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1
Nonsense Mutations in Close Proximity to the Initiation Codon
Fail to Trigger Full Nonsense-Mediated mRNA Decay
Ângela Inácio1, Ana Luísa Silva1, Joana Pinto1, Xinjun Ji2, Ana Morgado1, Fátima
Almeida1, Paula Faustino1, João Lavinha1, Stephen A. Liebhaber2 and Luísa Romão1,*
1Centro de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA),
Av. Padre Cruz, 1649-016 Lisboa, PORTUGAL, and 2Departments of Genetics and
Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
* Corresponding author
Mailing address: Centro de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge,
Av. Padre Cruz, 1649-016 LISBOA, PORTUGAL
Phone number: (+351) 21 751 9234
FAX number: (+351) 21 752 6410
E-mail: [email protected]
JBC Papers in Press. Published on May 25, 2004 as Manuscript M405024200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Running title:
Proximity of nonsense codon to AUG inhibits NMD
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SUMMARY
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades
mRNAs containing premature translation termination codons. In mammalian cells, a
termination codon is ordinarily recognized as ‘premature’ if it is located greater than 50-54
nucleotides 5’ to the final exon-exon junction. We have described a set of naturally occurring
human β-globin gene mutations that apparently contradict this rule. The corresponding β-
thalassemia genes contain nonsense mutations within exon 1 and yet their encoded mRNAs
accumulate to levels approaching wild-type β-globin (βWT) mRNA. In the present report we
demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are
intermediate between βWT mRNA and β-globin mRNA carrying a prototype NMD-sensitive
mutation in exon 2 (codon 39 nonsense; β39). Functional analyses of these mRNAs with 5’-
proximal nonsense mutations demonstrate that their relative resistance to NMD does not
reflect abnormal RNA splicing or translation re-initiation and is independent of promoter
identity and erythroid specificity. Instead, the proximity of the nonsense codon to the
translation initiation AUG constitutes a major determinant of NMD; positioning a
termination mutation at the 5’-terminus of the coding region blunts mRNA destabilization and
this effect is dominant to the “50-54 nt boundary rule”. These observations impact on current
models of NMD.
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INTRODUCTION
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that
rapidly degrades mRNAs carrying premature translation termination codons (1). Nonsense-
containing mRNAs targeted by NMD can be generated by naturally-occurring frameshift and
nonsense mutations, splicing errors, leaky 40S scanning, and by utilization of minor AUG
initiation sites (2, 3). A major function of the NMD pathway is to block the synthesis of
truncated proteins that could have dominant negative effects on cell function (2, 4).
Recent studies have shown that the NMD pathway in mammalian cells is linked to
splicing-dependent deposition of a protein complex 20-24 nucleotides (nt) 5’ of each exon-
exon junction (exon-junction complex; EJC). The EJC contains the general splicing activator
RNPS1, the RNA export factor Aly/REF, the shuttling protein Y14, the nuclear matrix-
localized serine-arginine-containing protein SRm160, the oncoprotein DEK, and the Y14
binding protein magoh. The interaction of magoh with Y14 may have a role in cytoplasmic
localization of mRNAs and in anchoring the NMD-specific factors Upf3 and Upf2 to the
mRNA (5-18). Previous published data have shown that Upf3 and Upf2 join the EJC in
different subcellular compartments: Upf3 (Upf3a and Upf3b) is loaded onto mRNAs in the
nucleus during splicing via interactions with components of the EJC. In contrast, Upf2 joins
the complex soon after cytoplasmic export is initiated (14, 19, 20). According to present
models, translating ribosomes displace EJCs from the open reading frame (ORF) during the
‘pioneer’ round of cytoplasmic translation (21). If, however, the mRNA contains a premature
termination codon located more than 50-54 nt 5’ of at least one EJC, complex components 3’
to the termination mutation will remain on the mRNA. The retention of one or more EJCs on
the mRNA trigger the NMD response by an as yet undefined mechanism. According to
current models, recognition of the nonsense codon as ‘premature’ involves a direct interaction
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of the phosphorylated Upf1 RNA helicase with EJC-bound Upf2 (20, 22, 23). Interactions
between Upf1 and other Upf factors may also be involved in this process (14, 19, 20, 23-26).
The interactions of the translation termination factors eRF1 and eRF3 with Upf1 and of eRF3
with Upf2 and Upf3 appear to provide a mechanistic link between NMD and translation
termination at the premature stop codon (reviewed in reference 1, 27).
Studies supporting the ”50-54 nt boundary rule” suggest that an exon-exon junction
serves as the major cis-acting NMD-regulatory element (28-30). While EJC regulated NMD
is supported by multiple lines of evidence, exceptions to the “50-54 nt boundary rule” have
been reported. These exceptions suggest that additional determinants may be involved in
determining the net stability of nonsense-containing mRNAs. For example, nonsense codons
positioned close to the initiation AUG of the TPI, immunoglobulin µ heavy chain, and
BRCA1 mRNAs, fail to specify NMD (31-33). For the TPI and immunoglobulin µ heavy
chain mRNAs, NMD appears to be circumvented by re-initiation of translation 3’ to the
nonsense codon. This mechanism of NMD-avoidance has also been speculated for nonsense
codons located early in the BRCA-1 transcript (33). Such re-initiation would allow the
ribosome to disrupt EJCs located 3’ to the nonsense mutation (31, 32). Nonsense codons in
the T-cell receptor (TCR)-β transcript also fail to elicit canonical NMD; nonsense codons
located more 5’ in the mRNA trigger robust NMD while nonsense codons closer to the
terminal exon-exon junction induce a much weaker NMD response (34). Finally, premature
termination codons located in different exons of the fibrinogen Aα-chain gene or at exon 2 of
the human ALG3 gene can avoid NMD (35, 36). The basis for NMD-avoidance by the
fibrinogen Aα-chain and ALG3 mRNAs remain undefined. Thus present evidence indicates
that determinants of NMD, at least in some systems, are more complex than would be
predicted on the basis of the single ”50-54 nt boundary rule”.
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We have previously reported that human β-globin mRNAs containing naturally-
occuring nonsense mutations in the 5’-part of exon 1 accumulate to levels similar to those of
normal β-globin transcripts (37). The aim of the present study is to investigate the mechanism
by which these mutant β-globin transcripts circumvent the full impact of NMD. Our results
reveal that this unusual NMD-behaviour reflects the proximity of the nonsense mutations to
the AUG initiation codon. Remarkably, the impact of this 5’ localization is dominant to the
“50-54 nt boundary rule”.
