FEBS Letters 580 (2006) 4449–4456
NF1 mRNA biogenesis: Effect of the genomic milieu in splicingregulation of the NF1 exon 37 region
Marco Barallea, Natasa Skokoa, Anna Knezevicha, Laura De Contia, Dario Mottia,Madhuri Bhuvanagiria, Diana Baralleb, Emanuele Burattia, Francisco E. Barallea,*
a International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italyb Department of Medical Genetics, Addenbrooke’s Hospital, Box 134, Hills Road, Cambridge CB2 2QQ, UK
Received 18 May 2006; revised 3 July 2006; accepted 5 July 2006
Available online 14 July 2006
Edited by Takashi Gojobori
Abstract We have studied the splicing regulation of NF1 exons36 and 37. We show that they not only require an intact exonSplicing Enhancer (ESE) within exon 37, but also need the geno-mic region stretching from exons 31 to 38. Any nucleotidechange in two exon 37 third codon positions disrupts the ESE.The extent of exons 36 and 37 skipping due to a mutated ESEdepends on the genomic context. This is a unique example ofwhat may be a more general phenomena involved in the tuningof pre-mRNA processing and gene expression modulation inthe chromosomal setting.� 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.
Keywords: NF1; Splicing; Genomic context; Exonic splicingenhancers
1. Introduction
Over the recent years it has become well established that pre-
mRNA splicing is accomplished by the combinatorial recogni-
tion of multiple degenerate signals, resulting in a network of
RNA–protein interactions across an exon and/or intron [1–
3]. Moreover, recent evidence indicates that alternative splicing
and possibly splicing itself is not only regulated by the relative
abundance of antagonistic factors but by a more complex pro-
cess involving the transcription machinery [4]. However, the
fact that in many cases splicing systems are not easy to model
[5] indicates that the determinants of its regulation are poorly
understood. It is obvious that a greater knowledge of the inter-
play between all the cis and trans-acting elements that modu-
late splicing is needed.
The NF1 gene has been the subject of extensive studies since
its identification as the defective locus causing Neurofibroma-
tosis type 1. In particular, comprehensive sequencing has been
carried out for diagnostic and research purposes [6–9].
Premature termination codon (PTC) induced skipping of
NF1 exon 37, that preserves an open reading frame, has been
previously reported in the case of the nucleotide substitutions
c.6792C > G (p.Tyr2264X) and c.6792C > A (p.Tyr2264X)
[10] (Fig. 1A). The phenomenon by which PTCs cause exon
skipping during pre-mRNA splicing in the nucleus is known
*Corresponding author. Fax: +39 040 3757361.E-mail address: [email protected] (F.E. Baralle).
0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu
doi:10.1016/j.febslet.2006.07.018
as nonsense altered splicing (NAS) [11,12]. However, in many
of these cases, it was found that these mutations disrupted an
exonic splicing enhancer (ESE) critical for exon inclusion [13].
In fact, mutations from other patients in the same area, a
c.6790_6791TTins and a c.6789_6792TTACdel (Fig. 1A), both
of which create PTC’s a few base pairs further down stream do
not result in the skipping of this exon [14]. These observations
suggested that the genomic variations were not subject to NAS
but were affecting a exonic Splicing Enhancer (ESE). Indeed,
in a recent report the splicing enhancer activities of the wild-
type and mutant putative ESE were tested in an heterologous
system by measuring the rescue of splicing of the 34 nucleotide
exon 2 of the pSXN minigene reporter construct [15]. In this
system, 15 nucleotides of the NF1 exon 37 WT sequence
(nucleotides c.6783–c.6796) were able to rescue splicing of
the alternative exon approximately 30% more than the mu-
tated sequences. Obviously, this is a limited indication on an
artificial system and a more detail molecular analysis was
desirable.
In this study we show that even a synonymous change at po-
sition c.6792 produces exon skipping conclusively demonstrat-
ing that altered splicing is not due to nonsense mutations at
this position but rather due to interference with a regulatory
region present in the NF1 exon 37, necessary for its definition.
We extensively mapped this element and show that it is not
only required for the definition of exon 37, but also that it con-
tributes towards the identification of the upstream exon 36, in
that the aberrant splicing of exon 37 affects the correct recog-
nition of exon 36. Intriguingly, we show that both these exons
need an extremely wide genomic context to obtain the in vivo
splicing pattern indicating that their correct definition relies, in
addition to the intrinsic properties of the exons themselves, on
the wider genomic environment within which the exons are
located.
