Báo cáo khoa học: Low U1 snRNP dependence at the NF1 exon 29 donor splice site doc

Low U1 snRNP dependence at the NF1 exon 29 donor
splice site
Michela Raponi
1,2
, Emanuele Buratti
3
, Elisa Dassie
1
, Meena Upadhyaya
4
and Diana Baralle
1,2
1 Department of Pathology, University of Cambridge, UK
2 Human Genetics Division, University of Southampton, UK
3 Department of Molecular Pathology, ICGEB, Trieste, Italy
4 Institute of Medical Genetics, Cardiff University, UK
The mechanisms involved in the inclusion of an exon
in the mature mRNA molecule are complex, with an
ever-increasing number of sequence and protein ele-
ments being involved in its definition [1,2]. At the most
basic level, canonical splice sites (SSs) are present at
the 5¢-ends and 3¢-ends of the exons, and other classic
splicing signals, the polypyrimidine tract and the
branch point, are present upstream of the 3¢SS [3,4].
To increase the overall fidelity of the splicing reaction,
additional enhancer and silencer elements are present
in the exons [exon splicing enhancers (ESEs); exon
splicing silencers (ESS)] and/or introns [intron splicing
enhancers (ISEs); intron splicing silencers (ISS)], allow-
ing the correct SSs to be distinguished from the
many cryptic SSs that have very similar signal

sequences [5–7]. In addition, it is now clear that many
other factors, such as RNA secondary structure, tran-
scription rates, genomic context, and external stimuli,
can profoundly affect the working of the splicing
machinery [8–11]. Not surprisingly, therefore, many
disease-causing splicing mutations described in the lit-
erature produce changes in these regulatory sequences
and processes [12,13]. It is the study of these new cases
that often provides novel insights into the basic mecha-
nisms of SS recognition by the spliceosome [14–16].
Neurofibromatosis type 1 (NF1) is a dominantly
inherited multisystem disorder with complete pene-
trance by age 5 years. Mutations in the NF1 gene
have been found to span the entire coding sequence.
In particular, a high incidence of splicing aberrations
Keywords
donor; NF1; splicing; U1 snRNP
Correspondence
D. Baralle, Human Genetics Division,
University of Southampton, Duthie Building
(Mailpoint 808), Southampton General
Hospital, Tremona Road, Southampton
SO16 6YD, UK
Fax: +44 0 238 079 4346
Tel: +44 0 238 079 6162
E-mail:
(Received 25 September 2008, revised 26
January 2009, accepted 30 January 2009)
doi:10.1111/j.1742-4658.2009.06941.x
Many disease-causing splicing mutations described in the literature produce

changes in splice sites (SS) or in exon-regulatory sequences. The delineation
of these splice aberrations can provide important insights into novel regula-
tion mechanisms. In this study, we evaluated the effect of patient variations
in neurofibromatosis type 1 (NF1) exon 29 and its 5¢SS surrounding area
on its splicing process. Only two of all nonsense, missense, synonymous
and intronic variations analyzed in this study clearly altered exon 29 inclu-
sion/exclusion levels. In particular, the intronic mutation +5g>a had the
strongest effect, resulting in total exon exclusion. This finding prompted us
to evaluate the exon 29 5¢SS in relation to its ability to bind U1 snRNP.
This was performed by direct analysis of the ability of U1 to bind to wild-
type and mutant donor sites, by engineering an in vitro splicing system to
directly evaluate the functional importance of U1 snRNA base pairing with
the exon 29 donor site, and by coexpression of mutant U1 snRNP mole-
cules to try to rescue exon 29 inclusion in vivo. The results revealed a low
dependency on the presence of U1 snRNP, and suggest that exon 29 donor
site definition may depend on alternative mechanisms of 5¢SS recognition.
Abbreviations
ATM, ataxia telangiectasia mutated gene; EDB, extra type III homology B or extra domain B; EMSA, electromobility shift analysis; ESE, exon
splicing enhancer; ESS, exon splicing silencer; ISE, intron splicing enhancer; ISS, intron splicing silencer; NF1, neurofibromatosis type 1;
SNP, single-nucleotide polymorphism; SS, splice site.
2060 FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS
has been reported in this gene, making it an ideal
model with which to study how this process is
involved in disease. Examples have included muta-
tions interrupting U1 snRNA binding [17,18] and
ESEs [19,20].
Given the high number of NF1 patients found to
carry single-nucleotide substitutions in NF1 exon 29
and its 5¢SS, we developed a functional assay to distin-
guish which genomic variants cause aberrant splicing.