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EXPERIMENTAL PROCEDURES
Plasmid constructs. The plasmids containing the normal (βWT), β15 [CD 15
(TGG→TGA)] or β39 [CD 39 (C→T)] human β-globin genes were previously described
(37). All variants were created within the 428 bp Nco I-Bam HI fragment of the β-globin gene
template by overlap-extension PCR (37). PCR reactions were performed by using Life
Technologies primers (Barcelona, Spain; see Table I). To create variants carrying DelA
(deletion of 69 bp between codons 3 to 27, exclusively), the PCR reaction # 1 was performed
by using L5’β-S and DelA-AS primers, and DNA template from the previously cloned βWT
gene. PCR reaction # 2 was performed by using DelA-S and R5’β-AS primers and the βWT or
�39 genes as template. To clone variant βWTseqhet and β39seqhet constructs, which contain a
different sequence between codons 3 and 27, from the second exon of the human α2 globin
gene (from codon 34 through codon 56), PCR reaction # 1 was performed by using Seqhet-S
and Seqhet-AS primers, and the hα2-globin gene as DNA template (pTet-Wt detailed in
reference 38). PCR reaction # 2 was performed by using 3’exon1-S and 3’exon2-AS primers
and the βWT or β39 gene DNAs. To obtain β62 and β62DelA variants carrying the nonsense
mutation (GCT→TAG) at codon position 62, PCR reaction # 1 was performed with ATG-S
and 62-AS primers and the previously cloned βWT or DelA gene DNAs. PCR reaction # 2 was
performed with 62-S and 3’exon2-AS primers and the βWT gene as DNA template. To clone
the double nonsense mutated gene β15nonβ39, carrying both nonsense mutations at codons
15 and 39, PCR reaction # 1 was performed by using L5’β-S and 15non39-AS primers and
the β39 gene DNA. PCR reaction # 2 was performed by using 15non39-S and R5’β-AS
primers and the same DNA template. To obtain the constructs βWT-CD55-63/64-73/74, β15-
CD55-63/64-73/74, β39-CD55-63/64-73/74, and β39DelA-CD55-63/64-73/74, the three
different mutations were introduced sequentially and the βWT-CD55, β15-CD55, β39-CD55
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and β39DelA-CD55 genes [βWT, β15, β39, or β39DelA genes carrying a missense mutation
that converts Met to Ile (AUG�AUA) at codon 55] were created first. Thus, PCR reaction #
1 was performed by using the L5’β-S and 55-AS primers, and the βWT, β15, β39, or β39DelA
gene DNAs. PCR reaction # 2 was performed by using the 55-S and the R5’β-AS primers and
the βWT DNA template. To create the βWT-CD55-73/74, β15-CD55-73/74, β39-CD55-73/74
and β39DelA-CD55-73/74 constructs [correspond to the βWT-CD55, β15-CD55, β39-CD55 or
β39DelA-CD55 human β-globin genes carrying an additional missense mutation that converts
the out-of-frame Met to Thr (AUG�ACG) at position 73/74] PCR reaction # 1 was
performed by using the L5’β-S and 73/74-AS primers, and the βWT-CD55, β15-CD55, β39-
CD55, or β39DelA-CD55 genes as DNA template. PCR reaction # 2 was performed by using
the 73/74-S and the R5’β-AS primers and the βWT DNA template. In all cases, PCR reaction #
3 was performed using a mix of PCR reactions # 1 and # 2 and primers sense from PCR
reaction # 1 and antissense from PCR reaction # 2. The obtained PCR products were digested
with Nco I-Bam HI and replaced into the plasmid containing the cloned normal human β-
globin gene previously digested with the same enzymes. The mutation at position 63/64
(ATG�ACG) was introduced into the genes βWT-CD55-73/74, β15-CD55-73/74, β39-CD55-
73/74, and β39DelA-CD55-73/74 by using the QuickChangeTM site-directed mutagenesis kit
as indicated by the manufacturers (Stratagene, Amesterdam, The Netherlands). Reactions
were performed with the 63/64-S and 63/64-AS mutagenic primers and the DNA template
from βWT-CD55-73/74, β15-CD55-73/74 β39-CD55-73/74 and β39DelA-CD55-73/74
plasmids. The correct Nco I-Bam HI globin gene fragment containing all mutations was
employed to substitute the original non-mutated Nco I-Bam HI fragment present in the native
gene of the p158.2 expression vector (37). To obtain plasmids used to transiently transfect
HeLa cells, a human �-globin gene fragment extending from the transcription initiation site to
200 bp 3’ to the polyadenylation site was amplified by PCR from the p158.2 plasmid, using
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LinkClaI-S and LinkNotI-AS primers (Table I). The 1806 bp Cla I - Not I human β-globin
fragment was sub-cloned into pTet2 plasmid, creating the pTet2-βWT plasmid [pTet2 plasmid
is derived from pTet-Splice vector (Life Technologies) by deleting a 126 bp fragment
upstream the Cla I site; the deletion was introduced by substituting the native 500 bp Xho
I/Cla I fragment by the 374 bp DNA fragment that was amplified using pTetXhoI1/15-S and
pTetClaI351/370-AS primers (Table I)]. The human β-globin variants β15, β17, β39, β62,
DelA, β39DelA, β62DelA, βWTseqhet, β39seqhet, or β15nonβ39 were cloned into the pTet2-
βWT vector previously obtained, by substituting normal Nco I-Bam HI fragment for the
corresponding mutated fragment, which had been previously cloned into the p158.2 vector
(see above). To obtain genes βWT(15:39) and β15(15:39) which present the original codon 15
at position 39, initially the restriction sites Hind III-Apa I were introduced between codons 14
and 15 into the Nco I-Bam HI fragment of the βWT and β15 genes, using the QuickChangeTM
site-directed mutagenesis kit as before. Reactions were performed with the following
mutagenic primers: WT15:39-S and WT15:39-AS (for βWT) or 15:39-S and 15:39-AS (for
β15) (Table I). After to obtain the correct mutagenized βWT and β15 clones, a 60 bp DNA
sequence of the ampicillin resistance gene (Ampr; 5’-
TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG
AACGT-3’) was introduced in both clones (βWT and β15) between Hind III/Apa I sites. The
sequence was previously obtained by PCR using HindIIIAmpr-S and ApaIAmpr-AS primers
(Table I), using the pGEM-3 plasmid (Promega) as DNA template. The cloned Nco I-Bam HI
quimeric fragments were transferred to the pTet2-βWT vector by substituting the normal Nco
I-Bam HI fragment. As the introduced DNA Ampr spacer revealed to contain one splicing
donor site with high consensus value (sequence CGGTAAGA), an additional mutagenesis
(T�A) was performed in order to inhibit the alternative splicing. The mutagenesis was
carried out in both constructs βWT(15:39) and β15(15:39), with the QuickChangeTM site-
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directed mutagenesis kit using the GA-S and GA-AS oligonucleotides (Table I). The pTet
plasmid containing the human α-globin gene (pTet-�wt) used as a control for transient
transfection efficiency was previously described (39).
Cell culture and transfections. Mouse erythroleukemia (MEL) cells were cultured in
RPMI medium with glutamax-1 (Invitrogen), supplemented with 10% fetal bovine serum.
Stable transfections were carried out with 40 µg of linearized plasmid DNA mixed with 2 µg
of linearized pGKpuro (plasmid containing the puromicin resistance gene). Electroporations
were performed using 3x107 cells per transfection, as previously described (37). Each
electroporated cell pool was expanded in selective medium during approximately 10-12 days,
by adding puromicin (Invitrogen) to 2.5 µg/mL. Erythroid cell differentiation was induced by
adding 2% (v/v) DMSO to the media of each transfection cell pool. Following five days, cell
pools were harvested for RNA extraction. MEL cells stably expressing the tet transactivator
(MEL/tTA; described in reference 39) were used for conditional expression of hβ-globin
genes (previously cloned into the pTet vector). For transient transfections, MEL/tTA cells
were split 1 day before transfection and cultured in minimal essential medium (MEM)
supplemented with 10% fetal bovine serum, and 100ng/ml tetracycline. Cells were washed
three times in cold Dulbecco phosphate-buffered saline (PBS) and ressuspended in cold
serum-free minimal essential medium at a concentration of 3 x 10 7 cells per ml. Two
micrograms of pTet2-βWT or each pTet2-β variant were added to the cell suspension and
mixed thoroughly. Electroporation was performed at 250 V, 1.180�F, and low resistance in a
BRL Cell-Porator system. For steady state mRNA quantification, cells were harvest after 12
hours transcription pulse. For mRNA stability analysis, cells were split into four 60 mm
diameter dishes. Cells were pulsed with β-globin mRNA for 12 hours by growth in Tet-
media. Following this period, transcription from the plasmid was then blocked by the addition
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of Tet to the media. Cells from each culture dish were harvest in different time points for
further analysis.
