The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells

Article (PDF Available)inEuropean Journal of HumanGenetics 15(11):1156-62 · December 2007with60 Reads
DOI: 10.1038/sj.ejhg.5201889 · Source: PubMed
Abstract
Nonsense-mediated mRNA decay (NMD) is a mechanism, which selectively degrades transcripts carrying premature termination codons (PTCs) and a variety of physiologic transcripts containing NMD-inducing features. In a recent study, we have found variable NMD efficiency among nasal epithelial cells obtained from cystic fibrosis (CF) patients. This variability was found for CF transmembrane conductance regulator (CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Here, we aimed to investigate the possibility that variability in NMD efficiency is a more generalized phenomenon and is not restricted to nasal epithelial cells. To investigate this possibility, we analyzed the NMD efficiency of both a CFTR constructs carrying the W1282X PTC and beta-globin constructs carrying the NS39 PTC, in HeLa and MCF7 cells. Variability in NMD efficiency was found for both constructs between the cells, such that in HeLa cells the NMD was highly efficient and in MCF7 the efficiency was significantly lower. Moreover, similar differences in the efficiency of NMD were found for five endogenous NMD physiologic transcripts. Altogether, our results demonstrate existence of cells in which NMD of all transcripts is efficient, whereas others in which the NMD is less efficient, suggesting that the efficiency of NMD is an inherent character of cells. Our results also suggest that variability in the efficiency of NMD is a general phenomenon and is not restricted to nasal epithelial cells. As NMD affects the level of many transcripts, variability in the NMD efficiency might play a role as a genetic modifier of different cellular functions.
4 Figures
ARTICLE
The efficiency of nonsense-mediated mRNA decay
is an inherent character and varies among
different cells
Liat Linde
1
, Stephanie Boelz
2,3
, Gabriele Neu-Yilik
2,3
, Andreas E Kulozik
2,3
and
Batsheva Kerem*
,1
1
Department of Genetics, Life Sciences Institute, The Hebrew University, Jerusalem, Israel;
2
Molecular Medicine
Partnership Unit, University of Heidelberg and European Molecular Biology Laboratory, Heidelberg, Germany;
3
Department for Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg,
Germany
Nonsense-mediated mRNA decay (NMD) is a mechanism, which selectively degrades transcripts carrying
premature termination codons (PTCs) and a variety of physiologic transcripts containing NMD-inducing
features. In a recent study, we have found variable NMD efficiency among nasal epithelial cells obtained
from cystic fibrosis (CF) patients. This variability was found for CF transmembrane conductance regulator
(CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Here, we
aimed to investigate the possibility that variability in NMD efficiency is a more generalized phenomenon
and is not restricted to nasal epithelial cells. To investigate this possibility, we analyzed the NMD efficiency
of both a CFTR constructs carrying the W1282X PTC and b-globin constructs carrying the NS39 PTC, in
HeLa and MCF7 cells. Variability in NMD efficiency was found for both constructs between the cells, such
that in HeLa cells the NMD was highly efficient and in MCF7 the efficiency was significantly lower.
Moreover, similar differences in the efficiency of NMD were found for five endogenous NMD physiologic
transcripts. Altogether, our results demonstrate existence of cells in which NMD of all transcripts is
efficient, whereas others in which the NMD is less efficient, suggesting that the efficiency of NMD is an
inherent character of cells. Our results also suggest that variability in the efficiency of NMD is a general
phenomenon and is not restricted to nasal epithelial cells. As NMD affects the level of many transcripts,
variability in the NMD efficiency might play a role as a genetic modifier of different cellular functions.
European Journal of Human Genetics (2007) 15, 11561162; doi:10.1038/sj.ejhg.5201889; published online 11 July 2007
Keywords: nonsense-mediated mRNA decay; premature termination codons; CFTR; b-globin
Introduction
Nonsense-mediated mRNA decay (NMD) is a translation
dependent post-transcriptional process in eukaryotic cells.
This mechanism selectively degrades transcripts carrying
premature translation-termination codons (PTCs), which
are generally located 45055 nucleotides upstream from
downstream exon exon junction. The purpose is to
prevent the synthesis of non-functional or potentially
deleterious proteins.
