the 100 nt 3⬘ TE, for cap-independent translation in vivo (Wang et al., 1999). This additional sequence can be replaced partially by a 60 nt
poly(A) tail, suggesting that it may function as a poly(A)
tail mimic (Guo et al., 2000). The in vivo-required sequence may bind additional factors that further promote
recruitment of ribosomes, or fortify the closed loop interactions in the more competitive in vivo conditions. The
additional sequence may also provide stability against
exonucleases that are absent in vitro (Jacobson and
Peltz, 1996).
Specific, long-range base-pairing between short sequences in loops separated by kilobases (Klovins et al.,
1998; Miller and Koev, 2000; van Marle et al., 1999), or
even on separate molecules (Mujeeb et al., 1998; Sit
et al., 1998), have been shown to control synthesis or
encapsidation of viral RNAs. Unlike the above examples,
we show a long-range base-pairing interaction that controls the universal process of translation. Moreover,
BYDV cap-independent translation also differs by not
requiring viral proteins, because reporter constructs behave similarly to viral genomic RNA. Interaction between
the 3⬘ UTR and the 5⬘ IRES may regulate cap-independent translation of HCV RNA, which is also nonpolyadenylated (Ito et al., 1998), but this interaction is proteinmediated and the effects on translation are of a lower
magnitude than that seen with the TE.
Base-pairing between UTRs was proposed for capindependent translation mediated by the 3⬘ UTR of Satellite tobacco necrosis virus (STNV) RNA, but covariation
mutagenesis did not support this hypothesis (Meulewaeter et al., 1998). It is possible that the particular
mutations caused unexpected structural changes, preventing restoration of a functional element in the double
mutants. Interestingly, the STNV sequence shows no
apparent similarity to the 3⬘ TE (Wang et al., 1997), yet
the 3⬘ UTR of its helper virus, TNV, is clearly similar
(Figure 2A).
The base-pairing between UTRs may also play a role in
replication. Mounting evidence indicates that replication
templates of some RNA viruses are circular. The 5⬘ UTR
of poliovirus RNA interacts with the 3⬘ end (via proteins)
to regulate initiation of (-) strand synthesis (Gamarnik
and Andino, 1998). Substantial complementary exists
between the UTRs of flaviviral RNAs (Hahn et al., 1987).
This base-pairing controls replication of Dengue virus
RNA in vitro (You and Padmanabhan, 1999).
Finally, this report provides evidence of a novel capindependent translation mechanism that resembles
translation of capped mRNAs by its requirement for ribosomal scanning from the 5⬘ terminus of the mRNA. Given
the large number of viruses that lack caps or poly(A)
tails or have potential base-pairing between UTRs (Hahn
et al., 1987), and given the numerous nonviral mRNAs
that undergo translational regulation via their 3⬘ UTRs
(Gunkel et al., 1998; Wickens et al., 2000), interactions
similar to those reported here may be widespread.
Experimental Procedures
mRNA Construction
RNAs were synthesized by in vitro transcription from plasmids with
T7 polymerase using Megascript (for uncapped RNAs) or mMessage
mMachine (for capped RNAs) kits (Ambion, Austin, TX). The in vitro
and in vivo reporter plasmids were derived by PCR from p5⬘UTRLUC-TE105 and p5⬘UTR-LUC-TE869, respectively (Guo et al., 2000).
For the 5⬘ truncations, a set of 5⬘ primers with 5⬘ EcoRI site extensions was made starting from the deletion boundaries indicated in
Figure 1C. The 3⬘ primer initiated at a site complementary to the 3⬘
end of the LUC ORF. The PCR products were cut with EcoRI (one
is an internal EcoRI site in LUC) and cloned into p5⬘UTR-LUC-TE105
or p5⬘UTR-LUC-TE869. The 3⬘ deletions in the 5⬘ UTR were made
similarly by a 5⬘ primer in the vector upstream of the 5⬘ UTR and a
set of 3⬘ antisense primers at the points of deletions shown in Figure
1C. The 3⬘ substitution bases were introduced in the 3⬘ primer.
The point mutations in Figures 2 and 3 were generated by PCR
using two complementary primers with the designated point mutation. In one tube, the sense mutagenic primer was paired with a
downstream antisense primer; in the other tube the antisense mutagenic primer was paired with an upstream sense primer. The two
overlapping PCR products were mixed as templates, and amplified
using the two flanking primers. The resulting mutated PCR product
was cut with appropriate restriction enzymes to replace the wildtype fragment. The K-SL stem loop, containing a BssHII site for
cloning, was introduced into different positions of the 5⬘ UTR by
the above mutagenesis methods. All constructs were verified by
sequencing on an ABI377 automated sequencer.
Translation and Replication Assays
Subsaturating levels (0.2–0.3 pmol) of transcript were translated in
wheat germ S30 extract (Promega), or 2–3 pmol of transcript were
electroporated in oat protoplasts, and products assayed, at least
in triplicate, for luciferase activity as in Guo et al., 2000. mRNA
half-life was approximated using the equation t1/2 ⫽ P(∞) • ln2/A
(Meulewaeter et al., 1998), where P(∞) is LUC in relative light units
(RLU) at 106 min; A is the slope (RLU/min) at t(0). For replication
studies, oat protoplasts were inoculated with 10 pmol of full-length
BYDV genomic RNA (or mutant versions) transcribed from SmaIlinearized pPAV6 (Allen et al., 1999). After 24 hr, RNA was extracted
and Northern blots performed as in Allen et al. (1999).
RNA Structure Probing
The 5⬘ UTR structure in Figure 1 was determined on transcript from
XbaI-digested p5⬘UTR-LUC-TE105. Twenty pmol of [␥-33P]ATP endlabeled RNA was purified on a 6% polyacrylamide, 7M urea denaturing gel and eluted, and T1 nuclease ladder generated all as described previously (Guo et al., 2000). The labeled RNA was probed
in 0.4 M imidazole solution containing 40 mM NaCl, 1 mM EDTA,
10 mM MgCl2 for 15 hr at 25⬚ C as in Vlassov et al. (1995). Probed
products were separated by 8% polyacrylamide, 7 M urea gel elec-
Molecular Cell
1108
trophoresis, and visualized with a STORM 840 Phosphorimager (Molecular Dynamics).
The 5⬘ UTR structure of full-length mRNA, and mutant versions,
(Figure 2D) transcribed from SmaI-linearized p5⬘UTR-LUC-TE105,
was analyzed by partial digestion with T1 nuclease (Guo et al., 2000)
under the same ionic conditions used for in vitro translation, followed
by reverse transcription using the 5⬘ end-labeled primer, CATAGCC
TTATGCAGTTGC (complementary to bases 72 to 90 in the LUC
ORF), as described by Merryman and Noller (1998).
Acknowledgments
The authors thank Gloria Culver for advice. Funding was provided
by NSF (MCB-9974590), and by Leopold Brown Fellowships to L.G
and E.M.A. This is paper J-19096 of the Iowa Agriculture and Home
Economics Experiment Station, project 3545.
Received January 4, 2001; revised March 7, 2001.
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