The adherent HeLa cells, stably expressing the tet transactivator (HeLa/tTA; described
in reference 39), were grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum. Transient co-transfections were performed with
the pTet-�wt (to control for transfection efficiency) and the appropriate pTet2-β plasmid,
using Lipofectin - Plus Reagent (Invitrogen), following the manufacturer’s instructions, using
1x105 cells per transfection and one microgram of each plasmid. Cells were harvest after one
day transcription pulse.
RNA isolation. Total RNA from transfected cells was prepared using the RNeasy total
kit (Qiagen) following the manufacturer’s instructions. Before analysis, RNA samples were
treated with RNase-free DNAse I (Ambion) and purified by phenol:chloroform extraction.
Primer extension analysis. Primer extension assays were performed using 5’end-
labeled primers: Hβexon3-AS or Hβ3’UTR-AS and Mα-AS (Table I). Both primers were
mixed, hybridized with 10 µg of MEL RNA at 50ºC during 5 hours, and extended with
reverse transcriptase Superscript II (Invitrogen), as described previously (37). Primer
extension products were analyzed by PhosphorImaging, using a Typhoon® Imager 8600
(Molecular Dynamics) with a 6 hours exposure period. One, ten, and fifteen micrograms of
RNA extracted from cells transfected with the normal β-globin gene were also transcribed to
show that the experiment was carried out in the presence of primer in excess. The intensity of
each band was quantified using the Image Master Phoretix® Software (Amersham Pharmacia
Biotech).
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Ribonuclease protection assays. Probes used were generated by in vitro transcription
of plasmids containing DNA fragments from hβ-globin, hα-globin (40), mα-globin (40), or
mGAPDH (Ambion). The hβ-globin probe is a 170 bp fragment encompassing last 20 bp of
intron 2, the entire exon 3 coding region, and first 21 bp of 3’UTR, which was generated by
PCR and inserted into the polylinker region of pGEM3. Amplification primers contained
restriction sites which facilitated insertion into the vector polylinker. To generate RNA
probes, each transcription vector was linearized and transcribed in the presence of α[32P]CTP
(400 ci/mmol, 10 mCi/ml; Amersham Pharmacia Biotech) using a Maxiscript SP6 kit, under
conditions recommended by the manufacturer (Ambion). Final concentration of ATP, GTP
and UTP were 0.5 mM, and that of CTP was 0.06 mM. One microgram of RNA was added to
20 µl of hybridization buffer (40 mM piperazine-N,N´-bis(2-ethanesulfonic acid) (pH 6.4), 1
mM EDTA (pH 8.0), 0.4 M NaCl, 80% formamide) supplemented with probes. Samples were
heated at 80ºC for 10 min, incubated overnight at 50ºC, and digested for 15 min at room
temperature in 200 µl of RNase assay buffer (300 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM
EDTA pH 8.0) containing 1 µl of RNase cocktail (500 mg of RNAse A/ml, 20.000 U of
RNase T1/ml; Ambion). Digestions were terminated by addition of 18 µl of an SDS-protease
K (10%:2mg/ml) mixture to each sample followed by incubation 20 min at 37ºC. RNA was
extracted, precipitated, dissolved in loading buffer, and resolved onto a 6 or 8 % acrylamide 8
M urea gel. Radioactivity in bands of interest was quantified by PhosphorImaging, using a
Typhoon® Imager 8600 (Molecular Dynamics).
RT-PCR analysis. Two micrograms of each total RNA sample were mixed with 2
pmoles of Hβ3’RNA-AS primer (Table I). The mixture was heated to 70ºC for 10 min and
quick chilled on ice. Four micriliters of 5X first strand Buffer (Invitrogen), 2 µl of 0.1 M DTT
(Invitrogen), 1 µl of 25 mM dNTP mix (25 mM each dATP, dGTP, dCTP and dTTP) and 0.1
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µl of Ribonuclease Inhibitor (Invitrogen) were added per sample. The contents were mixed
and reverse transcribed with 200 U of SuperscriptII (Invitrogen) at 42ºC for 2 min. Five µl
aliquots of cDNA product were PCR amplified in a 50 µl reaction volume, using 0.3 µl (1.5
U) of Amplitaq DNA polymerase (Applied Biosystems) for 30 cycles at 95 º C for 1 min, 54 º
C for 1 min, and 72º C for 2 min, with a final extension at 72ºC for 10 min. Reaction mixture
contents were: 37.3 µl of water, 5 µl of 10X buffer (15 mM MgCl2) (Applied Biossystems),
0.4 µl dNTP Mix (25 mM each dATP, dGTP, dCTP and dTTP), 15 picomoles of each primer
(Hβ5’RNA-S plus Hβ3’RNA-AS, Hβ5’RNA-S plus Hβ3’exon2-AS, or Hβexon2-S plus
Hβ3’RNA-AS; see Table I) and 0.3 µl of enzyme. 10 µl aliquots from each RT-PCR sample
were analysed by electrophoresis on 1% agarose gels.
Sucrose gradient polysome fractionation. Polysome gradients were carried out as
described (38). In brief, transiently transfected MEL cells were centrifuged at 1,000 x g for 5
min at 4°C. The cell pellet was washed with ice-cold PBS, lysed in TMK100 buffer (10 mM
Tris-HCl at pH7.4), 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 1% Triton X-100, and RNase
inhibitor 100U/ml - Promega), the nuclei were cleared at 10,000 x g for 10 min at 4°C, and
the supernatants (S10) were removed and layered onto the prepared 10%-to-50% linear
sucrose gradients and centrifuged at 40000 rpm for 85 min at 4°C. The gradients were
collected in 15 fractions from the top to the bottom by displacing them upwards with 60%
sucrose, and the gradient profile was monitored via UV absorbance at 254 nm with an ISCO
UA-5 detector (Lincoln, Nebr., USA). RNA was extracted from sucrose fractions and ethanol
precipitated. mRNA content was determined by RPA, using probes generated by in vitro
transcription of plasmids containing genomic DNA inserts for hβ-globin (38), and mGAPDH
(Ambion). The final protected fragments were resolved on a 6% acrylamide-8 M urea gel.
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Radioactivity in bands of interest was quantified by PhosphorImager analysis (Storm 840;
Molecular Dynamics).