1
Recently, it was found that NMD
also degrades a variety of physiologic transcripts, which
include transcripts with upstream open reading frame
(uORF), transcripts containing introns in the 3
0
untrans-
lated region (UTR) and transcripts derived from alternative
splicing.
2–6
Received 3 April 2007; revised 8 June 2007; accepted 8 June 2007;
published online 11 July 2007
*Correspondence: Professor B Kerem, Department of Genetics, The Life
Sciences Institute, The Hebrew University, Jerusalem, 91904 Israel.
Tel: þ 49 972 2 6585689; Fax: þ 49 972 2 6584810;
E-mail: kerem@cc.huji.ac.il
European Journal of Human Genetics (2007) 15, 11561162
&
2007 Nature Publishing Group All rights reserved 1018-4813/07
$30.00
www.nature.com/ejhg
In a recent study, we have shown that NMD efficiency
considerably varies among three cell lines derived from
nasal epithelium of cystic fibrosis (CF) patients.
7
We found
variability in NMD efficiency for CF transmembrane
conductance regulator (CFTR) transcripts carrying a dis-
ease-causing PTC, the W1282X mutation. Variability was
also found for five physiologic NMD substrates. These
included an alternatively spliced PTC-bearing transcript,
RPL3; transcripts with introns in their 3
0
UTR, SC35 1.6 kb
and SC35 1.7 kb (two splicing isoforms); a transcript with a
uORF, ASNS; and another bona fide NMD substrate with an
unknown NMD-inducing feature, CARS. Our results de-
monstrated the existence of nasal epithelial cells in which
NMD of all these analyzed transcripts was efficient and
others in which the NMD was less efficient. Furthermore,
downregulation of NMD increased the level of CFTR
nonsense transcripts and led to enhanced CFTR chloride-
channel activity in response to gentamicin.
Here we have investigated the possibility that variability
in NMD efficiency is a more generalized phenomenon, and
is not restricted to nasal epithelial cells. We demonstrate
that the same PTC can elicit NMD with variable efficiencies
when comparing two different epithelial cell lines, HeLa
and MCF7. HeLa cells are derived from cervical carcinoma,
whereas MCF7 are derived from breast adenocarcinoma.
Variability in NMD efficiency was found for two different
transcripts carrying disease-causing PTCs: CFTR constructs
carrying the W1282X nonsense mutation and b-globin
transcripts carrying the NS39 nonsense mutation. Varia-
bility was also found for five endogenous physiologic NMD
substrates: RPL3; SC35 1.6 kb; SC35 1.7 kb; ASNS; and
CARS. Altogether, our results suggest that the efficiency of
NMD is an inherent character of cells. Furthermore,
variability in the efficiency of NMD is a general phenom-
enon and is not restricted to nasal epithelial cells.
Materials and methods
Cell cultures
The human cell lines investigated in the study were of
epithelial origin: HeLa, which was derived from cervical
carcinoma and MCF7 from breast adenocarcinoma. The
cell lines were grown in DMEM supplemented with 10%
fetal calf serum.
Plasmid construction
Complete CFTR cDNA was inserted into pCMVbeta vector
(ATCC), in which sequences of the beta-gal were deleted.
We connected three PCR fragments generated from DNA
extracted from a healthy individual carrying wild-type
(WT) CFTR alleles. The PCR fragments contained exons 20
and part of its downstream intronic sequence (264 bp),
exon 21 and part of its flanking introns (240 bp at intron 20
and 289 bp at intron 21) and exon 22 and part of its
upstream intronic sequence (260 bp). The CFTR mutant
construct was generated using the same cloning approach
on DNA extracted from an individual homozygous for the
W1282X mutation. Both constructs were sequenced and
asides from the W1282X mutation no variation was
identified in all exons and 199 bp of each intron.
b-Globin WT and mutant (NS39) constructs were
described previously.
8
An elongated human b-globin gene
(wt300 þ e3) served as a control for transfection efficiency
in all experiments, as described previously.
9
Transfections
Transfections were performed by using an electroporation
protocol (described in Agami and Bernards
10
)orby
calcium-phosphate precipitation with standard methods.