Only two of the substitutions analyzed in this study
caused exon skipping in the range of 30–100% of the
total processed mRNA. Interestingly, the intronic
mutation +5g>a, at the 5¢ SS of exon 29, had the
strongest effect, resulting in total exon exclusion. This
finding prompted us to evaluate carefully the impor-
tance of 5¢SS definition in NF1 exon 29. In general,
the concept of 5¢SS definition itself has proven to be
surprisingly sensitive to sequence context, and several
papers have analyzed this issue in detail in the recent
past [21–27]. Indeed, although the importance of
U1 snRNA interaction with the 5¢SS is considered to
be one of the fundamental steps in SS definition
[28,29], the binding of this molecule through a straight-
forward RNA–RNA interaction has sometimes been
found to be dispensable for proper splicing [30,31],
and other accompanying factors may play an impor-
tant role therein [21,32,33]. From a clinical point of
view, this complexity is well reflected in the fact that
identical genomic variations around 5¢SSs can be
innocuous in one exon or cause disease-related skip-
ping in another [7]. For this reason, the susceptibility
of the NF1 exon 29 5¢SS to aberrant splicing was fur-
ther analyzed at the molecular level in order to delin-
eate the regulatory elements involved.
Results
NF1 exon/intron 29 mutations and evolutionarily
conserved splicing regulatory elements
The sequence variations identified in this study in
NF1 exon/intron 29 of patients with clinically defined

NF1 are shown in Fig. 1. Initially, in silico analysis of
splice regulatory sites was used to analyze the impact
Fig. 1. NF1 exon/intron 29 mutations and evolutionary conservation. NF1 exon 29 (upper-case) and partial intron 29 (lower-case) sequences,
showing the location of the nucleotide changes. The amino acid changes are in parentheses. Nucleotide positions that are not conserved
between humans and other placental mammals are underlined. Intron genomic sequence alignments are shown at the bottom. The 5¢SS
conserved sequences are boxed.
M. Raponi et al. Defective interaction between U1 snRNP and 5¢SS
FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS 2061
of each exonic variation on splicing elements. The
results where a significant variation was predicted
either to create or interrupt a putative splice regula-
tory site are reported in Table 1. In addition, evolu-
tionary conservation analysis was used to improve the
specificity of this ESE prediction tool, as suggested
recently by Pettigrew et al. [34]. In this analysis, the
potential evolutionary conservation of ESEs was
examined using the ucsc genome browser in all avail-
able placental mammals (chimp, mouse, rat, horse,
armadillo, cat, cow, and dog). This revealed that none
of the predicted ESE sequences interrupted by our
nine exonic variations were evolutionarily conserved
(compare Fig. 1 and Table 1). However, several exon-
ic variations were predicted to create new silencer
sites (Table 1).
Comparative genomic analysis among placental
mammalian species was also performed to evaluate the
conservation of the first 23 nucleotides of the intron 29
sequence (Fig. 1). Whereas the downstream sequence
(nucleotides +6 to +23) presents high variability
among the different species, the 5¢SS consensus

sequence is completely conserved, suggesting high
susceptibility to mutations of this region.
Table 1. Splicing Sequence Finder analysis for the nine exonic variations. New site refers to putative regulatory sequences created by the
variation. Broken site refers to putative regulatory sequences disrupted by the variation. Proteins predicted to bind the putative splicing regu-
latory sequences and the matrix sources are given.
Variation Matrix Protein Sequence Effect
c.5224C>T (Q1742X) ESE finder matrices SRp55 TAAGTA New site
RESCUE ESE examers CAAGTA Site broken
hnRNP motifs hnRNPA1 TAAGTA New site
Silencer motifs from Sironi et al. CTGTCTAA New site
c.5234C>G (S1745X) ESE finder matrices SRp40 CTTCAGC Site broken
SF2/ASF CAGCAGA Site broken
RESCUE ESE examers AACTTC Site broken
ACTTCA Site broken
CTTCAG Site broken
PESE octamers from Zhang & Chasing ACTTCAGC Site broken
c.5242C>T (R1748X) RESCUE ESE examers AGTGAA New site
PESE octamers from Zhang & Chasing CGAACA Site broken
GCGAACAA New site
c.5264C>G (S1755X) ESE finder matrices SRp40 GGGCAATG New site
SRp55 TGAGTC New site
RESCUE ESE examers CAATCA Site broken
AATCAG Site broken
Silencer motifs from Sironi et al. AATGAGTC New site
c.5290G>T (A1764S) ESE finder matrices SRp55 TGCTTC Site broken
Silencer motifs from Sironi et al. TTCTTCGG New site
c.5388T>A (C1796X) ESE finder matrices SRp40 TGAGAAG New site
RESCUE ESE examers TGAGAA New site
PESE octamers from Zhang & Chasing TGTGAAGC New site
ESE motifs Tra2 GAGAAG New site

c.5425C>A (R1809S) ESE finder matrices SF2/ASF CGGACCA New site
SF2/ASF CAGCTGG New site
PESE octamers from Zhang & Chasing CGCTGGG Site broken
CGGACCCG Site broken
GGACCCGC New site
ACCCGCTG New site
CCCGCTGG New site
GACCAG New site
ESE motifs Tra2 GAGAAG New site
c.5426G>T (R1809L) ESE finder matrices SF2/ASF CGCTGGG Site broken
SRp40 GGACCCTC New site
PESE octamers from Zhang & Chasing CGGACCCG Site broken
c.5427C>T (R1809R) ESE finder matrices SF2/ASF GACCCGT New site
SRp40 CCGCTGG Site broken
SF2/ASF CGCTGGG Site broken
Defective interaction between U1 snRNP and 5¢SS M. Raponi et al.
2062 FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS
Splicing analysis of NF1 exon\intron 29 variations
by the minigene approach
On the basis of these considerations, we conducted
in vivo splicing assays using a genomic portion of NF1
exon 29 and part of the flanking introns 28 and 29. A
schematic diagram of the hybrid minigene (pTB NF1–
29) used in transient transfection splicing assays is
shown in Fig. 2A. The transfection results for the
pTB NF1–29 hybrid minigenes carrying all exonic and
intronic nucleotide substitutions described in Fig. 1 are
shown in Fig. 2B.
Transfection of the normal wild-type pTB NF1–29
minigene in HeLa cells followed by RT-PCR amplifi-