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RESULTS
ββββ15 and ββββ17 mRNAs are more stable than the NMD-targeted ββββ39 mRNA. We
have previously reported that a set of thalassemic hβ-globin mRNAs bearing nonsense
mutations in exon 1 (β5, β15, and β17, respectively) accumulate to steady-state levels in vivo
that are significantly higher than the β-globin mRNA with a prototype NMD-sensitive
nonsense mutation (β39) (37, 41). This observation suggested that these nonsense-containing
β-globin mRNAs avoid the full impact of NMD. To firmly establish that this is the case, the
half-lives of two of the β-globin mRNAs with nonsense mutations in exon 1, β15 and β17,
were determined and compared to βWT and β39 mRNAs. Each β-globin gene (normal or
mutant) was cloned behind a tet-controlled promoter and transfected into a MEL cell line that
stably expresses the tet-transcriptional transactivator (MEL/tTA cells) (39). The cells were
transcriptionally pulsed for 12 hours with each β-globin mRNA, and the corresponding
mRNA decay rate was subsequently established. An example of this study is shown in figure
1A and a compilation of four independent experiments is shown in figure 1B. The half-life
(t1/2) of normal β-globin mRNA (βWT) in this system is 10 hours and that of the NMD-
sensitive β39 mRNA is 3.5 hours. These half-lives are in agreement with a recent study by
Couttet and Grange (42). The half-lives of the β15 and β17 mRNA were similar to each other
and were intermediate to the βWT and β39 mRNAs; t1/2 of β15 mRNA is ~6.3 hours, and t1/2 of
β17 is ~5.8 hours (Fig. 1B). These half-lives are consistent with the steady state levels of β15
and β17 mRNAs in erythroid cells (37) and suggest that hβ-globin mRNAs with nonsense-
mutations located in close proximity to the initiation AUG can escape the full impact of
NMD.
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Close proximity of nonsense mutations to the initiation AUG minimizes NMD of
hββββ-globin mRNA. The above data confirm that the β15 and β17 mRNAs are relatively
resistant to NMD. We hypothesized that resistance might reflect the positioning of the
nonsense codon relative to the initiation AUG. To test this hypothesis, a deletion was
introduced into the β39 mRNA that removed 69 bp between codons 3 – 27 (β39DelA). This
deletion relocates the β39 nonsense mutation to a position 16 codons from the AUG while
leaving the mutation within exon 2 and preserving the distance between the β39 nonsense
codon and the terminal (exon 2-3) splice junction (195 nt) (Fig. 2A). MEL cells were stably
co-transfected with equimolar amounts of β39DelA plus β39 or βWTDelA plus βWT and the
levels of the corresponding mRNAs were determined after the MEL cells were stimulated to
terminally differentiate (37) (Fig. 2B). The results from three independent experiments
revealed that the DelA deletion increased the level of β39 mRNA by 7.3-fold. This increase
was significantly greater than the 1.8-fold increase in the level of βWT mRNA. Thus,
shortening the distance between the β39 mutation and the AUG resulted in a significant and
preferential mRNA stabilization.
The stabilizing effect of DelA on the β39 mRNA was confirmed by a second
approach. MEL cells were stably transfected with each individual construct, the cells were
induced to erythroid differentiation, and the steady state level of each mRNA was assessed
relative to an internal control [mouse (m) α-globin mRNA] (Fig. 2C). Results from four
independent experiments indicate that β39DelA transcripts accumulate at about 84% of
βWTDelA transcript levels, while β39 transcripts show levels of accumulation at about 9% of
βWT (Fig. 2C). The studies support the conclusion that NMD is significantly abrogated when
β-globin nonsense mutations are in close proximity to the initiation AUG.
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The half-life of ββββ39DelA mRNA is similar to that of ββββ15 or ββββ17 mRNAs. Based on
steady state mRNA levels (Fig. 2), we hypothesized that DelA stabilizes the NMD-sensitive
β39 mRNA. To directly test this prediction, β39 and β39DelA genes were placed under
control of the tetracycline-regulated promoter. MEL/tTA cells transfected with the β39 and
β39DelA genes were transcriptionally pulsed for 12 hours and the decay rates of the β-globin
mRNAs were monitored over time (Fig. 3A). Results from three independent experiments
revealed a t1/2 of 5.2 hours for β39DelA mRNA (Fig. 3B). This half-life is equivalent to that
determined for the β15 or β17 mRNAs and significantly longer than that determined for the
β39 mRNA. These data confirm that DelA stabilizes the β39 mRNA.
The relative stability of ββββ-globin mRNAs with 5’-proximal nonsense mutations is
independent of promoter- and tissue-specificity. Work from others suggests that the decay
rate of nonsense-containing β-globin mRNAs can be impacted by an erythroid environment
(43). For this reason, we next compared the NMD profiles established in MEL cells (Figs. 2
and 3) with those in HeLa cells. HeLa cells were transiently co-transfected with equimolar
amounts of hα- and β39DelA-globin genes under control of the CMV promoter. Twenty-four
hours post-transfection, RNA was quantified by RPA using probes specific for the human α-
and β-globin mRNAs. The mRNA levels of βWT, β15, β39, and βWTDelA genes were assessed
in parallel (Fig. 4). Results from three independent experiments revealed accelerated decay of
β39 mRNA in non-erythroid cells; levels of β39 mRNA were 37% of βWT mRNA. In contrast,
mRNA with a 5’ proximal nonsense mutation (β15) accumulated to the same level as βWT.
Furthermore, deletion of sequences between codons 3-27 (Del A) of β39 mRNA increased its
accumulation in HeLa cells from 37% to 86% of βWT. Thus β15 mRNA evades NMD in the
non-erythroid (HeLa) as well as erythroid (MEL) cells, while β39 is NMD-sensitive in both
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settings and is stabilized by DelA. Since the stabilizing impact of DelA on β39 mRNA is
equivalent in MEL and HeLa cells, using either the homologous β-globin promoter or the
heterologous CMV promoter in erythroid and non-erythroid cells, the relative impact of NMD
on the various mutant mRNAs appears to be independent of promoter as well as tissue
specificity. Taken together, these data substantiate the stability of the 5’ nonsense mutations
and the stabilizing effect of deleting codons 3-27 from β39 mRNA.
Stabilities of ββββ39DelA and 5’-proximal nonsense mRNAs do not reflect activation
of abnormal splicing pathways. Mechanisms that might contribute to stabilization of the β-
globin mRNAs with 5’-proximal nonsense mutations were explored. We initially tested the
possibility that these mutations activated cryptic splicing pathway(s) with consequent
alteration in mRNA sequence and circumvention of the premature termination codon. This
possibility was of particular interest in light of the numerous cryptic splice-donor sites in exon
1 that can be activated in vivo (44-47). Representative hβ-globin genes were expressed in
transfected MEL cells and the encoded mRNAs were analysed by RT-PCR, using a set of
primers that encompass the full-length β-globin mRNA (Fig. 5A) (see Methods). A single
full-length product of 625 bp was amplified from all non-deletional transcripts and a 556 bp
fragment was amplified from cDNAs carrying DelA. In all cases, the cDNAs that were
amplified correspond to the expected sizes of normally spliced β-globin mRNA. These results
were confirmed by amplifying 5’ and 3’ halves of the hβ-globin cDNA from mRNA
transiently expressed in HeLa cells (Fig. 5B; see Methods). As expected, the 5’ half of the
cDNA generated a product of 351 bp for βWT, β39, and β15 transcripts and a fragment of 282
bp corresponding to the βWTDelA and β39DelA mRNA samples. Analysis of the 3’cDNA
fragment revealed the amplification of a 359 bp product in all samples (Fig. 5B). These
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results demonstrate normal splicing patterns for the transcripts containing the various
nonsense and deletion mutations.