Similar transcript levels were obtained by using either of
these transfection methods. The CFTR transfections were
performed with either WT or mutant construct together
with a plasmid encoding GFP (pEGFP, Clontech) for
normalization. The b-globin transfections were performed
with either WT or mutant construct, together with
wt300 þ e3 construct for normalization. Transfections were
performed using 1 or 2 mg of construct. The results showed
no difference in transcript level with the different amounts
of construct, excluding the possibility that transfection
efficiency led to variability in transcript levels among the
cell lines. Experiments were repeated at least three times.
RNA analysis
Total RNA was extracted using the RNeasy extraction kit
(Qiagen) or Tri-Reagent-LS (MRC). RNA-less and reverse-
transcriptase-less reactions were used as controls. For
quantification of CFTR W1282X transcript levels in HeLa
and MCF7 cells, which had been transfected with the CFTR
constructs, we performed real-time PCR in the LightCycler
(software version 3.5), using a FastStart DNA Master SYBR
Green I kit (Roche Diagnostics). A GFP plasmid was used as
a reference for transfection efficiency in each cell line. The
level of mRNA transcribed from the WT or W1282X CFTR
construct was normalized to the mRNA level of GFP.
Subsequently, the ratio between the normalized mRNA
level transcribed from the mutant and the WT constructs
following cycloheximide (CHX) treatment was calculated
and compared with this ratio in untreated cells. Sequence
of the primer pairs are as follows: for pCFTR: forward (exon
20) ATGGTGTGTCTTGGGA, reverse (exon 22) ACAAGGA
CAAAGTCAAGC; for pEGFP: forward GCAACTACAA
GACCCGC, reverse GTCGGCCATGATATAGACG. For each
pair of primers, a standard curve was performed and
annealing temperature was optimized in order to exclude
PCR artifacts. Moreover, we included in the analysis only
experiments in which the standard curves were as
expected, to ensure accurate quantification.
For quantification of b-globin NS39 transcript levels in
HeLa and MCF7 cells, transfected with the b-globin
constructs, we performed Northern Blots with 1.5 5 mgof
Variability in NMD efficiency
L Linde et al
1157
European Journal of Human Genetics
total cytoplasmic RNA according to standard protocols.
Northern blots of total cytoplasmic RNA of transfected cells
were probed with a b-globin-specific radiolabeled antisense
cRNA. The control b-globin plasmid (wt300 þ e3) was used
as a reference for transfection efficiency in each cell
line. The level of mRNA transcribed from the WT or NS39
b-globin construct was normalized to the mRNA level of
the b-globin wt300 þ e3 construct. Subsequently, the ratio
between the normalized mRNA level transcribed from the
mutant and the WT constructs following NMD inhibition
was calculated and compared with this ratio in untreated
cells.
The quantification of physiologic NMD substrates: RPL3;
SC35 1.6 kb; SC35 1.7 kb; ASNS; and CARS, was performed
as was previously described
7
and as mentioned in the
legend of Figure 3.
NMD inhibition
We indirectly inhibited NMD by treating the cells with
200 mg/ml CHX (Sigma) for 5 h. We directly inhibited NMD
by using short-interfering RNA (siRNA) directed against
UPF1. siRNA oligos for silencing UPF1 expression (Dhar-
macon) were those described by Mendell et al.
11
siRNA
oligo for luciferase (Dharmacon) was described by Gehring
et al.
12
Transfection conditions were described previously.
12
Experiments were repeated at least three times.
Western blots
Total proteins (2040 mg) were extracted from the different
cell lines and were subjected to SDS-PAGE as was described
previously.
12
These proteins were probed with antibodies
against UPF1 and tubulin.
Results
In order to investigate whether variability in NMD
efficiency is a general phenomenon or restricted to nasal
epithelial cells, we analyzed NMD efficiency in differently
derived epithelial cells, HeLa and MCF7. We analyzed the
efficiency of NMD for two different transcripts carrying
disease-causing PTCs: CFTR constructs carrying the CF-
causing PTC W1282X and the b-globin constructs carrying
the b-thalassemia-causing PTC NS39. We also studied five
endogenous physiologic NMD substrates: RPL3; SC35
1.6 kb; SC35 1.7 kb; ASNS; and CARS.
