Báo cáo khoa học: Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1 pdf

Polypyrimidine tract binding protein regulates alternative
splicing of an aberrant pseudoexon in NF1
Michela Raponi
1
, Emanuele Buratti
2
, Miriam Llorian
3
, Cristiana Stuani
2
, Christopher W. J. Smith
3
and Diana Baralle
1
1 Human Genetics Division, University of Southampton, UK
2 Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
3 Department of Biochemistry, University of Cambridge, UK
Pseudoexons are intronic sequences that are approxi-
mately the same length as exons (200 bp) with appar-
ently viable donor and acceptor splice sites but which
are not normally spliced in the mature mRNA tran-
script. Despite the known abundance of exon splicing
silencer regulatory elements within introns [1], it is not
possible to formulate general rules for pseudoexon
repression without remembering that splicing is very
much dependent upon local context [2,3]. Normal
exons need to encode the information for both protein
synthesis and RNA splicing, so one might expect dif-
ferences in pseudoexons with regard to several fac-
tors, including the distribution of splice consensus
sequences, splicing enhancers, splicing silencers and

secondary structures. In line with these considerations,
bioinformatics studies have observed enrichment of
exon splicing silencer elements within pseudoexons
relative to exonic splicing enhancer elements [4,5]. Fur-
thermore, it has been suggested that it is defective
splice sites rather than the lack of enhancers that
account for non-splicing of pseudoexon sequences,
although even perfect consensus sequences are not
always adequate for correct splicing [6]. To complicate
matters further, it has been recently suggested that
some pseudoexons are authentic exons whose inclusion
leads to efficient nonsense-mediated decay, such as the
pseudoexon located downstream of mutually exclusive
exons 2 and 3 of the rat a-tropomyosin gene, which
acts both as an alternative exon that leads to non-
sense-mediated decay and as a zero-length exon [7].
Nonetheless, in a clinical setting, pathological
pseudoexon sequences have been identified by simple
activation of cryptic splice sites which reinforce their
strength, de novo creation, the inactivation ⁄ activation
Keywords
intron; NF1; pseudoexon; PTB ⁄ nPTB;
splicing
Correspondence
D. Baralle, Human Genetics Division,
University of Southampton, Duthie Building
(Mailpoint 808), Southampton General
Hospital, Tremona Road, Southampton
SO16 6YD, UK
Fax: +44 2380794346

Tel: +44 2380796162
E-mail:
(Received 30 July 2008, revised
23 September 2008, accepted
9 October 2008)
doi:10.1111/j.1742-4658.2008.06734.x
In disease-associated genes, understanding the functional significance of
deep intronic nucleotide variants represents a difficult challenge. We previ-
ously reported that an NF1 intron 30 exonization event is triggered from a
single correct nomenclature is ‘c.293-279 A>G’ mutation [Raponi M,
Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294–295]. In this paper,
we investigate which characteristics play a role in regulating inclusion of
the aberrant pseudoexon. Our investigation shows that pseudoexon inclu-
sion levels are strongly downregulated by polypyrimidine tract binding pro-
tein and its homologue neuronal polypyrimidine tract binding protein. In
particular, we provide evidence that the functional effect of polypyrimidine
tract binding protein is proportional to its concentration, and map the cis-
acting elements that are principally responsible for this negative regulation.
These results highlight the importance of evaluating local sequence context
for diagnostic purposes, and the utility of developing therapies to turn off
activated pseudoexons.
Abbreviations
NF1, neurofibromatosis type 1; nPTB, neuronal polypyrimidine tract binding protein; PTB, polypyrimidine tract binding protein.
FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6101
of splicing regulatory elements, or manipulation of
RNA structures [8–14]. Buratti et al. [2] provide an
exhaustive table of pathological pseudoexon inclusion
events.
We previously reported a new intronic mutation
c.31-279A>G in neurofibromatosis type 1 (NF1)

intron 30 that results in creation of a new 3¢ splice site
and activation of a cryptic 5¢ splice site, leading to par-
tial inclusion of a pseudoexon [15]. The mutation
caused aberrant splicing from the mutated allele with
approximately 45% normal transcripts and 55%
containing the cryptic exon. Intriguingly, the same
c.31-279A>G variation has been found in the canine
NF1 gene sequence, where the strength of the potential
3¢ splice site was estimated to be as high as the
strength of the human pseudoexon 3¢ splice site. How-
ever, no canine expressed sequence tag sequences have
been reported that utilize this site. These data suggest
that the sequence of the pseudoexon itself and the
surrounding trans-acting factors may play a role in its
definition. In this work, we have investigated which
local context characteristics play a role in regulating
exonization of this sequence.
Results
Deletion analysis of pseudoexon sequences
In order to identify the splicing regulatory elements
responsible for pseudoexon exonization, we performed
a deletion analysis using a minigene carrying the
)279A>G mutation. The del_a, del_b and del_c
deletions are highlighted in Fig. 1A. The hybrid minig-
enes carrying each deletion were transiently transfected
into HeLa cells and the splicing outcome analysed
(Fig. 1B). The results showed that the pseudoexon was
only included in the minigene with the large central
deletion, del_b. Transfection of hybrid minigenes with
the del_a or del_c deletions resulted in complete