cation generated mostly transcripts of 580 bp contain-
ing exon 29, and a smaller proportion of transcripts of
239 bp lacking this exon. This leaky splicing of NF1
exon 29 in the minigene context was analogous to that
already reported in leukocytes from normal individuals
[35]. To further confirm the reliability of our system,
transfection of the minigene carrying the +19t>a sub-
stitution, which is considered to be a common single-
nucleotide polymorphism (SNP) [36], was undertaken,
and resulted in the same splicing outcome as for the
wild-type minigene. Moreover, transfection of the
minigene carrying the R1748X change gave the same
result as trasnfection of the wild-type minigene and as
that obtained when analyzing the RNA sample from
leukocytes available from the patient (data not
shown).
With regard to the variations found in patients, only
a minority of the substitutions observed in patients
resulted in low to moderate modifications of splicing
levels: R1809S and S1755X led to the highest amount
of exon 29 inclusion, whereas the Q1742X minigene
resulted in approximately 30% exon skipping with
respect to wild-type levels (Fig. 2B). Interestingly, the
strongest effect was observed for the minigene carrying
the intron 29 substitution +5g>a, which resulted in
100% exon skipping. It should also be noted that simi-
lar results were obtained when the minigenes were
transfected into COS cells (data not shown). We then
proceeded to characterize in more detail the impor-
tance of the NF1 exon 29 donor site in regulating

recognition of this exon.
Defective NF1 intron 29 5¢SS recognition
First, natural human 5¢SS sequences with similar
degrees of mismatching with the mutated donor site
were selected from the most up-to-date Homo Sapiens
Splice Sites Dataset, released by Sahashi et al. [37].
Interestingly, only 3.62% of the 1492 natural 5¢SSs
carrying +4g+5a (and thus containing the same in-
tronic mismatches with U1 snRNA as the mutated
donor site) present a cytosine as the mutated donor
site in position –2, whereas an adenosine is over-repre-
sented in this position (Fig. 3A). The adenosine
frequency is 89.14%, as opposed to 63.5% of the over-
all 189 249 human 5¢SSs [37]. In addition, the 542
functional 5¢SSs with both +5a and )2c rarely have a
guanosine in position +4, as for the mutated donor
site, whereas 64.31% of those sites have an adenosine
in this position (Fig. 3A). As the significant differences
Fig. 2. Hybrid minigene transient transfection assay. (A) Schematic representation of the hybrid minigene (pTB NF1–29) used in transient
transfection splicing assays. Minigene exons and introns are indicated as boxes and lines, respectively. The exon 29 sequence (white box)
was tested for splicing efficiency, using specific primers (arrows). Dotted lines show the two NF1 exon 29 alternative splicing possibilities.
(B) RT-PCR products from transfection experiments. The minigenes were transfected into HeLa cells, and RT-PCR analysis was performed
on a 2% agarose gel. RNA splicing variants corresponding to exon 29 inclusion (+) and exclusion ()) are shown. The mean levels of exon 29
inclusion, together with standard deviations (SDs) from three different experiments, are reported below.
M. Raponi et al. Defective interaction between U1 snRNP and 5¢SS
FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS 2063
between the group of functional 5¢SSs and the mutated
SS described here were in positions )2 and +4, we
decided to study the splicing effect of artificial muta-
tions in the NF1 exon 29 5¢SS to determine whether or

not a cooperative effect between those positions and
the +5 position exists. Therefore, )2c was replaced by
a purine both in the wild-type and in the +5g>a
pTB NF1–29 minigene.
The )2c>a substitution did not have any effect on
splicing, either in the wild-type or the +5g>a
pTB NF1–29 minigenes (Fig. 3B). Nevertheless,
according to several 5¢SS prediction programs
(Fig. 3C), the )2c>a substitution should have been
quite sufficient to restore the U1 complementarity of
the mutated +5g>a exon 29 donor site, thus allowing
5¢SS usage, as is the case in the functional 5¢SS donor
sequences described in Fig. 3A. Moreover, transfection
of the minigene carrying the artificial )2c>g substitu-
tion showed the same effect as the natural mutation
+5g>a, resulting in 100% exon 29 exclusion, even
though the available 5¢SS prediction programs were
not able to predict the dramatic effect of this substitu-
tion (Fig. 3B,C). These results suggested that other
factors may play a role in determining exon 29 recog-
nition besides U1 snRNA complementarity. Finally,
the only substitution in the mutant +5g>a 5¢SS that
was predicted to raise the SS score to 0.98 according
to the nnsplice program is represented by +4g>a
(Fig. 3C). We therefore replaced the +4g with adeno-
sine, both in the wild-type and in the +5g>a
minigenes. This time, in keeping with predictions,
although the +4g>a substitution alone had no effect
in improving wild-type exon 29 inclusion levels, the
double mutation +4g>a\+5g>a partially restored