ββββ39DelA and 5’-proximal nonsense mRNAs fail to undergo appreciable levels of
translation re-initiation. NMD can be abrogated if translation re-initiates downstream of the
nonsense codon (31). With this in mind, we investigated whether translational re-initiation
was occurring on the β-globin mRNAs with 5’-proximal nonsense mutations. Polysome
distributions were determined for βWT, β15, β39, βWTDelA, and β39DelA mRNAs expressed
in transiently-transfected MEL cells (Fig. 6A and B). Data revealed a normal polysome
profile for the βWT mRNA (ORF 146 codons) with a mean polysome size of 4 ribosomes.
The mean polysome size for the βWTDelA mRNA was 3 ribosomes, a size consistent with its
slightly shortened ORF (123 codons). In contrast, β15 and β39 mRNAs were both limited to a
mean polysome size of 1-1.5 ribosomes and β39DelA mRNA peaked at a mean polysome size
of 1 ribosome (Fig. 6C). These results support the conclusion that β15, β39, and β39DelA
mRNAs terminate translation at their respective nonsense codons without appreciable levels
of downstream re-initiation.
The question of translational re-initiation was further addressed by a second approach.
If residual re-initiation, even at low levels, did occur and was able to circumvent NMD, this
effect should be inhibited by inactivating putative initiating AUG codons located 3’ to the
nonsense codon. The most proximal hβ-globin AUGs in a favoured Kozak consensus (48) are
at positions 55 (in frame) and 73/74 (out of frame). A third AUG codon at 63/64 is in a less
optimal sequence context (49). All three putative reinitiation AUGs were mutated in cis
within the βWT, β15, β39, and β39DelA genes; Met-55 was converted to isoleucine (Ile)
(AUG�AUA), in cis to AUG�ACG substitutions at positions 63/64 and 73/74. HeLa cells
were transiently transfected with each of these constructs or with the parental βWT, β39DelA,
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β15, and β39 constructs. The expression of each β-globin mRNA was normalized to the
expression of a co-transfected hα-globin gene (Fig. 6D). The results demonstrate that the
combined array of missense mutations introduced at codons 55, 63/64, and 73/74 failed to
destabilize the β15 or the β39DelA mRNAs. These data, along with the polysome profiles,
lead us to conclude that the stability of the β15 mRNA and the stabilization of the β39 mRNA
by the DelA deletion, do not reflect translation re-initiation.
The proximity of nonsense codons to the translation initiation AUG is a major
determinant of NMD. The stabilization of the β39 mRNA by DelA could be explained by
two models. The first model reflects the repositioning the β39 mutation closer to the initiation
codon. The second model reflects deletion of a cis element within the DelA segment that is
important to the NMD pathway. The second model might also account for the relative
stabilities of β5, β15, and β17 mRNAs if each of the corresponding base substitutions altered
the structure of the putative NMD determinant. The merit of the second model was tested by
substituting the entire sequence between codons 3 and 27 in the β39 with a ‘neutral’ sequence
corresponding to exon 2 of the hα2-globin gene. The resulting β39seqhet gene and a control
βWTseqhet gene were separately stably transfected in MEL cells. Following erythroid
induction, hβ-globin mRNA levels were quantified relatively to the endogenous mα-globin
(Fig. 7A). Results from four independent experiments revealed that β39seqhet and β39
mRNAs accumulate to approximately the same levels when compared to the corresponding
βWT (31% and 28%, respectively) (Fig. 7A). These data suggest that sequences between
codons 3-27 in the β-globin mRNA do not contain an NMD-relevant determinant.
The lack of support for a NMD determinant in the first exon led us to consider in
further detail the model which proposes that the strength of NMD is impacted by the
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proximity of the nonsense mutation to the initiation AUG. This proximity effect was tested
by introducing DelA in a second NMD-committed β-globin mRNA, β62 (29). In this case
DelA moves the β62 nonsense codon to a position 39 codons from the AUG; ie the same
distancing as in NMD-sensitive β39 mRNA. The β62 and β62DelA constructs were stably
transfected into MEL cells in parallel with βWT, β15, or β39 genes. Transfected cell pools
were induced to differentiation and RNA levels were quantified in four experiments (Fig.
7A). The β62 transcripts are expressed at about 18% of normal and β62DelA mRNA
accumulates at about 15% of normal, and both of these transcripts have NMD-sensitivity
similar to β39 (Fig. 7A). These data support the conclusion that DelA stabilizes the β39
mRNA by bringing the nonsense codon into critical proximity with the AUG rather than by
removing an NMD determinant.
The relationship between the AUG-proximity of nonsense mutations to the strength of
NMD was confirmed in nonerythroid cells. HeLa cells expressing the tet transactivator
(HeLa/tTA; 39) were transiently co-transfected with each of seven β-globin constructs (βWT,
βWTseqhet, β15, β39, β39seqhet, β62, β62DelA) under control of the tetracycline-regulated
promoter. Cells were cultured in the absence of tetracycline for 24 hours to induce expression
and RNA levels were subsequently quantified. The expression of each β-globin gene was
normalized to a co-transfected hα-globin gene. The results of three studies are summarized in
figure 7B. When compared to the level of βWT mRNA, the β15 mRNA is expressed at 105%;
β39 mRNA at 38%; and β62 and β62DelA mRNAs at 44-49% of normal. The β39seqhet is
expressed at 40% of βWTseqhet. These data parallel those in MEL cells and reinforce the
conclusion that DelA stabilizes a nonsense-containing mRNA by repositioning a nonsense
codon close to the initiation AUG.
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As an additional test of the relationship of AUG proximity to NMD we increased the
distance between the β15 nonsense codon and the AUG. A heterologous 24 codon spacer was
inserted between codons 14 and 15 of β15 gene. As a control, the same insertion was made in
the βWT gene. This insertion moves the β15 nonsense mutation to a position 39 codons from
the AUG. HeLa/tTA cells were co-transfected with each of these hybrid constructs and the
hα-globin gene. Cells were grown for 24 hours in the absence of tetracycline and RNA levels
were subsequently quantified (Fig. 7B). These studies reveal that βWT(15:39) mRNA
accumulates at approximately 112% of normal (βWT) and β15(15:39) mRNA accumulates at
approximately 37% of βWT. These results further support the role of AUG-proximity in NMD
by demonstrating that increasing the distance between the β15 mutation and the AUG results
in mRNA destabilization.
Finally, we analysed if the impact of AUG-proximity on NMD is dominant over the
general “50-54 nt boundary” rule. Here, we introduced the β15 mutation in cis to the β39
(construct β15nonβ39). This gene was stably transfected in MEL cells and the level of
accumulation was determined after induction of differentiation. We find that the β15nonβ39
mRNA accumulates to approximately the same level as the β15 mRNA (respectively, 63%
and 60% of βWT; Fig. 7A). Concordant results were also obtained in HeLa cells, where
β15nonβ39 mRNA is at about 97% of βWT (Fig. 7B). These data unequivocally demonstrate
that the effect of the proximity of the nonsense mutation to the AUG is a dominant
determinant in the inhibition of the NMD pathway.
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DISCUSSION
Termination codons are recognized by the NMD apparatus as premature if they are
located more than 50-54 nt 5’ to the 3’-most exon-exon junction (28, 29). We have previously
reported that human β-globin mRNAs bearing nonsense mutations in the 5’-region of exon 1
accumulate to levels similar to those of wild type β-globin mRNA (37). This finding is in
apparent contradiction to the “50-54 nt boundary rule” as these mutations are substantially 5’
to the terminal exon 2-3 junction. In the present report we demonstrate that these mRNAs
with AUG-proximal nonsense mutations are in fact significantly more stable than prototype
NMD-sensitive β-globin mRNAs and that the proximity of nonsense mutation to the AUG
initiation codon comprises the basis of their relative NMD resistance. Thus, AUG-proximity
can over-ride the “50-54 nt boundary rule” in establishing the overall efficiency of NMD for a
particular mutant mRNA.
