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J Biol Chem, Vol. 274, Issue 32, 22452-22458, August 6, 1999
From the Department of Molecular Microbiology and Immunology,
School of Medicine, University of Missouri at Columbia,
Columbia, Missouri 65212
How premature translation termination codons
(PTCs) mediate effects on nuclear RNA processing is unclear. Here we
show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of
P4-generated pre-mRNAs of the autonomous parvovirus minute virus of
mice caused a decrease in the accumulated levels of doubly spliced R2
relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within
nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of
the large intron splice sites and by mutation of the AUG that initiated
translation of R1 and R2. In contrast to the PTC at nt 385, the reading
frame-dependent effect of the PTC at nt 2018 depended
neither on the initiating AUG nor the normal termination codon for NS2;
however, it could be suppressed by a single nucleotide deletion
mutation in the upstream NS1/2 common exon that shifted the 2018 PTC
out of the NS2 open reading frame. This suggested that there was
recognition and communication of reading frame between exons on a
pre-mRNA in the nucleus prior to or concomitant with splicing.
Premature termination codons
(PTCs)1 have been shown to
result in decreased levels of PTC-containing mRNAs in many
organisms, and there is an increasing appreciation of the effect of
PTCs on RNA levels in mammalian cells. In some of these cases, PTCs have been shown to affect nuclear-associated mRNA abundance by a
process termed nonsense-mediated decay, which has been suggested to
degrade fully spliced mRNAs in the nucleus, possibly during mRNA export (reviewed in Refs.1 and 2).
PTCs have also been implicated in altering nuclear RNA processing
events other than decay in mammalian cells, which results in both
intron retention and exon skipping, suggesting that PTCs may influence
splice site selection (1). In two cases, exon skipping of the
66-nucleotide exon 51 of fibrillin FBN1 RNA (3) and exon skipping and
intron retention for the P4-generated pre-mRNAs that generate the
nonstructural proteins of the autonomous parvovirus minute virus of
mice (MVM) (4, 5), the effects of PTCs on RNA processing have been
shown to be reading frame-dependent.
MVM is an autonomous parvovirus that is organized into two overlapping
transcription units that produce three major classes of RNA (6-8) (see
Fig. 1). Transcripts R1 and R2 are generated form a promoter (P4) at
map unit 4 and encode the viral nonstructural proteins NS1 and NS2,
respectively, whereas the R3 transcripts are generated from a promoter
at map unit 38 (P38) and encode the viral capsid proteins (6, 9). Both
NS1 and NS2 play essential roles in viral replication and cytotoxicity
(10), and so maintenance of their relative steady state levels, which is controlled at least in part by alternative splicing, is critical to
the viral life cycle (Refs. 11-13; reviewed in Ref. 14). All MVM
mRNAs generated during infection or following transfection are very
stable (15), and no viral proteins are known to participate in the
alternative splicing of MVM pre-mRNAs (reviewed in Ref. 14).
There are two types of introns in MVM P4-generated transcripts (14)
(see Fig. 1). An overlapping downstream small intron, which undergoes
an unusual pattern of overlapping alternative splicing using two donors
(D1 and D2) and two acceptors (A1 and A2) (16), is located between nt
2280 and 2399 and is common to both P4- and P38-generated transcripts.
An upstream large intron, located between nt 514 and 1989, is
additionally excised from a subset of P4-generated pre-mRNAs to
generate R2 mRNA. This upstream intron utilizes a nonconsensus
donor at nt 514 and has a weak polypyrimidine tract at its 3' splice
site (13).
The NS2-specific exon is a 290-nt alternatively spliced exon that is
translated in two open reading frames (ORFs). In singly spliced R1,
this region utilizes ORF3 to encode NS1; in doubly spliced R2, this
exon utilizes ORF2 to encode NS2 (Fig.
1). We have previously shown that PTCs in
the NS2-specific exon caused a decrease in the accumulated levels of R2
relative to R1, although the total accumulated levels of R1 plus R2
remained the same. This decrease was a consequence of the artificially
introduced translation termination signal acting in cis
rather than the absence of a functional viral gene product and
was shown to be evident in nuclear RNA and independent of RNA
stability, suggesting an effect on nuclear splicing (4, 5). Although
perhaps not directly comparable with PTCs that cause genetic diseases
in higher organisms, analyses of PTCs introduced into viral genes such
as MVM have proven to be informative models for the effects of PTCs on
RNA accumulation (4, 5).