In our previous study, we analyzed the efficiency of NMD
for endogenous CFTR transcripts carrying the W1282X
nonsense mutation, in nasal epithelial cells derived from
CF patients. As HeLa and MCF7 cells do not express CFTR
gene, we transfected them with CFTR constructs carrying
the W1282X PTC, which is located 455 nucleotides
upstream to the final exon exon junction, or the normal
sequence (WT) (Figure 1a). We examined the levels of
transfected W1282X transcripts following treatment with
CHX, a general inhibitor of translation elongation and as
NMD is a translation-dependent mechanism, CHX treat-
ment leads indirectly to inhibition of NMD.
13
A GFP
plasmid was used as a reference for transfection efficiency
in each cell line. The levels of mRNA transcribed from the
WT or W1282X CFTR construct was normalized to the
mRNA level of GFP. Subsequently, the ratio between the
normalized mRNA levels transcribed from the mutant and
the WT constructs following CHX treatment was calculated
and compared with this ratio in untreated cells. The
analysis showed that the fold increase differed between
the cell lines such that in MCF7, a modest increase in the
level was observed (1.370.03-fold), whereas in HeLa cells,
the increase was B2.5 higher (3.270.27-fold) (Figure 1b).
These results suggest that in MCF7 cells, the NMD of CFTR
transcripts carrying the W1282X PTC is inefficient, as the
transcript level showed only a marginal increase following
the NMD indirect inhibition. The results further indicate
that the NMD efficiency of CFTR might vary between
different epithelial cell lines.
We then analyzed the efficiency of NMD of b-globin
constructs carrying the NS39 PTC or the normal sequence
(WT) (Figure 2a), in HeLa and MCF7 cells. The levels of the
a
b
Figure 1 Effect of CHX treatment on the level of CFTR mRNA
transcribed from constructs carrying the W1282X PTC. (a) A scheme of
the WT (upper panel) and W1282X (lower panel) constructs, which
contained all CFTR exons (marked in the boxes by numbers) and part
of the intronic sequences between exons 20 22. The CMV promoter
is marked by a thick horizontal arrow. (b) Real-time PCR analysis of
CFTR transcripts following CHX treatment. The level of mRNA
transcribed from CFTR constructs carrying either the normal sequence
or the W1282X PTC was normalized to the mRNA level of GFP. The
ratio between these normalized levels following CHX treatment was
calculated and compared with the ratio in untreated cells. The fold
increase in the level of CFTR W1282X transcripts is shown as
mean7SEM.
Variability in NMD efficiency
L Linde et al
1158
European Journal of Human Genetics
NS39 transcripts were analyzed following transfection of
the constructs and NMD inhibition. As a reference for
transfection efficiency, we co-transfected the b-globin
constructs with another b-globin construct, which in-
cluded the normal sequence and a 300 bp exon
(wt300 þ e3, control). The level of mRNA transcribed from
b-globin constructs, carrying either the normal sequence or
the NS39 PTC, was normalized to the mRNA level of the
control construct. Subsequently, the ratio between the
normalized mRNA level transcribed from the mutant and
the WT constructs following NMD inhibition was calcu-
lated and compared with this ratio in untreated cells.
Following CHX treatment, no increase in the level of NS39
transcripts was observed in MCF7 cells, whereas in HeLa
cells a significant increase (4.470.20-fold) was observed
(Figure 2b), similar to the variable effect of CHX on the
level of CFTR W1282X transcripts in these cell lines.
Subsequently, we inhibited NMD in these cells by siRNA
duplexes against UPF1, a key factor that operates in the
NMD machinery. As a control, we analyzed the effect of
luciferase siRNA duplexes. Western blot analysis showed a
sequence-specific and a very efficient downregulation of
UPF1 expression in both HeLa and MCF7 cells (Figure 2c).