2. Materials and methods
2.1. Plasmid constructionThe hybrid minigene constructs were prepared using the PTB plas-
mid [16] by inserting wild-type and mutated DNA fragments amplifiedwith PTBEX37F and PTBEX37R primers. The same sense mutation(6792C > T) was created by site directed mutagenesis. Minigenes carry-ing NF1 genomic sequences1 from exons 36 to 38 and from 34 to 38were made by amplifying wild-type and mutated genomic DNA with
1 GenBank Accession Number for NF1 Sequence NM_000267.
blished by Elsevier B.V. All rights reserved.
Fig. 1. Nonsense or same sense mutations at position 6792 of the NF1 gene behave in an identical manner. (A) Previously described mutations (grey)in the exon 37 of patients suffering from NF1 that give rise to PTC (denoted by X) and their effect on the splicing of this exon. The sequence shownhere only represents part of exon 37. (B) Schematic representation of the PTB hybrid minigene carrying the NF1 exon 37 along with intronic flankingsequence. Black, shaded and white boxes represent alpha globin, fibronectin and NF1 exons respectively. Below are the RNA products generated bythe splicing assay for the wildtype, 6792C > G nonsense mutation and the 6792C > T same sense mutation hybrid minigenes. Two products are seenon the agarose gel electrophoresis, the 239 bp band represents the RNA transcript lacking NF1 exon 37 (�Ex37) and the 342 bp band represents thetranscript that includes NF1 exon 37 (+Ex37).
4450 M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456
the following forward primers: EX36BAMHI EX34BAMHI and thereverse primer EX38XHO. The amplicon was subsequently cloned inthe pcDNA3 vector (Invitrogen). PCR site directed mutagenesis wasperformed to create the nucleotide substitutions reported, makinguse of the internal cassette inside the minigene defined by the uniquerestriction enzymes sites EcoRI–AfeI.
The minigenes extending from NF1 exons 31–38 were constructedby cloning the genomic segment NF1 34–38 (amplified with EX34-BAMHI and EX38XHO) into SmaI site of PUC19 (Biolabs) vectorwhose BamHI site had being destroyed. The vector was then openedwith BamH1 and SphI, into which the genomic fragment ampli-fied with the oligos EX31BAMH1/SPH1R and subsequently digestedwith BamH1 and SphI, was cloned. The genomic fragment thus con-taining exons 31–38 was then sub-cloned into pcDNA3 vector Bam-HI/XhoI. The nucleotide substitutions of interest in the ESE regionwere introduced by excising the DNA fragment flanked by therestriction enzymes EcoRI–AfeI substituting it with the identicalfragment, bar the nucleotide transition of interest. All constructswere designed to maintain an open reading frame in the wild-typecontext.
The correct sequence of all constructs was checked by a CEQ2000sequencer (Beckman Coulter, Fullerton CA) according to the manu-facturers instructions.
2.2. Primers
PTBEX37F TTCATATGTAGCTACCAAGATCA
PTBEX37R TTCATATGTAACTTATTCTCAAT
EX36BAMH1
ACGTGGATCCATGGATTTGCATTCCAATATAATC-
CAT
EX34BAMH1 ACGTGGATCCATGGAAGAGACCAAG-
CAAGTTTTGA
EX38XHO TGCACTCGAGTCACTTGTCATTGAATATA-
CGGAGA
EX31BAMH1 ACGTGGATCCATGAGTATTGAATTGA-
AACACCTTTG
Fig. 2. The importance of the genomic context on NF1 exon 37 definition. (A) In scale schematic representation of the genomic region encoding NF1exons 30–39. Agarose gel electrophoresis showing RT-PCR products obtained with a minigene. (B) Composed of exons 36–38. The upper bandcorresponds to the transcript that includes all three exons as depicted on the right. The lower band was a mixture of transcripts, where although all ofthem skipped exon 37 several showed an incorrect use of splice sites (depicted by the black box) in the graphical representation. (C) Composed ofexons 34–38. The wildtype construct results in a transcript that carries all five exons. The nonsense and same sense mutations result in a three bandprofile where the upper band represents correct assembly of the five exons, the intermediate band lacks exon 37, and the lower band lacks both exons36 and 37. (D) Agarose gel electrophoresis showing RT-PCR products obtained from RNA prepared from EBV lymphoblast derived from a normalindividual (WT) or from a NF1 patient carrying the c.6792C > G substitution in one allele. The splicing pattern of the latter shows three bands, theupper one corresponds to the correct mature mRNA derived from the normal and to some extent from the mutant allele. The intermediate band lacksexon 37 and the lower band lacks both exons 36 and 37. (E) Comparison of RT-PCR products obtained with the use of minigenes composed of exon34–38 with those composed of exons 31–38. From the gel we can see that the more genomic sequences are present the more correctly spliced mRNA ispresent with the wider context resembling the chromosomal situation (compare gels D with E). (F) Graphical representation of the position of thespurious splice sites used from the transcripts lacking exon 37 (Fig. 2B). **indicates correct splice site, *splice site recognized as such by the software‘‘Splice Site Prediction by Neural Network Site’’ (http://www.fruitfly.org/seq_tools/splice.html). Bold uppercase indicates exonic sequence, lowercaseintronic/denotes the splice junction.