Efficient inclusion of the NS2-specific exon as an internal exon
in vivo and consequent excision of the upstream large intron from P4-generated pre-mRNA to generate R2 require an internally redundant, bipartite exon splicing enhancer (ESE) comprised of 5' and
3' elements within the NS2-specific exon (17). The function of this ESE
is sensitive to the presence of PTCs (5). A nonsense but not a missense
mutation in the NS2 open reading frame at nt 2018 within the 5' element
of the ESE affected definition of the NS2-specific exon by interfering
with the ability of the bipartite ESE to strengthen interactions at the
upstream large intron polypyrimidine tract. A PTC at nt 2018 alone
resulted in retention of the upstream intron without a net decrease in
the total accumulated P4-generated product. When the PTC at nt 2018 was
combined with a mutation in the 3' element of the bipartite enhancer,
the NS2-specific exon was skipped. These effects were independent of
RNA stability and were shown to be reading frame-dependent;
single nucleotide deletions in front of the 2018 mutation, which
removed the PTC from the NS2 open reading frame, fully suppressed its
effect (5).
Although the NS1/NS2 shared exon is a 5' terminal exon, PTCs at nt 385 within this exon have also been shown to result in a decrease in the
accumulated level of doubly spliced R2 relative to singly spliced R1,
in a manner independent of the stability of R2 or R1 (4). In this
report we show that the effect of the PTC at nt 385 was evident within
nuclear RNA, was dependent on a PTC and not a missense transversion
mutation at this position, and could be suppressed by improvement of
the large intron splice sites. The effects of the 385 PTC and a PTC at
nt 2018 were at least partially additive, suggesting that they operated
by at least partially independent mechanisms. This was further
underscored by the observation that the effect of the PTC at nt 385 depended upon the AUG that initiated translation of R1 and R2, whereas the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor on the normal termination
codon for NS2. The effect of the PTC at nt 2018 could, however, be
suppressed by a single nucleotide deletion mutation in the upstream
NS1/2 common exon that shifted the 2018 PTC out of the NS2 ORF,
suggesting that there was communication of reading frames between
exons. Our observations are most consistent with a model in which
reading frame can be recognized on a pre-mRNA molecule in the
nucleus, prior to or concomitant with splicing.
Mutant Construction--
Construction of p385UTT and
p2018TAA has been previously described (4).
The mutants p385CAA, p385TAA, and pSTOP(
p385UTT+2018TAA, pCSD385UTT, p2T385UTT, p4T385UTT,
pCSD20188TAA, pAUG(X)2018TAA, pAUG(X)385UTT,
pAUG(XXX)385UTT, p2018D1( Transfection and RNA
Isolation--
Murine A92L cells, the normal tissue
culture host for MVM(p), were grown as described previously (6)
and transfected with wild type and mutant
MVM plasmids, using either DEAE-Dextran (4) or
LipofectAMINE-Plus reagent (as described in the LipofectAMINE-Plus reagent kit manufactured by Life Technologies, Inc.). RNA was typically
isolated 48 h post-transfection, after lysis in guanidinium thiocyanate, by centrifugation through CsCl exactly as described previously (15). RNase protection assays RNase protection assays were
performed as described previously (15), using an
[ A Nonsense but Not a Missense Mutation at nt 385 in the NS1/NS2
Common Exon of P4-generated Pre-mRNAs Resulted in a Reduced
Accumulated Level of R2 Relative to R1, Likely Because of Effects on
Excision of the Downstream Large Intron--
Introduction of an ochre
(TAA) PTC at nt 385 in the NS1/NS2 common exon of P4-generated
pre-mRNA, either by point mutagenesis or by insertion of a 15-nt
linker with overlapping PTCs in all three reading frames, led to a
significant inversion in the accumulated levels of doubly spliced R2
relative to singly spliced R1 in total RNA, compared with that
generated by wild type, although the total P4-generated product (R1+R2)
was unchanged (Fig. 2, A and B). This result was
similar to that previously seen when a PTC was introduced at nt 2018 in
the internal NS2-specific exon of P4-generated pre-mRNA (4, 5) but
of somewhat greater magnitude. A missense transversion (CAG) at nt 385 showed no such effect (Fig. 2, A and B). When the
PTCs at nt 385 and 2018 were combined, an even greater decrease in the
accumulated level of R2 relative to R1 was seen (Fig. 2, A
and C). That the effects of the two mutations were at least
somewhat additive suggested that the mechanisms behind their effects
were at least partially independent, a possibility that is further
supported by the observations described below. The effect of the PTC at
nt 385 was also evident in pure preparations of nuclear RNA, suggesting
that the effect of this PTC was manifest within the nucleus (Fig.