The mRNA quantification results showed a similar effect to
a
b
c
d
Figure 2 Effect of NMD inhibition on the level of b-globin mRNA transcribed from constructs carrying the NS39 PTC. (a) A scheme of the WT
(upper panel) and NS39 (lower panel) constructs, which contained all b -globin exons (marked in the boxes by numbers) and part of the intronic
sequences. The CMV promoter is marked by a thick horizontal arrow. (b) Northern blot analysis of b-globin transcripts following CHX treatment. The
level of mRNA transcribed from b-globin constructs carrying either the normal sequence or the NS39 PTC was normalized to the mRNA level of the
control construct. The ratio between these normalized levels following treatment was calculated and compared with the ratio in untreated cells. (c)
Western blot analysis in HeLa (left panel) and MCF7 (right panel) following sequence-specific downregulation of UPF1. (d) Northern blot analysis of b-
globin transcripts following UPF1 downregulation. mRNA quantification was performed as described in (b). The fold increase in the level of b-globin
NS39 is shown as mean7SEM.
Variability in NMD efficiency
L Linde et al
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European Journal of Human Genetics
that observed following CHX treatment. Although in HeLa
cells, a significant increase was observed (2.970.27-fold),
in MCF7 cells, no increase was found (the difference
between 43 and 31% or 0.7-fold increase following UPF1
downregulation is within the experimental variability)
(Figure 2d). It is important to note that most of the
knowledge regarding NMD mechanism comes from studies
of the NS39 b-globin construct in HeLa cells. The increase
in the level of NS39 b-globin transcripts found in our study
in HeLa cells (B3 to 4.5-fold) following UPF1 down-
regulation is similar to that found in previous studies.
8,14,15
Together, these results show that the NMD efficiency of
b-globin transcripts might vary between different cell lines,
similar to the variability found for the CFTR in these cells.
Specifically, both the CFTR and b-globin transcripts were
more efficiently degraded in HeLa than in MCF7 cells.
We further extended our analysis in HeLa and MCF7 cells
and studied the efficiency of NMD for physiologic NMD
substrates following CHX treatment or downregulation of
UPF1. The NMD physiologic substrates included RPL3;
SC35 1.6 kb and SC35 1.7 kb; ASNS; and CARS. For all five
analyzed transcripts, the results show an increase in the
level following CHX treatment, which widely varied
between the cells (Figures 3a e and 4). In MCF7 cells, the
increase in the level of each analyzed transcripts was
significantly lower than that found in HeLa cells. Similar
results were found following UPF1 downregulation (Figures
3f j and 4 and Figure 4a with Figure 4b). It is interesting to
note that the consistent results, which were obtained by
NMD inhibition using CHX treatment or UPF1 siRNA in
HeLa and MCF7 cells, emphasize that variable uptake or
metabolism of CHX between these two cell lines is not
likely the basis for variability in NMD efficiency. The 0.7-
fold increase following UPF1 downregulation in MCF7 cells
for RPL3 and SC35 1.7 kb transcripts is within the
experimental variability, and indicates no increase in the
level of these transcripts. It is important to note that the
fold increase following CHX treatment was significantly
higher than that observed following UPF1 downregulation
(compare Figure 3a e with Figure 3f j and Figure 4a with
Figure 4b). We conjecture that this effect probably resulted
from the incomplete NMD inhibition following UPF1
downregulation. Altogether, the results from CHX treat-
ment and from UPF1 downregulation show variability in
af
b
c
d
ej
i
h
g
Figure 3 Effect of NMD inhibition on the transcript level of
physiologic NMD substrates. The level of each analyzed transcript was
measured by real-time PCR and normalized to that transcribed from
GAPDH and the ratio between these normalized levels following
treatment was calculated and compared with the ratio in untreated
cells. (a e) Effect of indirect NMD inhibition by CHX on the level of
RPL3 (a), SC35 1.7 kb (b), SC35 1.6 kb (c), ASNS (d), and CARS (e)
transcripts. (f–j) Effect of direct NMD inhibition by UPF1 down-
regulation on the level of RPL3 (f), SC35 1.7 kb (g), SC35 1.6 kb (h),
ASNS (i), and CARS (j) transcripts. The increase in the level is shown as
mean7SEM.
Variability in NMD efficiency
L Linde et al
1160
European Journal of Human Genetics
NMD efficiency between HeLa and MCF7 cells. The results
further show that the NMD in MCF7 is relatively less
efficient than in HeLa cells.
Discussion
Our results provide evidence that the efficiency of NMD is
an inherent character of cells and may vary among
different cells. Previous studies showed variability in
NMD efficiency only for transcripts carrying disease-
causing PTCs among cells derived from different tissues.