The AUG-proximity effect studied in the current report is based on the hβ-globin
mRNA model. The study stems from observations originally made in patients with specific β-
thalassemic mutations. The cell culture studies currently reported appear to accurately
recapitulate our prior in vivo observations. The effect of AUG-proximity on NMD mechanism
in β-globin mRNA is independent of promoter sequence and is not restricted to erythroid
cells. Thus, it is likely that these findings are not restricted to the globin genes or to the
erythroid cell environment and may in fact constitute general determinants of the NMD
pathway.
To our knowledge, the AUG-proximity effect that we report represents a newly
recognized parameter of the NMD response. However, a number of apparent exceptions to the
“50-54 nt boundary rule” have been previously reported by others. The mechanisms involved
in these individual cases of NMD-resistance include translation re-initiation downstream the
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nonsense codon (TPI and immunoglobulin µ heavy chain mRNAs; 31, 32) and the presence
of a sequence cis-acting element that confers immunity to the “50-54 nt boundary rule” (TCR-
β mRNA; 34). In additional cases the rule is apparently circumvented although the underlying
mechanism responsible for inhibition of the NMD response remains undefined (BRCA1,
fibrinogen Aα-chain and ALG3 mRNAs; 33, 35, 36). We propose, based on the likelihood
that the current findings are not limited to the β-globin mRNA, that the NMD-resistance in
some of these model systems reflects the AUG-proximity effect.
The observed lack of cell-type specificity for NMD phenotypes in the β-globin
mRNAs is consistent with the understanding that NMD constitutes an essential surveillance
process operating in all tissues. Nonetheless, a comparison of results shown in figures 2C and
4 suggests that NMD of human β-globin mRNA may be somewhat more efficient in
terminally-differentiated erythroid cells when compared to HeLa cells. This may be
contributed by the observation of Stevens et al. (43) that the degradation of nonsense-mutated
β-globin mRNAs in erythroid cells is accomplished by a tissue specific endonuclease with
preference for UG dinucleotides (43). Thus, while specialized decay pathways may exist in
the erythroid compartment, the general conservation of the NMD phenotypes that we observe
in MEL and HeLa cells suggests that the AUG-proximity effect reflects a general attribute of
the NMD pathway.
The relevance of the AUG-proximity of a nonsense codon to NMD is consistent with
prior studies on 5’ ORF structure in NMD in other systems (50). For example, thrombopoietin
mRNA is normally insensitive to NMD, but can be rendered NMD-sensitive by extending the
length of its upstream ORF. These data led to the suggestion that mammalian mRNAs with
naturally occurring short upstream ORFs may have a general resistance to NMD (50). These
data, in conjunction with our present study, would support a model in which termination of
translation in close proximity to the initiation codon results in a resistance to NMD. This
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notion combined with the knowledge that the NMD pathway is translation-dependent, may
lead to a further proposal that the observed NMD-insensitivity of mRNAs with a short 5’
ORF, whether natural or resulting from a nonsense mutation, can mechanistically reflect
ineffective and/or brief translation. In this regard, it is interesting to note that the smallest
naturally occurring eukaryotic ORF that produces significant levels of protein product
comprises 24 codons (51). Based on our results and those published by Zhang et al. (28), this
boundary for human β-globin mRNAs appears to map between codons 17 and 21/22 (β17 is
the 3’-most mutation that fails to trigger full NMD and the β21/22 mutation is the 5’-most
mutation able to fully commit mRNA to the NMD pathway; 28, 37). Thus, the reported
minimal size of an ORF for effective translation roughly correlates with the boundary
between NMD-resistant and NMD-sensitive mutations.
The apparent concordance between the minimal length of an ORF for effective protein
synthesis and the minimal length for effective NMD may be relevant to disease
pathophysiology. In the case of thalassemia, the production of truncated proteins via nonsense
mutations can have a dominant-negative effect on red cell structure and function (52, 53). C-
terminal truncated proteins, if produced at significant levels, can block proteolytic pathways
in differentiating erythroblast and/or can interfere with critical aspects of the assembly and
function of hemoglobin tetramers (53). The NMD pathway is generally considered to clear
cellular mRNAs that can encode such ‘toxic’ protein fragments. However, mutant β-globin
mRNAs with AUG-proximal nonsense mutations are relatively stable. This apparent defect in
NMD surveillance would theoretically place the cell in jeopardy. Nevertheless, we note that
the thalassemic phenotype of heterozygotes and homozygotes carrying AUG-proximal
nonsense mutations do not appear to be any more severe than in patients carrying more distal
nonsense mutations that are effectively targeted by NMD. The lack of a detectable dominant-
negative effect in these patients may reflect the inefficiency with which mutant mRNAs with
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short 5’ ORFs are translated. Thus, the inability of the NMD pathway to effectively clear
mRNAs with 5’-proximal nonsense mutations may be mitigated, at least in part, by the
inability of these mRNAs with very short ORFs to be effectively translated.
The relative NMD resistance of mRNAs with AUG-proximal nonsense-mutations can
be related to critical steps in translational biochemistry. The short ORF may result in a
temporal overlap between translational initiation and termination. The ribosome may arrive
at the premature termination (nonsense) codon before it has discharged its initiation factors
and/or stabilized interactions with elongation/termination factors. This effect would result in
interference with elongation/termination reactions that may be central to the NMD pathway.
Present evidence indicates that the translation termination process is intimately linked with
the NMD pathway. Analyses in other systems have shown that during the process of
translation of short upstream ORFs, interactions involving ribosomal or ribosome-associated
factors including but not limited to releasing factors, can inhibit translation elongation as well
as termination (54-57). In the case of NMD, Upf1 must interact with eRF1 and eRF3 to
terminate translation and, in the proper setting, to interact with additional factors to trigger
NMD (reviewed in reference 1, 27). Therefore, it is possible that the dynamics of the mRNP
remodelling necessary for NMD cannot occur or is defective in this compressed environment
of the AUG-proximal nonsense-mutated mRNAs. The present study further supports the
notion that NMD is a multifaceted process that reflects the overall complexity of eukaryotic
translational biochemistry.
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ACKNOWLEDGEMENTS
This work was partially supported by Fundação para a Ciência e a Tecnologia
(PRAXIS/P/SAU/63/96, POCTI/MGI/34105/2000 and Programa Plurianual/CIGMH), and by
NIH grant RO1-HL 65449 (SAL). J. Pinto, A.L. Silva, A. Morgado, F. Almeida and A. Inacio
were supported by Fellowships from Fundação para a Ciência e a Tecnologia.
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FIGURE LEGENDS
FIGURE 1. The stabilities of hββββ-globin mRNAs with nonsense mutations at codons 15
and 17 (ββββ15 and ββββ17, respectively) are intermediate between ββββWT and a ββββ-globin mRNA
fully committed to NMD (ββββ39). MEL cells stably expressing the tet transactivator (MEL tTA
cells) were transiently transfected with βWT, β15, β17 or β39 genes under the transcriptional
control of a tTA-regulated promoter. Cells were pulsed with the respective β-globin mRNAs
for 12 hours by transfer to Tet(-) media and then transferred back to Tet(+) media for analysis
of decay rates. Total mRNA was isolated at various time intervals during the transcriptional
chase period and analysed by RPA.