2D), as was previously reported for the PTC at 2018 (5).
Subtle improvements of the large intron splice sites could suppress the
effect of the PTC at nt 385, consistent with the suggestion that this
effect was nuclear. As shown in Fig. 3 (A and B),
improvement of either the large intron 5' splice site to consensus or
the large intron 3' splice site polypyrimidine tract by as few as two
additional pyrimidines, restored the accumulated levels of R2 relative
to R1 to near wild type levels. Improvement of the large intron 5'
splice site to consensus also suppressed the phenotype of the PTC at
2018 (Fig. 3, A and C); we have previously
reported that improvements of the 3' polypyrimidine tract also
suppresses the effect of the PTC at nt 2018 (5). Improvement of the
large intron 3' splice site polypyrimidine tract by four pyrimidines resulted in splicing of the P4 product almost exclusively to R2 (Fig.
3B, sixth lane).
That the effects of the PTCs can be overcome by improvements of the
large intron splice sites, which do not appear in the final spliced R2
mRNA (Fig. 3), further suggests that the effects of the PTCs are
independent of effects on RNA stability, as has been shown previously
(4, 5). Taken together, these results suggested that PTCs in either
exon of R2 can interfere with the nuclear excision of the upstream
large intron from P4-generated pre-mRNAs.
The Effect of the PTC at nt 385, but Not That of the PTC at 2018, Could Be Suppressed by Mutations in the Initiating AUG--
The
accumulated level of R2 relative to R1 was affected by a PTC but not by
a missense transversion at nt 385, and we have previously shown that
the effect of a PTC at nt 2018 was reading frame-dependent,
i.e. it was suppressed by a frameshift mutation in front of
nt 2018 (5). Therefore, we chose to ask whether the effects of the PTCs
were linked to translation in the cytoplasm by determining whether
their effect was dependent on the initiating AUG that begins the
reading frame for NS1 and NS2. Although such mutations may conceivably
have other effects on RNA processing, similar mutations have been used
to suppress the effects of PTCs in the triosephoshate isomerase gene
RNA in order to link the PTC effects to cytoplasmic translation (19).
As can be seen in Fig. 4, destruction by point mutagenesis of the
initiating AUG at nt 260 suppressed the effect of the PTC at nt 385 but
not the effect of the PTC at nt 2018. Translation reinitiation has also
been shown to abrogate nonsense-mediated decay of the triosephoshate isomerase gene RNA (19). There are also two internal AUG codons in
frame in the NS1/NS2 shared exon (at nt 474 and 504), and the effect of
the 2018 PTC was also resistant to mutation of all three AUGs.
Interestingly, the triple AUG mutations were less effective than the nt
260 mutation alone in suppressing the effect of the PTC at nt 385 (Fig.
4). This may have been because nt 474 and 504 lie close to the large
intron donor at nt 514 and because mutations at these sites may have
interfered with an exonic signal required for splicing of the large
intron. That there was a reduction in the accumulated levels of R2
relative to R1 in RNA generated by a mutant in which the three AUGs
were destroyed but no PTC had been introduced (pAUG(XXX)) is consistent
with this possibility (Fig. 4). Thus, nonsense codon-mediated reduction
of the accumulated levels of R2 relative to R1 caused by the PTC at nt
385 but not at nt 2018 was dependent upon the reading-frame initiating
AUG at nt 260.