M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456 4451
SPH1R GTTGGAATATCTCTCATGCATGCCT
Sp34F ACAGAAGCTTTGTTGGAGATC
Sp38R CTCATCAAGCTGCACAGCC
2.3. Cell culture, transfection, and RT-PCR analysisTransfections of the human cervical carcinoma (HeLa) cells were
carried out as previously described [17]. Total RNA was then preparedfrom the transfected cells using RNAwiz (Ambion). Analysis of thealternatively spliced products was performed on each cDNA by PCRamplification with the primers previously described, in the case of hy-brid minigenes [16], T7 and SP6 in the case of the 36–38 minigene,Sp34F and Sp38R in the case of the lymphoblasts and Sp34F withSP6 in the case of the minigenes 31 or 34–38.
2.4. EBV patient transformed lymphoblastsEBV transformed lymphoblasts from a patient suffering from NF1
and previously identified to carry the c.6792C > G mutation [7] wereobtained from the European Collection of Cell Cultures ECACC.
3. Results
3.1. c.6792C > A and c.6792C > G mutations result in exon
skipping due to the disruption of an exonic regulatory
element
To test the hypothesis that mutations c.6792C > A and
c.6792C > G in NF1 exon 37 were affecting a splicing regula-
tory region in NF1 exon 37 and not causing NAS, wild-type
and mutated NF1 exon 37 sequences along with flanking intro-
nic sequence were inserted in the previously reported PTB
hybrid minigene (Fig. 1B). Surprisingly, we observed that the
wild-type sequence is not efficiently processed (Fig. 1B) in this
context as only approximately 50% of exon inclusion could be
observed. This was unexpected, as in many other cases the
minigene reflected accurately the splicing pattern of the endog-
enous gene [16,18,19]. In any case, it was still possible to see
4452 M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456
the deleterious effect on splicing of the disease causing muta-
tion 6792C > G as this nucleotide transition in the minigene re-
sulted in complete skipping of NF1 exon 37. The fact that also
the same sense (translationally silent) substitution 6792C > T
(Fig. 1B) introduced at this position caused total exon skipping
indicated that the aberrant processing of mRNA carrying
c.6792C > A or c.6792C > G observed in the patients is indeed
due to the disruption of a ESE and not to NAS.
3.2. The importance of the genomic context and the peculiar
genomic organization in the NF1 exon 34–38 region
The poor performance of the hybrid minigene exon 37 wild-
type construct indicated that efficient definition of NF1 exon
37 could need additional flanking intronic and/or exonic NF1
sequences localized within the exon-rich genomic cluster in
which it is found (Fig. 2A). We then constructed a minigene
with additional genomic sequences spanning the region from
NF1 exons 36–38 (Fig. 2B). In this case the wild-type construct
showed efficient processing of exon 37 whilst the 6792C > G
(nonsense mutation) and 6792C > T (translationally silent)
mutant constructs showed significant exon skipping. This re-
sult confirms that, as observed with the PTB minigenes, the
translationally silent mutation acts in the same way as the non-
sense mutation. However, this expanded context was not com-
pletely satisfactory in mimicking the natural effects of these
mutations. In fact, an intriguing and albeit unexplained obser-
vation was made when the transcripts lacking exon 37 (lower
bands Fig. 2B) were analysed. Cloning and sequencing these
bands showed that the junction between exons 36 and 38
was not always derived from clean skipping of exon 37 but
in addition to the normal 36 and 38 splice sites customarily
used there was an heterogeneous collection of unusual splice
sites usage (Fig. 2F). This was true for mRNA lacking exon
37 obtained from wild-type and 6792C > G constructs.