Fig. 3. NF1 intron 29 5¢SS recognition. (A) Nucleotide frequency (%) at human natural (true) 5¢SSs with similar degrees of mismatching with
U1 snRNA as the +5g>a mutated donor site. The 5¢SS consensus sequence and the intron 29 +5g>a 5¢SS sequences are shown. Nucleo-
tide positions in the 5¢SSs are numbered. (B) Point mutations were introduced into the IVS29 5¢SS, and the resulting minigene variants were
transfected into HeLa cells. The splicing pattern of these mutants was analyzed by RT-PCR on a 2% agarose gel, and the mean levels of
exon 29 inclusion, together with standard deviations from three different experiments, are shown below. (+) and ()) indicate exon 29 inclu-
sion and exclusion, respectively. The additional band below the (+) band is due to heterodimer formation. (C) Table showing the 5¢SS score
calculated by the maximum entropy method (ME), the Neural Network website (NN), the Shapiro and Senapathy matrix (S&S), and hydrogen
bond base pair formation for the wild-type and mutant IVS29 donor sites.
Defective interaction between U1 snRNP and 5¢SS M. Raponi et al.
2064 FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS
exon 29 inclusion levels (Fig. 3B). In addition, the
+4g>a and +7t>a substitutions together rescued exon
inclusion when inserted in the )2c>g minigene, a result
that is generally in keeping with program predictions
(Fig. 3C). These results are in support of U1 snRNP
dependency when the 5¢SS sequence is sufficiently
altered. However, reproduction of similar complemen-
tarity using mutant U1 snRNPs did not result in exon
inclusion in the original IVS29 context (see below).
Interaction between U1 snRNP and IVS29 donor
sites
We then decided to investigate the ability of all these
donor sites to bind U1 snRNA/U1 snRNP directly,
under splicing conditions. In these assays, we used a
sequence from the ATM gene as a positive control that
was previously shown to bind U1 snRNP with high
affinity [15].
Synthetic oligonucleotides of all these sequences
(Fig. 4A) were end-labeled with a-
32

P and UV-cross-
linked with a HeLa nuclear extract that was treated
with RNAse H in the presence of a specific antisense
oligonucleotide in order to inactivate the endogenous
U1 snRNP molecules (Fig. 4B, minus lanes). A mock-
inactivated sample obtained using a random oligonu-
cleotide was also used as a control (Fig. 4B, plus
lanes). Figure 4B shows that, under splicing condi-
tions, all IVS29 donor sites bound U1 snRNA with
substantially reduced efficiency as compared to the
ATM wild-type sequence (compare the intensities
between lane 2 and lanes 4, 6, 8, and 10 in Fig. 4B).
Although this experiment ruled out a strong and direct
interaction between U1 snRNA and the IVS29 donor
site sequences, there also remained the possibility that
U1 snRNP might still be recruited to the IVS29 donor
site by other indirect interactions. Moreover, the much
stronger signal for the ATM SS than for the NF1 SS
could have simply reflected the higher number of com-
plementary nucleotides in this oligonucleotide with
respect to the IVS29 oligonucleotides.
In order to address these questions, we then per-
formed an electromobility shift analysis (EMSA) of the
RNAÆprotein complexes assembled on the ATM oligo-
nucleotide and each of the IVS29 oligonucleotides,
using nuclear extract depleted or mock-depleted of
U1 snRNA. Figure 4C shows, as expected, that in the
Fig. 4. UV crosslinking and EMSA analysis. (A) Sequences of the
RNA oligonucleotides used in these experiments: ATM wild-type
(WT), IVS29 WT, IVS29 +5A, IVS29 +4A+5A, and IVS29 ) 2A+5A.

(B) The reactivity of each 5¢-end-labeled single-stranded oligonucleo-
tide with U1 snRNA following RNAÆRNA UV crosslinking analysis is
shown. For each RNA oligonucleotide, this reaction was performed
using U1 snRNP-depleted HeLa nuclear extract ()) and mock-
depleted extract (+). The RNAÆRNA crosslinked complexes were
separated by EMSA, and the region where the U1 snRNAÆRNA
complexes migrate is indicated by an open box. (C) EMSA analysis
of proteinÆRNA complexes that are formed on each of these oligo-
nucleotides in the presence of U1 snRNP-depleted HeLa nuclear
extract () ) and mock-depleted extract (+). The region involved in
the U1 snRNPÆRNA interaction is indicated by an open box.
M. Raponi et al. Defective interaction between U1 snRNP and 5¢SS
FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS 2065
case of the wild-type ATM oligonucleotide, only one
major complex was formed in the presence of the
mock-U1-depleted nuclear extract and that formation
of this complex was completely abolished after RNa-
se H treatment (compare lanes 1 and 2 in Fig. 4C). In
the case of the IVS29 oligonucleotides, a series of
complexes were also assembled on each oligonucleo-
tide, most of them occurring outside the region defined
by the ATM–U1 snRNA complex (see open box).
Moreover, no major effect on any of these complexes
could be observed following U1 snRNP removal from
the nuclear extract (Fig. 4C; compare lanes 3 and 4, 5
and 6, 7 and 8, and 9 and 10).
In vitro analysis of the functional relationship
between U1 snRNP and the NF1 IVS29 donor site
In order to functionally test the dependency of the
NF1 exon 29 5¢SS and its ability to base-pair with