The Effect of the PTC at nt 2018 Is Not Dependent upon the Normal
Stop Codon in R2 but Rather upon Communication between the Two
Exons--
The effect of the PTC at nt 2018 has previously been shown
to be reading frame-dependent; a single nucleotide mutation
at nt 2011 that shifted the R2 reading frame into the open NS1 reading frame prior to the 2018 PTC could suppress its effect (5). That the
2018 PTC could not be suppressed by destruction of the initiating AUG
codon suggested that the R2 reading frame must be marked from another
point. As shown in Fig. 5 (A and B), this marker
is not the normal termination codon for the major isoform of NS2. (The
major isoform of NS2 is encoded by an R2 molecule that uses the small
intron pair D1/A1 and is found in approximately 75% of the MVM
mRNAs.) When the NS2 major isoform TAA codon was mutated so that
the NS2 open reading frame was fused in-frame into the capsid gene VP1
open reading frame that terminated at nt 4552, the effect of the 2018 PTC on the accumulated levels of R2 relative to R1 remained unchanged
(p2018TAASTOP(
Studies with the T-cell receptor gene have suggested that a spliceable
downstream intron was required for some effects of PTCs on RNA
processing (20). This was not the case, however, for the PTC at MVM nt
2018. Mutations that disabled both the outside small intron donor and
the acceptor (D1 and A2; Fig. 1) prevent excision of the small intron
(probably because the remaining internal D2-A2 pair that is separated
by only 59 nt is too close to be spliced together (14, 21)); however,
the upstream intron is still excised at wild type efficiency in this
mutant (Fig. 5, A and C). When a PTC was
introduced at nt 2018 in this small intron-disabling mutant, the full
effect of the PTC on the accumulated levels of R2 relative to R1 was
still evident (p2018TAAD1( A PTC transversion mutation but not a missense transversion
mutation at nucleotide 385 in the 5'-terminal NS1/2 common exon of
P4-generated pre-mRNAs of the autonomous parvovirus MVM resulted in
an increased level of the singly spliced R1 mRNA relative to doubly
spliced R2 mRNA in the nucleus of transfected cells. This effect
could be suppressed by improvement of the splice signals of the large
intron, suggesting that the effect on MVM RNA processing was at the
level of excision of the large intron. Mutation of the initiating AUG
at nt 260 also suppressed the effect of the 385 PTC, suggesting a link
to the establishment of the reading frame of this 5'-terminal exon.
These results help place the MVM P4-generated pre-mRNAs in a
growing list of examples in which PTCs can either directly or
indirectly affect nuclear RNA processing in an open reading
frame-dependent manner (1, 22).
We have previously shown that a PTC at nt 2018 in the downstream
internal NS2-specific exon of MVM P4-generated pre-mRNA also inhibited excision of this large intron, likely by virtue of
interfering with the function of an ESE within the NS2 specific exon
(5). In that case, the effect of the PTC on splicing was shown to be dependent upon an intact open reading frame. Here we show further that
the effect of the PTC at nt 2018, although reading
frame-dependent, was not affected by alterations of either
the protein translation initiating AUG or terminating TAA signals,
which have been shown to be important for PTC effects in other systems
(1, 19, 20, 22). Surprisingly, the effect could be suppressed by a frameshift mutation in the 5' terminal NS1/2 common exon of R2 message,
suggesting that the effect of the 2018 PTC was dependent upon
communication of reading frame between the two exons in the nucleus,
prior to the completion of the splicing process.
PTCs have been shown to have significant effects on RNA processing
associated with the nucleus of mammalian cells (1, 20). In some cases
PTCs have been shown to trigger nucleus-associated nonsense-mediated
decay; in other cases PTCs have been shown to affect other nuclear
processes that lead to altered steady state RNA levels (1). We have
recently shown that the PTC at nt 2018 likely affects splicing of MVM
P4-generated pre-mRNA by interfering with the nuclear function of
an ESE within the internal NS2-specific exon of MVM RNA (5). These
observations are most consistent with "nuclear scanning"-type
models of reading frame recognition (1, 23). Because our results
suggest that recognition of the PTC at nt 2018 is not influenced by the
initiating AUG and yet can be suppressed by a frameshift mutation in an
upstream exon, such a model would have to accommodate recognition of
frame between the upstream NS1/2 common exon and the NS2-specific exon prior to splicing. On the other hand, because the PTC at nt 385 is
suppressible by mutation of the initiating AUG, it is formally possible
that the effect of this PTC could be linked to cytoplasmic translation.