To better assess the influence of the flanking genomic se-
quences on NF1 exon 37 splicing, a further set of minigenes
was then assembled comprising NF1 exons 34 through to exon
38 (Fig. 2C). The wild-type construct when transfected in Hela
cells mimicked what has being previously reported to occur in
human tissues [8,20], with proper processing of the exons and
an extremely small amount of transcript lacking NF1 exon 37.
Again, the results show that as with the other minigenes the
translationally silent mutation 6792C > T acts in the same
way as the nonsense mutation 6792C > G, causing exon 37
skipping. In addition, a third type of mRNA processing is also
observed, namely one in which both the exons 36 and 37 are
skipped. This, to a smaller extent, was also observed in EBV
transformed lymphocytes obtained from a patient suffering
from NF1 and carrying the c.6792C > G substitution in hetero-
zygosis with a normal allele (Fig. 2D). The bands resulting
from the processing of the minigene and the lymphoblasts were
sequenced and, in contrast with those derived from the minig-
enes made from exons 36 to 38, this time all of them resulted
from correct splice site usage.
Although exons 34–38 are sufficient for correct wild-type
processing of the minigene we observed that the mutated con-
struct did not imitate exactly the splicing pattern obtained with
the patient lymphoblast. Extending the minigene to exons 31
(31–38 constructs) caused a decrease of the exon 36–37 double
skipping (Fig. 2E). This is a shift towards the splicing pattern
observed in lymphoblasts derived from a NF1 patient carrying
the c.6792C > G mutation (Fig. 2D), further underlining the
long-range interplay of splicing regulatory elements within this
exon cluster.
3.3. Definition of NF1 exon 37 regulatory element of splicing
We have then carried out a study to define the ESE and the
interplay with other exon 37 sequences. Extensive site directed
mutagenesis was undertaken of the bases between positions
c.6790 and c.6797 with all three possible nucleotide substitu-
tions (Fig. 3). We observed that any nucleotide transition at
positions c.6790, 6791, 6793, 6794, 6796, and 6797 had no ef-
fect on splicing. However, every nucleotide change at positions
c.6792 and c.6795 led to aberrant splicing, resulting in a three
band profile where the upper band represents correct assembly
of the five exons, the intermediate band lacks exon 37 and the
lower band lacks both exons 36 and 37. This observation is
also valuable for future diagnostic procedures as any nucleo-
tide variation at position c.6795 will very likely be a disease
causing mutation.
3.4. Exon 37 definition influences exon 36 recognition
In previous reports of upstream exon skipping the defect was
always located in the downstream exon splice sites [21–23].
This is the first report of decreased downstream exon definition
due to an ESE disruption affecting the upstream exon defini-
tion. In order to distinguish if this was a direct effect of disrup-
tion of the ESE or an indirect effect in as much as the exon 36
recognition is dependent on exon 37 definition we analyzed the
splicing pattern resulting from a minigene spanning exon 34–
38 with the donor site of exon 37 mutated by a
IVS37 + 1G > T mutation. From Fig. 4 we can see that this re-
sults in two transcripts, one in which only exon 37 is skipped
and a second where both exons 36 and 37 are skipped. These
results indicate that the definition of exon 36 is dependent on
that of 37. The severity of which the definition of exon 37 is
affected reflects on exon 36 definition as shown by comparing
the splicing pattern of transcripts resulting from disruption of
ESE (6792C > G) with the splicing pattern caused by the more
drastic donor site disruption (IVS37 + 1G > T).
3.5. The exon regulatory element in NF1 exon 37 offsets the
presence of a non-consensus 3 0 splice site
Exonic or intronic cis-acting regulatory regions are often de-
scribed on exons with weakly defined 5 0 and 3 0 splice site [1,24].
The ESE that we have characterized may thus be necessary be-
cause of the extremely weak definition of NF1 exon 37 3 0 splice
site that does not compare favorably to consensus (Fig. 5).