(A) Representative RPA analysis. Expression constructs are indicated above the
respective autoradiographs, and hours of chase are noted below the respective lanes. RNase
protection bands corresponding to hβ-globin mRNA and to the constitutively expressed
mGAPDH mRNA (loading control) are indicated. The intensities of the hβ-globin mRNA
bands were quantified and normalized relative to GAPDH mRNA band.
(B) Decay rates of ββββ-globin mRNAs. The data generated from the RPA studies (A,
above) are recorded in the graph. To calculate the β-globin mRNA half-lives, each data time
point was expressed as a ratio of hβ-globin:mGAPDH mRNA and normalized to the average
value of all time points from a single transfection. The ratios were then re-normalized to the
average initial time point from all transfections (time 0 = 1). Each point represents the mean
+/- Standard Deviation from four independent experiments. Linear regression analysis was
performed by standard techniques. The half-lives (t1/2) of the mRNAs are indicated.
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FIGURE 2. Shortening exon 1 stabilizes the ββββ39 mRNA.
(A) Physical maps of the hββββ-globin constructs. The closed rectangles, open
rectangles, and lines depict UTRs, exons and introns, respectively. The vertical line represents
the position of the nonsense mutation. The name of each construct is indicated on the right.
(B) Comparison of ββββWT and ββββ39 mRNA expression with expression of the
corresponding set of DelA mRNAs. MEL cells were co-transfected with equimolar amounts
of the βWT and β39 gene with (DelA) and without (Non-del) the exon 1 deletion. Primer
extension analysis is as in Methods. The identity of each end-labelled cDNA product is
indicated on the right of the gel and the level of each DelA mRNA normalized to the level of
its corresponding intact mRNA is represented at the bottom of the corresponding lane (mean
of three independent experiments). The standard deviations (SD) are indicated.
(C) Representative primer extension analysis of the ββββWT, ββββ39, ββββWTDelA, and
ββββ39DelA mRNA expression in transfected MEL cells normalized to endogenous mαααα-
globin mRNA. hβ-globin exon 3 and mα-globin specific end-labelled oligonucleotides
(described in Materials and Methods and Table I) were hybridized with RNA, and extended
with reverse transcriptase (see Methods). Human reticulocyte RNA (hRet) and untransfected
differentiated MEL mRNA (t-) were also reverse transcribed to mark the positions of hβ- and
mα-globin cDNAs, respectively (indicated on the right). The mean PhosphorImager
quantification of the hβ-globin mRNA (hβ) normalized to the level of endogenous mα-globin
mRNA (mα) and calculated as a percentage of the corresponding nonsense-free transcript
(from four independent experiments) is shown at the bottom of the respective lanes. The
standard deviations (SD) are indicated.
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FIGURE 3. Decay rate of ββββ39DelA mRNA in tTA/MEL cells. The tet-regulated promoter-
driven β39DelA construct was transiently transfected into tTA/MEL cells and the decay of the
pulsed mRNA was followed over time (hours, bottom) by RPA analysis.
(A) Representative RPA analysis.
(B) Compilation of data from three studies (means and standard deviations). The
half-life (t1/2) of the β39DelA mRNA was determined by regression analysis.
FIGURE 4. The impact of NMD on the various ββββ-globin mRNAs is cell-type and
promoter independent. HeLa cells were transiently co-transfected with a tet-regulated β-
globin (specified above each respective lane) and normal hα-globin gene. After 24 hours of
transcription the RNA from transfected and untransfected (t-) HeLa cells was isolated and
analysed by RPA using specific probes for hβ- and hα-globin mRNAs (see Methods).
Increasing amounts of RNA from HeLa cells transfected with normal hβ- and hα-globin
genes (indicated above the lanes by a triangle) were also analysed to demonstrate that the
experimental RPA was carried out in probe excess and was in the linear range of detection.
The level of mRNA from each β-globin allele was normalized to the level of hα-globin in
order to control for variations in the cell transfection efficiency and RNA recovery.
Normalized values were then calculated as the percentage of βWT-globin mRNA; the values
below each lane represent three independent experiments. Mean values and standard
deviations (SD) are shown.
FIGURE 5. The structures of ββββ39DelA, ββββ15 and ββββ39 mRNAs indicate that the
corresponding transcripts are normally spliced.
(A) Structural analysis of full-length hββββ-globin mRNAs stably expressed in MEL
cells. The identity of each gene is indicted above the respective lane. RNA from untransfected
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(t-) cells was used as the negative control for RT-PCR reactions. Plasmid DNA carrying the
human βWT gene constitutes the positive PCR control. Molecular weight marker (M) is the
100 bp DNA ladder (Invitrogen). RT-PCR products were resolved on an ethidium bromide
stained agarose gel. Primers (Table I) for RT and PCR reactions are represented in the
diagram below the gel. A single full-length product of 625 bp was amplified from all non-
deletional transcripts and a 556 bp fragment was amplified from cDNAs carrying deletion
between codons 3 and 27. The 1605 bp fragment was amplified from plasmid DNA (pDNA)
containing the human βWT-globin gene.
(B) Structural analysis of mRNA transiently expressed in HeLa cells. The top gel
picture shows the analysis of amplification between exon 1 and exon 2; a single 351 bp
product was amplified from all non-deletional transcripts and a 282 bp fragment was
amplified from cDNAs carrying delA. The 482 bp fragment was amplified from plasmid
DNA containing the human βWT-globin gene. The lower gel shows the analysis between exon
2 and exon 3; a single 359 bp product is observed for each construct. The 1209 bp fragment
was amplified from plasmid DNA containing the human βWT-globin gene. Plasmid DNA
(pDNA) and water comprise the positive and negative RT-PCR controls. M represents the 100
bp DNA ladder marker. Primers localization (Table I) for RT and PCR reactions are
represented on the bottom of each ethidium bromide stained agarose gel.
FIGURE 6. hββββ-globin mRNA and its nonsense derivatives are localized to polysome
peaks consistent with the sizes of their predicted ORFs.
(A) Representative sucrose gradient profile. The absorbance profile (OD254) of the
gradient is shown. The top of the gradient is to the left; the positions of absorbance peaks
corresponding to pre-ribosomal RNPs, 40S, 60S, and 80S, and polysomes (2-, 3-, 4-, 5-, 6-,
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and 7-somes) are indicated. The 15 fractions collected for subsequent analysis are identified
below the gradient profile.
(B) Representative RPA to detect the globin RNAs across the polysome gradient.
MEL cells were transiently transfected with the β-globin genes. Each gradient fraction was
assessed for human β-globin and mouse GAPDH mRNAs by RPA with the corresponding
32P-labeled riboprobes; hβ-globin and mGAPDH protected mRNAs fragments are shown.
(C) Polysome distribution of human ββββWT-globin, ββββ15, ββββ39, ββββWTDelA or ββββ39DelA
mRNAs. The contents of hβ-globin mRNAs in each fraction (see panel B) were quantified by
PhosphorImager analysis (data concerning βWTDelA and β39DelA mRNAs not shown in
panel B). The amount of each mRNA species in each fraction is depicted as a percentage
(ordinate) of the total for the corresponding species across the gradient.