This seems unlikely, however, because the large intron begins only 129 nt downstream of the PTC at nt 385. The observation that the effects of
the two PTC mutations together are additive suggests that they may
function somewhat differently.
Although the mechanisms of PTC action in the nucleus are not well
understood, their effect has provoked a growing appreciation that the
recognition of reading frame in the nucleus of mammalian cells can
influence RNA processing events (1, 3). How such recognition can occur
is not known; however, there have recently been some interesting
observations that suggest that an appropriate apparatus may be in
place. Ribosomal proteins and RNAs, elongation factor subunits eIF2A
(20) and eIF4E (24), and aminoacyl tRNAs have recently been detected in
the nucleus of mammalian cells (25). The U5 snRNP, which binds to exon
sequences adjacent to 5' and 3' splice sites and has a role in
juxtaposing exons together during splicing (26, 27), contains a 116-kDa
protein that is both essential for splicing and closely related to the
ribosomal translocase EF-2 (28). In addition, a 200 S ribonuclear
protein particle that contains pre-mRNA and splicing factors but is
much larger than the 60 S splicesome identified in extracts competent for splicing in vitro has been identified in mammalian cells
(29). Our previous observations (4, 5) and those in this manuscript taken together provide strong evidence for the existence of such an
exon-scanning reading frame recognition mechanism in the nucleus of
mammalian cells that can interfere with the processing of
PTC-containing RNAs.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
PTC, premature
termination codon;
MVM, minute virus of mice;
nt, nucleotide(s);
ORF, open reading frame;
ESE, exon splicing enhancer;
WT, wild type.
A Premature Termination Codon in Either Exon of Minute Virus of
Mice P4 Promoter-generated Pre-mRNA Can Inhibit Nuclear Splicing of
the Intervening Intron in an Open Reading
Frame-dependent Manner*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Genetic map of MVM. The three major
transcript classes and protein-encoding open reading frames are shown.
The two promoters (P4 and P38) are indicated by arrows. The
large intron, the small intron, and the NS2-specific exon are
indicated. The nonconsensus donor (ncD) and the poor
polypyrimidine tract (poor (Py)n) of the large intron are
also shown. The bottom diagram shows nucleotide locations,
and the probe B (nts 1854-2378) used for RNase protection assays was
as described fully under "Experimental Procedures."
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
were constructed by m13-based oligonucleotide mutagenesis as described
previously (4). Mutant oligonucleotides were homologous to the viral
DNA except at the nucleotides that were to be altered. pSTOP() is a
deletion of two nucleotides at 2380 and 2381. The changes made in
p385CAA and p385TAA are shown in Fig.
2A. pAUG(X) was constructed by
cutting the wild type
(WT) clone of MVM at the NcoI site at nt 260 and chewing
back with mung bean nuclease to create a deletion between nt 261 and
265 (which deletes the initiating AUG at nt 260). To generate pAUG(XXX)
and pAUG(XXX)2018TAA, mutations of the Kozak consensus
sequences (18) at nt 474 and 504 were initially generated by m13-based
oligonucleotide mutagenesis (the 474 mutation changes the WT sequence
5'-GACATG-3' to 5'-TACATT-3'; the 504 mutation
changes the WT sequence 5'-GAAATG-3' to
5'-CCAATT-3'), and then the m13 fragment
bearing these mutations was subcloned into pAUG(X) and
pAUG(X)2018TAA, respectively. All the final clones of the
above mutants were sequenced to confirm that only the desired mutations
were introduced, and the absence of the P38 promoter product R3 for
these mutants confirms that no NS1 was made.
View larger version (39K):
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Fig. 2.