For example, a different increase was obtained in HeLa and
T cells stably transfected with TCR-b construct carrying a
PTC, following treatment with CHX.
16
Variability in NMD
efficiency was also found among chondrocyte, lympho-
blasts and bone cells of a Schmid metaphyseal chondro-
dysplasia patient carrying a PTC in the collagen X gene.
17
Recently, an additional study reported variability in NMD
efficiency for transcripts carrying a deletion in the ESCO2
gene, which generates a PTC, among different tissues from
two unrelated Roberts syndrome fetuses.
18
Variability in
NMD efficiency of CYH2 transcripts carrying a PTC among
different yeast strains was also reported.
19
The results
presented in the current study together with the results
from our recent work
7
show, for the CFTR and the b-globin
transcripts carrying a disease-causing PTC and for five
physiologic NMD substrates, that there are cells, such as
HeLa, CFP15a, and CFP22a, in which NMD of all
transcripts was efficient and other cells, such as MCF7
and CFP15b, in which the NMD was less efficient. It is
important to note that there are cells derived from the
same tissue between these two cell groups, suggesting that
the observed variable NMD efficiency between them does
not reflect tissue-specific variability. Moreover, these find-
ings strongly indicate that besides the position of the PTC
relative to the exonexon junction suggested previously in
other studies,
1
there are additional factors, which play a
role in regulating the efficiency of NMD. Hence, variability
in NMD efficiency probably results from interactions
between elements in transcripts, which are subject to
NMD and factors in the NMD machinery or regula-
tion. Single-nucleotide polymorphisms in genes encoding
the NMD factors and their regulators, in factors from the
exon junction complex, or in other translation termina-
tion factors can lead to variable efficiency of NMD among
cells. Different expression levels of one or more of these
factors might also contribute to the variability in NMD
efficiency.
It is important to note that variability in NMD efficiency
might have a role as a genetic modifier of different cellular
functions. Our previous study showed an important role
for NMD efficiency in the response to readthrough
treatment and demonstrated the effect of variable NMD
efficiency on the response. Following treatment with
50 200 mg/ml gentamicin, concentration-dependent chloride
efflux was detected in CFP15a cells, whereas in IB3-1 cells
no significant activation was observed. Only following
downregulation of UPF1 together with high gentamicin
concentrations (100 and 200 mg/ml), an impressive CFTR
activation was observed in IB3-1 cells, whereas in CFP15a
cells downregulation of UPF1 or UPF2 in the presence of
gentamicin at all concentrations, resulted in an improved
chloride-channel activity compared with gentamicin
alone. Variable efficiency of NMD can also affect disease
severity in the cases of truncated proteins either by gain-
of-function or dominant-negative effects. This may explain
the phenotypic variability among patients of Duchenne
muscular dystrophy (severe) and Becker muscular dystro-
phy (mild), carrying the same PTC in the dystrophin
gene.
20
Variable efficiency of NMD can also alter the
pattern of inheritance of mendelian diseases. A well-known
example is the b-thalassemia in which PTCs at the 5
0
of the
b-globin gene trigger NMD and thus lead to a recessive trait
of b-thalassemia,
21,22
whereas PTCs at the 3
0
does not
generally trigger NMD and thus lead to dominant form of
the disease.
23 25
To summarize, our study provide evidence for variable
NMD efficiency among cells from the same cell type, and
even from the same tissue, as a general phenomenon and
an inherent character of a given cell. Thus, NMD might
have a role as a genetic modifier and hence the outcome of
many studies in human genetic diseases has to be
considered in this light.
Acknowledgements
We thank T Danieli, M Goldberg, and YS Oren for useful advice on the
CFTR plasmid constructions. The work was partially supported by a
grant from Yissum to Batsheva Kerem and a DFG Grant to Andreas E
Kulozik.
a
b
Figure 4 Combined presentation of the effect of NMD inhibition
on the transcript level of physiologic NMD substrates. (a) Effect of CHX
treatment. (b) Effect of UPF1 downregulation. The increase in the level
is shown as mean.