(D) The stability of the ββββ15 or ββββ39DelA mRNAs is unaffected by elimination of
potential re-initiating AUGs. The three potential reinitiating AUGs at codons 55, 63/64, and
73/74, were converted to coding triplets in each of the index mRNAs. Shown is a
representative RPA of RNA isolated from HeLa cells untransfected (t-) or transiently co-
transfected with βWT, β15, β39, β39DelA, βWT-CD55-63/64-73/74, β15-CD55-63/64-73/74,
β39-CD55-63/64-73/74 or β39DelA-CD55-63/64-73/74 human globin genes along with an
equal quantity of the hα-globin gene. The positions of the hβ- and α-globin protected
fragments are indicated to the right of the autoradiograph. Levels of hβ-globin mRNA were
quantified relative to the hα-globin mRNA, and these values are shown beneath each
respective lane [average and standard deviations (SD)] normalized to the expression level of
the βWT gene. All assays were carried out in riboprobe excess. For each case, three
independent experiments were performed.
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FIGURE 7. NMD-resistance of human ββββ-globin mRNAs carrying a 5’-proximal
nonsense mutation reflects the proximity of the nonsense codon to the initiating AUG.
(A) Analysis of mRNA expression in terminally differentiated MEL cells. MEL
cells were stably transfected with the hβ-globin construct specified above each lane. After
erythroid induction, the normalized steady-state levels of β-globin mRNA were determined
from either transfected or untransfected (t-) MEL cell pools by RPA using specific probes for
hβ- and mα-globin (see Methods). The protected bands corresponding to the hβ- and mα-
globin mRNA are indicated. The level of mRNA from each β-globin gene was normalized to
the level of endogenous mα-globin in order to control for RNA recovery and erythroid
induction. Normalized values were then calculated as the percentage of βWT (left panel), or of
βWTseqhet (central panel) globin mRNAs. The right panel contains an analysis of 10 and 15
µg of RNA from MEL cells transfected with normal β-globin gene (indicated by a wedge) to
demonstrate that the RPA was in linear range. The values at the bottom of each lane represent
a mean of four independent experiments, and are plotted below the autoradiograph with
standard deviations.
(B) Analysis of mRNA expression in HeLa cells. HeLa cells were co-transfected
with the β-globin constructs specified above each lane and the hα-globin gene to normalize
the expression levels for transfection efficiency. The β-globin and α-globin genes are both
driven by the tetracycline-regulated promoter. Twenty-four hours after transfection, steady-
state total RNA from either transfected or untransfected (t-) HeLa cells was isolated, analysed
by RPA using specific probes for hβ- and α-globin mRNAs, and quantified as indicated
above. The percentage mRNA values (indicated on the bottom) were plotted for each
construct, and standard deviations from three independent experiments are shown.
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Table I. DNA oligonucleotides used in the current study
Primer* Sequence (5’�3’) Genomic position Cloning
L5’β-S TAAGCCAGTGCCAGAAGAG β 5’ flanking
DelA-AS CAACCTGCCCAGGGCCAGGTCCACCATGGTG β Exon 1
DelA-S CACCATGGTGCACCTGGCCCTGGGCAGGTTG β Exon 1
R5’β-AS TCCCATTCTAAACTGTACCC β Intron 2
Seqhet-S GACCATGGTGCACCTGCTGTCCTTCCCCACCACC(a) Compound β/α
Seqhet-AS TACCAACCTGCCCAGGGCCTTAACCTGGGCAGAGCC(a) Compound β/α
3’exon1-S GCCCTGGGCAGGTTGGTA β Exon 1/Intron 1
3’exon2-AS TCAGGATCCACGTGCAGC β Exon 2
ATG-S GACACCATGGTGACAATG β 5’ UTR/Exon 1
62-AS TTGCCATGCTACTTCACCTTAGG(b) β Exon 2
62-S CCTAAGGTGAAGTAGTAGCATGGCAA(b) β Exon 2
15non39-AS CTTGCCTCACAGGGCAG(b) β Exon 1
15non39-S CTGCCCTGTGAGGCAAG(b) β Exon 1
55-AS GGTTGCCTATAACAGCATC(b) β Exon 2
55-S GATGCTGTTATAGGCAACC(b) β Exon 2
73/74-AS GCCAGGCCGTCACTAAAG(b) β Exon 2
73/74-S CTTTAGTGACGGCCTGGC(b) β Exon 2
63/64-S GGTGAAGGCTCACGGCAAGAAAGTG(b) β Exon 2
63/64-AS CACTTTCTTGCCGTGAGCCTTCACC(b) β Exon 2
LinkClaI -S CCATCGATACATTTGCTTCTGACACAACTG(c) β 5’UTR
LinkNotI-AS ATAGTTTAGCGGCCGCAGGGATGAATAAGGCATAGGC(c) β 3’ flanking
pTetXhoI1/15-S CCCCCTCGAGTTTACCACT(c) pTet-Splice
pTetClaI351/370-AS CCATCGATACGGTTCACTAAACGAGCTC(c) ptet-Splice
WT15:39-S GCCGTTACTGCCCTGAAGCTTGGGCCCTGGGGCAAGGTGAAC(d) β Exon 1
WT15:39-AS GTTCACCTTGCCCCAGGGCCCAAGCTTCAGGGCAGTAACGGC(d) β Exon 1
15:39-S GCCGTTACTGCCCTGAAGCTTGGGCCCTGAGGCAAGGTGAAC(d) β15 Exon 1
15:39-AS GTTCACCTTGCCTCAGGGCCCAAGCTTCAGGGCAGTAACGGC(d) β15 Exon 1
HindIIIAmpr-S TTTAAGCTTTACATCGAACTGGATCTCAA(c) Ampr
ApaIAmpr-AS AAAGGGCCCACGTTCTTCGGGGCGAAAAC(c) Ampr
GA-S GATCTCAACAGCGGAAAGATCCTTGAGAG(b) Ampr Spacer
GA-AS CTCTCAAGGATCTTTCCGCTGTTGAGATC(b) Ampr Spacer
Primer extension assay
Hβexon3-AS CACTTTCTGATAGGCAGCCTGC β Exon 3
Hβ3’UTR-AS GAAAGCGAGCTTAGTGATAC β 3’UTR
Mα-AS CAGCCTTGATGTTGCT Mouse α Exon 1
RT-PCR assay
Hβ3’RNA-AS GCAATGAAAATAAATGTTTTTTATTA β 3’UTR
Hβ5’RNA-S ACATTTGCTTCTGACACAAC β 5’UTR
Hβexon2-S TGATGGCCTGGCTCACCTG β Exon 2
Hβ3’exon2-AS GATCCACGTGCAGCTTGTCA β Exon 2 *Primer designations reflect the corresponding utilization; S means sense and AS means antissense. (a)The underlined sequences anneal to the hα-globin gene; (b)the underlined sequences correspond to the introduced mutation; (c)the underlined sequences correspond to the restriction site indicated in the primer designation; (d)the underlined sequences correspond to the Hind III-Apa I restriction sites.
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Paula Faustino, Joao Lavinha, Stephen A. Liebhaber and Luisa RomaoAngela Inacio, Ana Luisa Silva, Joana Pinto, Xinjun Ji, Ana Morgado, Fatima Almeida,
nonsense-mediated mRNA decayNonsense mutations in close proximity to the initiation codon fail to trigger full
published online May 25, 2004J. Biol. Chem.
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