U1 snRNA, we next set up an in vitro splicing system
based on the PY7 tropomyosin plasmid (see Fig. 5A
for a schematic diagram). Initially, a preliminary
in vitro splicing analysis was performed to confirm
that, even in these very different conditions, the +5A
and +4A+5A mutations had a similar effect to that
observed for the IVS29 mutants in the pTB context.
The results of this analysis confirmed that these in vitro
splicing substrates were spliced as observed in the
minigene splicing system (Fig. 5B).
An in vitro splicing reaction was then performed in
the presence of 50 ng of an antisense oligonucleotide
(U1AS) aimed at blocking U1 snRNA interactions. In
this respect, it should be noted that the simple incuba-
tion of HeLa nuclear extract with an antisense oligo-
nucleotide against the 5¢-end U1 snRNA has
previously been reported to activate the endogenous
RNase H activity, with consequent inactivation of this
factor [38]. In our case, this was confirmed by incuba-
tion of approximately 150 lg of commercial HeLa
nuclear extract with increasing concentrations of
U1AS oligonucleotide, followed by phenol/chloroform
extraction of total RNAs and reverse primer extension
analysis using a labeled oligonucleotide localized in
U1 loop 2 [39]. The results of this analysis showed
that, after 30 min, the quantity of intact U1 snRNA
was reduced by more than 90% in the presence of
12.5 ng of U1AS oligonucleotide (Fig. 5C, lane 2), and
that it became undetectable after addition of 25 ng of
oligonucleotide (Fig. 5C, lane 3). The U1-depleted mix

was then used to test whether the absence of
U1 snRNP could affect the in vitro recognition of the
IVS29 donor site. As shown in Fig. 5D, addition of
Fig. 5. In vitro splicing of NF1 5¢SS in the absence of U1 snRNP. (A) Schematic diagram of the hybrid NF1 exon 29 construct used in the
in vitro splicing assays. The NF1 exon/intron sequence present in these construct is shown in full. The open box represents tropomyosin
exon 3, and the black line the remaining 94 nucleotide tropomyosin intron. The position of the T7 promoter used to transcribe this RNA is
also indicated. (B) In vitro splicing efficiency after 2 h of each RNA template carrying the IVS29 wild-type (WT) donor sequences and the
IVS29 +5A and IVS29 +4A+5A mutants. A schematic diagram of the different RNA species is given on the right. (C) Reverse primer exten-
sion analysis of the effects of endogenous RNase H activity on U1 snRNP in the presence of an antisense oligonucleotide (U1AS) targeted
against the 5¢-end of U1 snRNA. Lane 1: no oligomer. Lanes 2–5: increasing oligomer concentrations. The positions of wild-type uncleaved
U1 snRNA, U1 (+1), and of the cleaved RNase H products, U1 (+7/+11), are indicated. (D) RT-PCR analysis of an in vitro splicing assay using
the IVS29 wild-type construct in the presence of 50 ng of U1AS oligonucleotide (+U1AS) or of a mock oligonucleotide (+mock).
Defective interaction between U1 snRNP and 5¢SS M. Raponi et al.
2066 FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS
the U1AS oligonucleotide had no effect on the splicing
efficiency of the exon 29 donor site with respect to the
addition of a mock oligonucleotide (Fig. 5D; compare
lanes 1 and 2). It should be noted that the ability of
U1AS addition to inhibit splicing efficiency was previ-
ously confirmed using other two intron/three exon
in vitro substrates (data not shown).
Rescue of U1 complementarity in the +4 and
+5 positions of IVS29 using mutant U1 snRNA
sequences
One potential criticism of these in vitro binding and
splicing experiments is that the RNA substrates used
in these assays lacked regions that are necessary to sta-
bilize U1 snRNP binding to the donor site. To investi-
gate this possibility, we determined whether rescue of
exon inclusion from the +5g>a mutant could be

obtained following in vivo complementation experi-
ments with mutant U1 snRNAs designed to function-
ally complement different mutated positions of this
5¢SS. In this respect, it should be noted that in other
NF1 settings, such as an NF1 exon 3 donor site carry-
ing a +5g>c disease-causing mutation, this strategy
was particularly efficient in rescuing donor site muta-
tions [17]. Therefore, three U1 snRNA mutants were
engineered to achieve this in the IVS29 donor site con-
text. The first was designed to complement specifically
the +5a position (U1+5A), the second was designed
to bind specifically at both the +4g and +5a
(U1+4G+5A) positions, and the third was designed
to bind with full complementarity to the +5g>a
intron 29 5¢SS (U1–2C+4G+5A+7U).
Complementation experiments were thus attempted
by cotransfecting the +5g>a IVS29 splice site with
U1+5A, U1+4G+5A and U1–2C+4G+5A+7U
mutant U1 snRNPs and the +4g>a+5g>a IVS29
mutant with just the U1+5A mutant U1 snRNP (see
Fig. 6A for a schematic diagram of the predicted
interactions between these mutant donor sites and the
wild-type and engineered U1 snRNA molecules). The
results in Fig. 6B show that U1+4G was incapable of
A
B
C
Fig. 6. U1 complementarity experiments. (A) Base-pairing between
IVS29 +5g>a, IVS29 +4g>a+5g>a and IVS29 )2c>g donor sites
and either U1+5A, U1+4G+5A, U1–2C+4G+5A+7U, or U1+4G. Ver-