Effects of nonsense and missense mutations in
either the first or second exons of NS2 pre-mRNA.
A, sequences of the codons at nt 385 (UTT
indicates the insertion of the universal translation termination
cassette at nt 385; see text and "Experimental Procedures") and
2018 in wild type MVM and mutants are shown underneath their
appropriate map positions (deviations from the wild type sequence are
underlined), together with quantitations of the R2/R1 ratio
obtained by RNase protection analysis with probe B for each mutant. All
the values are the average of at least three separate experiments.
Standard deviations are indicated in parentheses.
B and C, RNase protection analysis of total RNA
using probe B (see Fig. 1), of RNA generated by wild type MVM
(WT), mutants (as described in text), or mock-transfected,
as designated at the top of each lane. The identities of the
protected bands are shown on the left and explained under
"Experimental Procedures." *, undigested probe B. D, RNase protection analysis of nuclear RNA using probe B
(Fig. 1) of RNA generated by wild type MVM(WT) or p385UTT as designated
at the top of each lane. The identities of the protected
bands are shown on the left as explained under
"Experimental Procedures." Nuclear fractions were determined to be
>95% pure as explained under "Experimental Procedures." Nuclear
RNA from equivalent cell amounts were loaded for each sample.
)A2(), and
p2018TAASTOP() were generated by combining the individual mutations using standard molecular cloning techniques. The changes made
in the mutants are shown in Figs. 2A,
3A, 4A, and
5A. All the final clones of the above mutants were
sequenced to confirm that only the desired mutations
were introduced.
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Fig. 3.
Improvement of either the weak
donor (5' splice site) or the weak polypyrimidine tract (3' splice
site) of the upstream intron can suppress the effects of nonsense
mutations in either the first or second exons of NS2 pre-mRNA.
A, sequences of the codons at nt 385 (UTT
indicates the insertion of the universal translation termination
cassette at nt 385; see text and "Experimental Procedures") and
2018, as well as sequences of the large intron donor and polypyrimidine
tract, in wild type MVM and mutants are shown underneath their
appropriate map positions (deviations from the wild type sequence are
underlined), together with quantitations of the R2/R1 ratio
obtained by RNase protection analysis with probe B for each mutant. All
the values are the average of at least three separate experiments.
Standard deviations are indicated in parentheses.
wt, wild type sequence. B and C, RNase
protection analysis of total RNA using probe B (see Fig. 1) of RNA
generated by wild type MVM (WT), mutants (as described in text), or
mock-transfected as designated at the top of each lane. The
identities of the protected bands are shown on the left and
explained under "Experimental Procedures." *, undigested probe B. A
doublet is occasionally generated (and here shown) by the R2M species
of RNA generated from the CSD2018 mutant. The reason for this banding
pattern is not yet known.
-32P]UTP-labeled, SP6-generated antisense MVM RNA
probe from MVM nucleotides 1854-2378 (probe B). This probe extends
from before the acceptor site of the large intron to within the small
intron common to all MVM RNAs and distinguishes P4-RNA species using the large intron acceptor and either of the alternative small intron
donors (designated M for the major splice donor (D1) at nt 2280 and m
for the minor splice donor (D2) at nt 2317 (15)). Thus probe B can
distinguish between unspliced, minor, and major forms of both R1 and
R2. For analysis of RNA produced after transfection with
p2018TAA, p1T2018TAA, p2T2018TAA,
p4T2018TAA, p385TAA+2018TAA, p2T385UTT, p4T385UTT, pCSD2018TAA,
pAUG(X)2018TAA, pAUG(XXX)2018TAA, p2018TAAD1()A2(), and
p2018TAASTOP(), antisense RNase protection probes
homologous to the 2018 and polypyrimidine tract mutations being
analyzed were constructed and used. Because in all cases the relative
ratio of bands within an individual lane was the important result,
rigorous attempts were not made to equilibrate the amounts of specific
RNA between lanes. This would be expected to vary from sample to sample
depending upon transfection efficiencies and does not effect the
comparison of relative ratios within a sample. RNase protection
products were analyzed on a Betagen-scanning phosphorimage analyzer,
and molar ratios of MVM RNA were determined by standardization to the
number of uridines in each protected fragment. Nuclear RNA was isolated
after the pelleting of nuclei twice through sucrose exactly as
described previously (4). The nuclear RNA fractions were determined to
be >95% pure as monitored by the relative absence of mature rRNA and
the presence of rRNA precursors in these preparations by ethidium
bromide staining following gel electorophoresis (data not shown), also
as described (4).