To evaluate the effect of ESE disruption in relation to the
strength of the 3 0 splice site we tested a minigene construct with
an improved 3 0 splice site, created by the nucleotide transition
IVS36-A > C, carrying the mutation c.6792C > G that caused
aberrant splicing. Fig. 5 shows that the double mutant
(c.6792C > G and IVS36-A > C) has a splicing pattern identi-
cal to the wild-type, with full inclusion of exons 36 and 37.
4. Discussion
We have studied the effect on splicing of naturally occurring
and site directed mutations in NF1 exon 37. Our work, in
accordance with the greater enhancer activity of the wild-type
Fig. 3. Definition of the ESE. Schematic representation of the minigene used in this assay with nucleotides c.6790–c.6797 in NF1 exon 37 highlightedin their codon triplets. The lower panel shows the effect on RNA processing of changing each nucleotide present between position c.6790 and c.6797of the NF1 gene, into the three alternative nucleotides for that position. It can be observed that every nucleotide change at positions c.6792 and c.6795led to aberrant splicing, resulting in a three band profile where the upper band represents correct assembly of the five exons, the intermediate bandlacks exon 37 and the lower band lacks both exons 36 and 37.
Fig. 4. Effect of decrease of NF1 exon 37 definition on exon 36 recognition. To test this phenomena we have decreased exon 37 definition by ESEdisruption (6792C > G) and the more drastic 5 0ss disruption (IVS37 + 1G > T). Upper panel shows a schematic representation of the minigenescarrying the ESE 6792G > C mutation or the IVS37 + 1G > T mutation that disrupts the donor site of NF1 exon 37. Lower panel shows the effect onRNA processing of disrupting NF1 exon 37 definition. We can observe that the greater the skipping of exon 37 the more exon 36 is also skipped.
M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456 4453
sequence over the mutated sequence [15], shows that the dis-
ruption of a ESE as the cause of the inefficient inclusion in
NF1 mRNA of exon 37 carrying the c.6792C > G and
c.6792C > A mutations found in patients [10,14]. In fact, even
the same sense variation, 6792C > T results in the skipping of
NF1 exon 37.
Exon 37 is localized in an exon-rich genomic cluster and is
badly recognized by the splicing machinery if it is placed with
limited flanking intronic sequences in a minigene with heterol-
ogous genomic context. It needs to be embedded in the region
spanning NF1 exons 34–38 to become efficiently recognized
(Fig. 6A–C, wildtype constructs). Indeed, a blanket mutagen-
esis analysis carried out in the 34–38 context shows that
changes in 2 out of the 8 nucleotide positions analyzed (specif-
ically the C’s in the ACAAC motif) produced aberrant tran-
scripts: exon 37 skipping and the double skipping of exons
36 and 37, indicating unequivocally that this region contains
a regulatory element important for correct exon definition. It
is interesting to note that both the c.6790_6791TTins and a
c.6789_6792TTACdel observed in patients that did not cause
exon skipping (Fig. 1A) preserve the sequence ACAACA. sim-
ilar to the A/C splicing regulatory element previously reported
[25]. Widening the genomic context further from NF1 exons 31
to 38 partially offsets the importance of the exon 37 ESE
reducing the skipping of both exons 36 and 37 in the minigene
carrying the c.6792C > G mutation. Indeed, if we consider that
in lymphoblasts obtained from a patient carrying the
c.6792C > G mutations the splicing pattern observed derives
equally from both the normal and mutant alleles, as has been
shown for c.6792C > A [14] (Fig. 2D) and hence the contribu-
tion to the top band is predominantly from the non-mutated
allele it follows that the pattern resulting from the 31 to 38
minigene carrying the c.6792C > G mutation is practically
identical to that of the chromosomal setting (Fig. 6, compare
lines C, D and E in the c.6792C > G minigene constructs/lym-
phoblast allele column).
Regarding exon 36, as far as we know this is the first time
that upstream exon definition is shown to be at least in part
dependant on an ESE in the downstream exon, in as much
as this element is necessary for definition of exon 37 whose
Fig. 5. The ESE is necessary due to the weak definition of exon 37 splice sites. Schematic representation of the minigenes carrying the wild-type 3 0ssor improved 3 0 splice site (IVS36-3A > C). Splice site strength was evaluated using the ‘‘Splice Site Prediction by Neural Network Site’’ (http://www.fruitfly.org/seq_tools/splice.html)./ denotes intron exon junction. The nucleotides 6790–6798 in NF1 exon 37 are highlighted in their codontriplets. The lower panel shows the effect on RNA processing of improving the 3 0 splice site. The aberrant splicing previously observed with themutations 6792C > G that disrupted the ESE is no longer present (lanes 1 and 2).