tical lines indicate Watson–Crick base pairs, and circles indicate
wobble base pairs. (B) These U1 variants were cotransfected into
HeLa cells together with pTB NF1–29 minigenes carrying different
5¢SS changes, and the splicing pattern was analyzed by RT-PCR on
a 2% agarose gel with respect to inclusion (+) and exclusion ())of
this exon. The mean levels of exon 29 inclusion, together with
standard deviations from three different experiments, are shown
below. (C) Amplification of each mutant U1 cDNA, to check for
comparable and correct expression in the transfected cells. ()) indi-
cates cotransfection of the minigene with the U1empty vector.
Minus RT controls confirmed that plasmid DNA was not being
amplified (data not shown).
M. Raponi et al. Defective interaction between U1 snRNP and 5¢SS
FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS 2067
rescuing the IVS29 )2c>g minigene, and that both
U1+5A and U1+4G+5A were incapable of rescuing
the IVS29 +5g>a minigene, whereas U1–
2C+4G+5A+7U had a very weak effect. In addition,
U1+5A had only a weak positive effect with regard to
promoting exon 29 inclusion in the IVS29
+4g>a+5g>a minigene (Fig. 6B). These results indi-
cate a situation where, even in vivo, the expression of
U1 mutants carrying more extensive U1 snRNA com-
plementarity for the IVS29 donor site mutants than
U1–IVS29 donor site wild-type complementarity are
very inefficient at promoting donor site recognition. As
a control, correct and comparable expression levels of
the U1+5A, U1+4G+5A, U1)2C+4G+5A+7U
and U1+4G constructs were confirmed by amplifying
their cDNA with specific oligonucleotides following

their transfection into HeLa cells (Fig. 6C).
Discussion
In this study, we found that only a minority of the
mutations found in NF1 patients in NF1 exon 29 and
in its 5¢SS led to significantly altered levels of exon
skipping. This was a rather surprising observation, as
in silico analysis of the sequences disrupted by these
mutations was predicted to alter several putative splic-
ing regulatory sequences. However, other studies have
shown that analysis of polymorphic variations and
their putative effects on splicing can show a discrep-
ancy between splicing defect, protein binding, and ESE
predictions [40–43].
The only substitution that can be considered with
certainty to be an aberrant splicing mutation is an
IVS29 +5g>a intronic substitution, which led to
100% exon skipping. In this respect, it should be noted
that a missense mutation, c.5546g>a (R1849Q), in
NF1 exon 29 affecting the 5¢SS has also been described
previously [36,44].
One of the most surprising findings of this analysis
concerns the different splicing outcomes, strikingly
divergent from in silico predictions, observed for the
nucleotide substitutions in position )2 (Fig. 3). For
example, the )2c>g substitution, which generally has
no effect on exon skipping unless a guanosine is in the
)1 position and a weak 5¢ consensus is present, in our
case had a dramatic effect on splicing when inserted in
the wild-type pTB NF1–29 minigene. This may well be
because the )2 position is also affecting some other

context-specific element. This speculation is reinforced
by the results obtained when adenosine was placed in
the )2c position in the +5g>a pTB NF1–29 minigene;
splicing remained completely abolished, instead of hav-
ing a compensatory effect, as expected from the overall
in silico predictions. Indeed, our mutational data sug-
gest that each position in the 5¢SS of exon 29 is critical
for the splicing outcome. This result, and the low num-
ber of natural 5¢SSs with guanosine in position +4
and adenosine in position +5, strongly suggests the
existence of a mutual relationship between these two
positions, as previously suggested from a human–
mouse comparative genomic analysis by Carmel et al.
[45], and that positions +4 and +5 are less tolerant to
base combinations that differ from the consensus
sequence.
This situation could, however, be similar to a recent
example in NF1 involving the 5¢SS of exon 3. In this
case, an IVS3 +5g>c mutation caused aberrant splic-
ing and disease, whereas the same sequence in IVS1
and IVS7 of the NF1 gene did not affect splicing. Fur-
ther mechanistic studies of this region showed that
hnRNP H binding at the 5¢SS inversely correlated with
U1 snRNP binding to this site, and thus with the path-
ological effect of this intron 3 mutation [14]. It should
be noted, however, that the IVS29 donor site presents
a distinct difference with respect to this situation. In
fact, the IVS3 +5g>c mutation could be rescued suc-
cessfully by a U1 snRNP molecule carrying a compen-
satory mutation in position +5 [17], whereas, in our