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Fig. 4.
The nonsense-mediated reduction of R2
mRNA depends on the initiating AUG for mutations in the first exon
but not the second exon of NS2 pre-mRNA. A,
sequences of the codons at nt 385 (UTT indicates the
insertion of the universal translation termination cassette at nt 385;
see text and "Experimental Procedures") and 2018, as well as the
presence (wt) or mutation ( ) of AUG codons at nt 260, 474, and 504 in wild type MVM and mutants are shown underneath their
appropriate map positions (deviations from the wild type sequence are
underlined), together with quantitations of the R2/R1 ratio
obtained by RNase protection analysis with probe B for each mutant. All
the values are the average of at least three separate experiments.
Standard deviations are indicated in parentheses.
wt, wild type sequence. (), mutation. B, RNase
protection analysis of total RNA using probe B (see Fig. 1) of RNA
generated by wild type MVM (WT), mutants (as described in
text), or mock-transfected as designated at the top of each
lane. As described under "Experimental Procedures," because these
experiments were designed to examine the relative ratio of bands within
a sample, the individual samples were not equilibrated for amounts of
MVM-specific RNA. The identities of the protected bands are shown on
the left and explained under "Experimental Procedures."
*, undigested probe B. Note the absence of the P38-generated R3
products in the AUG mutants. P38 promoter activity depends upon the
viral NS1 protein.
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Fig. 5.
The nonsense-mediated effect of
p2018TAA is dependent upon a communication between open
reading frames in the first and second exons but does not require a
spliceable downstream intron or the normal stop codon.
A, sequences of the codon at nt 2018, the presence
(wt) or mutation ( ) of donor D1 and acceptor A2 of the
small intron and the presence of a frameshift (by deletion of a single
nt) at nt 505 (fs) in wild type MVM and mutants are shown
underneath their appropriate map positions (deviations from the wild
type sequence are underlined), together with quantitations
of the R2/R1 ratio obtained by RNase protection analysis with probe B
for each mutant. All the values are the averages of at least three
separate experiments. Standard deviations are indicated in
parentheses. wt, wild type sequence.
fs, frameshift. (), deletion of two nucleotides at 2380 and 2381. B, RNase protection analysis of total RNA using
probe B (see Fig. 1) of RNA generated by wild type MVM (WT),
mutants (as described in text), or mock-transfected as designated at
the top of each lane. The identities of the protected bands
are shown on the left and explained under "Experimental
Procedures." *, undigested probe B. C, RNase protection
analysis of total RNA using probe B (see Fig. 1), of RNA generated by
wild type MVM (WT), mutants (as described in text), or
mock-transfected as designated at the top of each lane. The
identities of the protected bands for WT and p2018TAA (the
two left lanes) are shown on the left and
explained under "Experimental Procedures." *, undigested probe B. The top, middle, and bottom bands
shown for pD1()A2() and p2018D1()A2() (the two right
lanes) represent forms of R1, R2, and R3, respectively, that are
unspliced in the small intron region and are smaller than the
corresponding bands generated by the wild type and 2018 mutant because
the wild type protection probe B used for these experiments was not
entirely homologous in the small intron region of these mutants.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)). The 2018 PTC was, however, suppressed
by a single nucleotide deletion at nt 505 in the upstream NS1/2 common
exon which shifts the PTC into the NS1 (ORF) in the final R2 spliced
product (Fig. 5, A and B). This result suggested
that there was communication of reading frame between the two exons
before final splicing of the intervening intron.
)A2(); Fig. 5, A
and C). Thus the 2018 PTC did not require a prior splicing event downstream for its effect.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed. Tel.: 573-882-3920;
Fax: 573-882-4287; E-mail: pinteld@health.missouri.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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