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L Linde et al
1161
European Journal of Human Genetics
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Variability in NMD efficiency
L Linde et al
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European Journal of Human Genetics
    • Protein extraction, quantification, western blot analysis, and co-immunoprecipitation (Co-IP) assays were performed as described previously (GÖRL et al. 2001). To inhibit translation, cycloheximide (CHX, Sigma) was used to treat samples for 3h and the final concentration was 200 g/ml (LINDE et al. 2007).
    [Show abstract] [Hide abstract] ABSTRACT: Nonsense-mediated RNA decay (NMD) is a crucial post-transcriptional regulatory mechanism that recognizes and eliminates aberrantly processed transcripts, and mediates the expression of normal gene transcripts. In this study, we report that in the filamentous fungus Neurospora crassa, the NMD factors play a conserved role in regulating the surveillance of NMD targets including PTC-containing transcripts and normal transcripts. The circadian rhythms in all of the upf1-3 knockout strains, which are principle NMD factors, were aberrant. The upf1 knockout strain displays a shortened circadian period, which can be restored by constantly expressing exogenous UPF1. UPF1 regulates the circadian clock by modulating the splicing of the core clock gene frequency (frq) through spliceosome and spliceosome-related Arginine/serine-rich splicing factors, which partly account for the short periods in the upf1 knockout strain. We also demonstrated that the clock genes including WC-1, WC-2 and FRQ are involved in controlling the diurnal growth rhythm, and UPF1 may affect the growth rhythm by mediating the FRQ protein levels in the daytime. These findings suggest that the NMD factors play important roles in regulating the circadian clock and diurnal growth rhythms in Neurospora.
    Article · Jun 2017
    • Class V: mutations that reduce CFTR protein levels, often by affecting splicing and generating both aberrant mRNA transcripts and a reduced amount of normal mRNA transcripts[4,5]. With this type of mutation it is sometimes challenging to establish their effect on the overall CFTR function and therefore their involvement in the disease, which has a direct and significant impact on genetic counseling[7]. Class VI: mutations that include those that decrease the retention and stability of CFTR at the cell surface.
    [Show abstract] [Hide abstract] ABSTRACT: Cystic Fibrosis has attracted much attention in recent years due to significant advances in the pharmacological targeting of the basic defect underlying this recessive disorder: the deficient functional expression of mutant CFTR chloride channels at the apical membrane of epithelial cells. However, increasing evidence points to the reduced efficacy of single treatments, thus reinforcing the need to combine several therapeutic strategies to effectively target the multiple basic defect(s). Protein-repair therapies that use potentiators (activating membrane-located CFTR) or correctors (promoting the relocation of intracellular-retained trafficking mutants of CFTR) in frequent mutations such as F508del and G551D have been put forward and made their way to the clinic with moderate to good efficiency. However, alternative (or additional) approaches targeting the membrane stability of mutant proteins, or correcting the cellular phenotype through direct effect upon other ion channels (affecting the overall electrolyte transport or simply promoting alternative chloride transport) or targeting less frequent mutations (splicing variants, for example), have been proposed and tested in the field of CF. Here, we cover the different strategies that rely on novel findings concerning CFTR interactome and signalosome through which it might be possible to further influence the cellular trafficking and post-translational modification machinery (to increase rescued CFTR abundance and membrane stability). We also highlight the new data on strategies aiming at the regulation of sodium absorption or to increase chloride transport through alternative channels. The development and implementation of these complementary approaches will pave the way to combinatorial therapeutic strategies with increased benefit to CF patients. This article is protected by copyright. All rights reserved.
    Full-text · Article · Sep 2015
    • The concept of this approach has been proven to be effective in cystic fibrosis and in Duchenne muscular dystrophy (Linde & Kerem, 2008; Keeling et al, 2012 ). Specifically, nonsense suppression has been found to be more effective in patients with naturally less efficient NMD than in those with more efficient NMD (Linde et al, 2007a). NMD inhibitors such as 5-azacytidine may thus increase the abundance of mRNA substrates that would be targeted by compounds that are currently being developed for the induction of translational readthrough (Lee & Dougherty, 2012).