case, cotransfection with a modified U1 designed
to complement specifically the +5g>a position was
incapable of rescuing the IVS29 +5g>a minigene
(Fig. 6B).
It is interesting to note that a recent analysis of
mutations occurring within the TCIRG1 gene also
showed lack of U1 rescue of a +5g>a mutation
occurring in the donor site of exon 14, but successful
recovery for a +4a>t mutation in for exon 2 [46]. In
addition, an early analysis in yeast showed the impor-
tance of the +5G residue for the correct identification
of the position of the 5¢SS cleavage, suggesting that
this is not related to base pairing with U1 but to the
sequence composition itself [47]. These results suggest
that, in a growing number of cases, U1 snRNP com-
plementarity may not necessarily represent the most
important step in the initial 5¢SS definition, although
one potential criticism of this view is the observation
that past studies have reported a rather mixed
response of mutant U1 snRNP carrying compensatory
substitutions for different mutations in the same 5¢SS
[48].
In fact, this lack of suppression by mutant
U1 snRNPs may simply be due to the fact that, for
some unknown reason, not all mutant U1 snRNPs are
equally effective at compensating for particular nucleo-
tide substitutions. However, it may also suggest that
other factors besides the interaction with U1 snRNP
Defective interaction between U1 snRNP and 5¢SS M. Raponi et al.
2068 FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS

may be more important in the definition of the IVS29
donor site. For example, it is well known that U6
interacts with the fifth nucleotide of the intron [49],
and it is well established that base pairing between U6
and the donor site can affect SS recognition [50,51].
An alternative explanation may involve the effect of
other cis-acting elements, as has been hypothesized to
be the case for other U1-independent model systems.
[52]. As a side note, it is important to note that for the
recognition of the other end of the exon, represented
by the 3¢SS sequence, it has long been known that the
absence of some key splicing factors, such as U2AF,
can be overcome by an excess of SC-35 protein [53].
Moreover, it has been recently reported that introns
with unconventional polypyrimidine tracts are unaf-
fected by U2AF inactivation, suggesting that, in some
of these cases, there may be additional mechanisms of
3¢SS identification other than that traditionally
described [54]. Taken together, these results suggest
that both 5¢SS and 3¢SS alternative splicing assembly
pathways may coexist within the eukaryotic cell, and
further work will be required to clarify this issue.
From a clinical point of view, the practical conse-
quence of our work resides in the conclusion that most
substitutions that occur within or near the NF1
exon 29 donor site may have profoundly adverse con-
sequences on its inclusion levels, even in cases where
they are not predicted to disrupt U1 snRNA inter-
actions.
Experimental procedures

GenBank access
The human NF1 exon\intron 29 sequence can be derived
from the genomic NF1 sequence (GenBank accession
number: AY796305).
Hybrid minigene splicing assay
To generate the hybrid minigene constructs, human geno-
mic DNA was amplified from normal and mutated exon 29
to generate fragments that contain the exon along with the
intronic flanking sequence, using the following oligonucleo-
tides: NF29-F, 5¢-ttcattcatatgaccatttgaatatacaatggt-3¢; and
NF29-R, 5¢-aagtaacatatgatggagaaaggacatatat-3¢. Both oli-
gonucleotides carry an Nde1 site in their 5¢-ends, and they
were used to clone the product into a modified version of
the a-globin-fibronectin extra type III homology B or extra
domain B (EDB) minigene, in which the alternatively
spliced EDB exon has been removed to generate a site for
the insertion of the genomic sequence under study [55]. For
analysis of the splicing pattern in this hybrid minigene
expression system, 1 lg of each minigene plasmid was
transfected into HeLa and COS cells with Invitrogen
lipofectamine reagent. RNA extraction and RT-PCR
analysis were performed as previously described [56], using
primers complementary to sequences in the flanking
fibronectin exonic sequence.
Mutant U1 snRNA cotransfection experiments
The parental U1 snRNA clone was pG3U1 (WT-U1), a
derivative of pHU1 [57]. We created the variants U1+4G,
U1+5A, U1+4G+5A and U1–2C+4G+5A+7U by site-
directed mutagenesis using pGEM Hind R (5¢-aagctatttagg
tgacactatagaa-3¢) as a reverse primer, and U1+4G_Bgl2F

(5¢-ccaagatctcatacctacctggcag-3¢), U1+5A_Bgl2F (5¢-ccaag
atctcatatttacctggcag-3¢), U1+4G+5A_Bgl2F (5¢-ccaagatct
catatctacctggcag-3¢), and U1-2C+4G+5A+7U_Bgl2F (5¢-
ccaagatctcaaatctaccgggcaggggaga-3¢), respectively, as the
forward primers. Primers carry the appropriate restriction
site in order to replace the sequence between the BglII and
HindIII sites with mutant clones. We transfected HeLa cells
with the lipofectamine reagent, with 1 lg of each minigene
plasmid, and with 0.8 lg of the U1 snRNA coding plas-
mids. The expression of the transfected U1 variant minig-
enes was tested by amplifying the cDNA with specific
oligonucleotides, using pGEM Q (5¢-atcgaaattaatacgactca
-3¢) as a forward primer and U1_QR (5¢-ctgggaaaac
caccttcgt-3¢) as the reverse primer.
RNase H digestion to inactivate U1 snRNA
Approximately 300 lg of commercial HeLa nuclear extract
(CilBiotech, Mons, Belgium, approximate concentration
15 lgÆlL
)1
) were digested with 5 units of RNase H (USB)
at 37 °C for 30 min, according to the manufacturer’s
instructions, in a 60 lL final reaction volume. In order
to inactivate U1 snRNP, a small oligonucleotide (5¢-ccagg
taagtat-3¢, U1AS oligonucleotide) was added to the reaction
mixture at a final concentration of 5 ngÆlL
)1
, and a mock-
depleted extract was prepared by adding a random oligo-
nucleotide. In order to assess the endogenous RNase H
inactivation of U1 snRNP, different concentrations of