4454 M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456
definition is in turn necessary for the correct recognition of
exon 36 by the splicing machinery. In previous reports of up-
stream exon skipping the defect was always located in the
downstream exon splice sites [21–23]. These results highlight
the dynamic setting of the splicing process, where early-and-
late transcribed sequences condition the definition of specific
exons located at considerable distance. The experimental data
therefore point to a particular long-range combinatorial con-
trol of exon definition in this cluster. The recognition of splice
sites involves crosstalk between multiple, at times relatively
weak, interactions that contribute to establish complexes defin-
itively committed to splicing [1–3]. Our work shows that there
is a much larger long distance network across exons and in-
trons than previously thought, which contributes to correct
pre mRNA processing. In fact, the more genomic sequences
present in the minigenes, the greater amount of correctly pro-
cessed transcript is produced, even in the versions of the mini-
gene where the ESE is disrupted (Figs. 2 and 5). This type of
control may not be restricted to NF1 exon 37. Although we
do not yet fully understand the intricate mechanisms of the
interactions involved in this cluster it is possible that up and
down stream sequences may modify the transcription kinetic
and/or the spliceosome composition. For example, it has been
shown that in the human thrombopoietin gene processing of a
small intron present within the last exon of the gene, is depen-
dent on processing of previous introns [26]. Furthermore, the
effect of this type of long distance interactions was highlighted
by Fededa et al. in a recent study [27] on splicing in the Fibro-
nectin gene where the processing undergone by an upstream
alternatively spliced exon has an influence on the pattern of
alternative splicing of downstream exons.
Another interesting observation related to our finding re-
gards the overlapping of codon usage and splicing regulatory
elements. The latter represents an additional constraint during
the evolution of an exon sequence. Synonymous variations are
assumed to be functionally neutral both in clinical diagnosis
and when measuring evolutionary distances between species
because they do not change the peptide sequence. However,
a recent study [28] has shown that synonymous variations in
CFTR exon 12 cannot evolve neutrally, but are significantly
constrained by splicing requirements. In fact, roughly one
quarter of the synonymous changes studied in CFTR exon 9
[29] and CFTR exon 12 [28] have an effect on splicing. A sim-
ilar situation is observed, albeit in a limited setting, with the
synonymous changes introduced during the mutational analy-
sis on the NF1 exon 37 where the only two nucleotide transi-
tions that result in a same sense mutation (c.6792C > T and
c.6795C > T) result in aberrant splicing. Our study shows that
a weak 3 0 splice site, like the one flanking exon 37, conditions
the evolution of the exon that must maintain exonic splicing
regulatory sequences to ensure its inclusion in mature mRNA.
We have tested this hypothesis experimentally by improving
the NF1 exon 37 3 0 splice site. The introduction of a single
nucleotide transition IVS36-3A > C overrides the need for a
functional ESE in NF1 exon 37. From an evolutionary bearing
this means that any nucleotide variation, in an improved con-
text, and hence amino acid changes may occur without altering
the pre-mRNA processing.
Fig. 6. Quantification of transcripts from Figs. 1 and 2 by Image J (http://rsb.info.nih.gov/ij/) shows the influence of the genomic context induplicating the in vivo effects (E). Wild-type exon 37 alone with a small portion of flanking intronic regions is not sufficient to yield the observed>99% inclusion in normal lymphoblasts (compare lines A and E). However, this result can be readily achieved when larger portions of this genomicregion are included in the genomic construct (compare lines C and D with E). In addition, an increased genomic context also provides a closercorrespondence with regards to the exon skipping effects of the 6792C > G substitution In fact, only the construct carrying NF1 exons 31–38 togetherwith a mutated exon 37 (D) yields a splicing pattern that reflects the observed percentage in a patient’s lymphoblast that carry the C6792Gsubstitution in one allele (compare D and E). Quantification in the minigene 36–38 of the bands lacking exon 37 was performed on the totalaberrantly spliced bands.
M. Baralle et al. / FEBS Letters 580 (2006) 4449–4456 4455
Acknowledgements: This work was supported by grants from TelethonOnlus Foundation and by a European community grant (EURAS-NET) to FEB.
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