    [Show abstract] [Hide abstract] ABSTRACT: Nonsense-mediated RNA decay (NMD) is an RNA-based quality control mechanism that eliminates transcripts bearing premature translation termination codons (PTC). Approximately, one-third of all inherited disorders and some forms of cancer are caused by nonsense or frame shift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. 5-azacytidine is an analogue of the naturally occurring pyrimidine nucleoside cytidine, which is approved for the treatment of myelodysplastic syndrome and myeloid leukemia. Here, we reveal that 5-azacytidine inhibits NMD in a dose-dependent fashion specifically upregulating the expression of both PTC-containing mutant and cellular NMD targets. Moreover, this activity of 5-azacytidine depends on the induction of MYC expression, thus providing a link between the effect of this drug and one of the key cellular pathways that are known to affect NMD activity. Furthermore, the effective concentration of 5-azacytidine in cells corresponds to drug levels used in patients, qualifying 5-azacytidine as a candidate drug that could potentially be repurposed for the treatment of Mendelian and acquired genetic diseases that are caused by PTC mutations.
    Full-text · Article · Oct 2014
    • Altogether, these results highlight the importance of the NMD and UPR mechanisms to maintain homeostasis under normal conditions and in genetic diseases caused by PTCs. Variability in NMD efficiency is found in cell lines, tissues, and among individuals, suggesting that the efficiency of NMD is an EMBO Molecular Medicine Vol 6 | No 5 | 2014 ª 2014 The Authors EMBO Molecular Medicine UPR modulates readthrough treatment Yifat S Oren et al inherent feature of cells (Holbrook et al, 2004; Linde et al, 2007a) that can be modulated by genetic traits (sequence variations) (Tarpey et al, 2007), environmental conditions (hypoxia, amino acid starvation, etc.) (Wang et al, 2011b), or aberrant cellular regulation (tumorigenicity) (Wang et al, 2011a ). Our results showing a feedback loop between NMD and the UPR suggest that either the variability in UPR activation results from variability in NMD efficiency or vice versa.
    [Show abstract] [Hide abstract] ABSTRACT: One-third of monogenic inherited diseases result from premature termination codons (PTCs). Readthrough of in-frame PTCs enables synthesis of full-length functional proteins. However, extended variability in the response to readthrough treatment is found among patients, which correlates with the level of nonsense transcripts. Here, we aimed to reveal cellular pathways affecting this inter-patient variability. We show that activation of the unfolded protein response (UPR) governs the response to readthrough treatment by regulating the levels of transcripts carrying PTCs. Quantitative proteomic analyses showed substantial differences in UPR activation between patients carrying PTCs, correlating with their response. We further found a significant inverse correlation between the UPR and nonsense-mediated mRNA decay (NMD), suggesting a feedback loop between these homeostatic pathways. We uncovered and characterized the mechanism underlying this NMD-UPR feedback loop, which augments both UPR activation and NMD attenuation. Importantly, this feedback loop enhances the response to readthrough treatment, highlighting its clinical importance. Altogether, our study demonstrates the importance of the UPR and its regulatory network for genetic diseases caused by PTCs and for cell homeostasis under normal conditions.
    Full-text · Article · Apr 2014
    • fect the level of the mRNA, although the precise ''rules'' governing nonsensemediated mRNA decay (NMD) have not been completely defined. In different cell lines harboring the CFTR W1282X mutation, NMD efficiency was shown to correlate with response to the stop codon readthrough compound gentamicin, as measured by restored chlorine channel activity. [22] Down regulating NMD by reducing UPF1 or UPF2 levels increased chloride channel activity in response to gentamicin in cell culture, consistent with the notion that transcript levels may act as a disease modifier. The applicability of modifying NMD for DMD has yet to be investigated. The activity, safety, and pharmacokinetic findings from
    [Show abstract] [Hide abstract] ABSTRACT: Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. ClinicalTrials.gov NCT00264888.
    Full-text · Article · Dec 2013
    • Beside the common F508del variant, our CF patients carry missense and nonsense alterations in the CFTR gene. Indeed, different studies have shown that the effect of nonsense mutations on CFTR mRNA levels are very variable [16] and nonsense-mediated decay (NMD) is addressed as the major mechanism for rapid degradation of aberrant transcripts harboring premature termination codons. Interestingly, microRNAs have recently been defined as important regulators of the NMD efficiency [17].
    [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-β1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.
    Full-text · Article · Apr 2013
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