U1AS oligonucleotide were added directly to 5 lL of HeLa
nuclear extract and incubated for 30 min at 37 °C. Total
RNA was then isolated by phenol/chloform extraction and
ethanol precipitation. The uncleaved and cleaved forms of
U1 snRNA were visualized by reverse primer extension,
using a protocol described by Mccullough & Berget [39].
The primer used for the reverse transcription reaction
corresponds to the U1 loop 2 region of U1 snRNA (5¢-cgg
agtgcaatg-3¢). Primer extension products were run on a 6%
polyacrylamide sequencing gel before being dried and
exposed overnight with X-Omat autoradiographic film
(Kodak).
M. Raponi et al. Defective interaction between U1 snRNP and 5¢SS
FEBS Journal 276 (2009) 2060–2073 ª 2009 The Authors Journal compilation ª 2009 FEBS 2069
EMSA and UV crosslinking analysis
EMSA reactions were performed in a final volume of 20 lL
(binding buffer: 20 mm Hepes, pH 7.9, 72 mm KCl, 1.5 mm
MgCl
2
, 0.78 mm magnesium acetate, 0.52 mm dithiothreitol,
3.8% glycerol, 0.75 mm ATP, and 1 mm GTP), by mixing
36 lg of HeLa nuclear extract with each labeled RNA
oligonucleotide (present at a final concentration of 6 nm).
Samples were incubated at room temperature for 15 min,
and were then loaded onto a 4% native acrylamide gel in
0.5· TBE that was run at a constant 150 V for 2 h at 4 °C.
The gel was then dried and exposed to Kodak XAR autora-
diographic film. In order to UV-crosslink U1 snRNA to the
RNA oligonucleotides, the reaction mixture used for the
band-shift analysis was subjected to irradiation at 254 nm

for 15 min, using a UV crosslinker (Euroclone, Milano,
Italy). The mixture was then digested using proteinase K
(Sigma, Milano, Italy) at a final concentration of 2 lgÆlL
)1
for 30 min at 37 °C before being loading onto the gel.
Site-directed mutagenesis
Mutations were introduced by the two-step PCR method
[58], using primers carrying each substitution, and the
flanking primers used were NF29-F and NF29-R
(sequences available upon request).
Bioinformatics predictions
The sequence environment of all acceptor sites was
analyzed using Splice Site Prediction by Neural Network,
nnsplice [59] (itfly.org/seq_tools/splice.html/).
Maximum entropy scores were obtained using software
based on the maximum entropy principle, maxentscan [60]
( />html/). The Shapiro and Senapathy scores [61] were
obtained using the software available at http://ast.
bioinfo.tau.ac.il/SpliceSiteFrame.htm. The hydrogen bond
scores were calculated at />rna/html/hbond_score.php [22]. Comparative genomic anal-
ysis was undertaken with the help of the UCSC genome
browser ( [62]. Prediction of the
presence of a splicing motif sequence was performed using
the web-based splicing sequences finder software program
( [63,64].
In vitro splicing assays
The NF1 IVS29-based in vitro splicing system was prepared
by amplifying part of this exon/intron sequence to join it to
the tropomyosin-based sequences of the PY7 plasmid [65].
The sense primer used to achieve this contained a T7 pro-

moter at the 5¢-end, in order to allow capped RNA synthe-
sis from the amplified products and either the wild-type
exon 29 donor sequence or those carrying the +4A and
+4A+5A mutations. The in vitro splicing assay conditions
used in these experiments have been extensively described
elsewhere [66]. U1 snRNP inactivation was achieved by
adding 50 ng of U1AS antisense oligonucleotide (5¢-
ccaggtaagtat-3¢) to the 20 lL splicing mixture. Following
RNA extraction, the spliced/unspliced mRNA products
were amplified by RT-PCR, using a sense primer that
contained the exon sequence of either NF1 exon 29 or the
b-globin sequence and a common antisense primer that was
localized on the tropomyosin exon 3 sequence. For this rea-
son, no splicing intermediates could be detected. RT-PCR
was performed in conditions that gave a linear relationship
between the input RNA and the PCR products over an
extended range of PCR conditions (from four-fold less
to four-fold more than the amounts used for the published
figures).
Acknowledgements
We wish to thank the families and physicians who
participated in this study, Professor Charles ffrench-
Constant for support and advice, Professor F. E.
Baralle for his support and for helpful discussions, and
P. Zago for her help in the minigene transfection
assays. D. Baralle and M. Raponi are supported by
grant funding from Action Medical Research and
EURASNET. E. Buratti is supported by the Telethon
Onlus Foundation (GGP06147) and by EC grant
EURASNET-LSHG-CT-2005-518238.

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