pseudoexon exclusion.
Pulldown analysis of deletion mutants del_a
and del_c
Affinity purification analysis was then performed in
order to define putative trans-acting regulatory factors
that might be lost ⁄ gained with the del_a and del_c
deletions. Figure 1C shows the general protein binding
profiles of wild-type (WT), del_a and del_c RNAs
bound to adipic acid dehydrazide beads and incubated
with total HeLa nuclear extract. Coomassie staining
of the SDS–PAGE gel showed a very prominent
change in the binding profile of protein bands migra-
ting in the 60 kDa range (boxed area, Fig. 1C). In
particular, the intensity of these bands was greatly
increased in the del_a mutant but absent in del_c.
Direct sequencing of these bands using MS identified
them as polypyrimidine tract binding protein (PTB)
hnRNP I. Analysis of the WT lane band yielded only
the PTB protein (accession number AAH04383). On
the other hand, the del_a mutant yielded three
sequences: NP_114368 corresponding to PTB iso-
form c, NP_114367 corresponding to PTB isoform b,
and AAP35465 corresponding to PTB itself. No other
clear differences in Coomassie intensities were seen
between the profile binding patterns of WT, del_a and
del_c RNAs in independent experiments (data not
shown). In order to confirm this observation and to
extend this analysis to specific splicing factors, western
blot assays were with an array of specific antibodies.
Figure 1D shows that the western blot experiments

confirmed the variation in PTB binding ability to the
WT, del_a and del_c RNAs. As in the Coomassie gel,
PTB binding was increased for the del_a mutant,
suggesting that its binding might be responsible for the
increased exon skipping in the del_a mutant. In con-
trast, PTB showed reduced binding to del_c. Binding of
other well-known splicing factors, such as SRp55,
ASF ⁄ SF2, SC35 and hnRNP A1, A2 and C showed no
reproducible variations between the RNAs, even though
dedicated bioinformatics tools such as ESEfinder
( and Splicing Rainbow
( had
suggested their possible involvement. The only excep-
tion was hnRNP H, which appeared to bind in the
del_a mutant but not to the WT or del_c RNAs. The
observation that this protein did not binding to the WT
RNA (unlike PTB) suggested that it does not represent
a natural regulator of pseudoexon inclusion under
normal conditions. We therefore decided to concentrate
on characterizing the functional effects of PTB alone.
Functional effects of PTB/nPTB knockdown on
pseudoexon inclusion
RNA interference experiments were undertaken in
order to determine whether PTB acts as a negative reg-
ulator of pseudoexon splicing. As nPTB, the neuronal
homologue of PTB, has been demonstrated to func-
tionally compensate for PTB [16] and is upregulated
when PTB is removed, we decided to investigate the
effect of PTB and PTB + nPTB knockdown on
pseudoexon inclusion. In this experiment, the WT

minigene (pNF1, c.31-279A>G) was cotransfected into
HeLa cells with siRNA against PTB and nPTB. As
shown in Fig. 2A, PTB plays an important role as a
PTB pseudoexon repression M. Raponi et al.
6102 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS
negative splicing regulator for the WT pseudoexon
construct. WT pseudoexon inclusion levels increased
moderately when just PTB was knocked down, but
double knockdown of both PTB and nPTB caused a
prominent increase in pseudoexon inclusion levels
(from 41% to 81%).
To confirm that PTB acts as a negative regulator of
pseudoexon splicing, we then undertook cotransfection
experiments of pNF1 c.31-279A>G with increasing
amounts of a PTB expression minigene (Fig. 2B). As
expected, PTB overexpression antagonized pseudoexon
inclusion in this case and in also contexts where either
PTB or nPTB were previously knocked down by siRNA
treatment (Fig. 2C). Interestingly, in all cases, high con-
centrations of PTB expression vector almost completely
abolished the levels of pseudoexon inclusion, despite the
presence of the c.31-279A>G mutation. The same result
was also obtained by overexpressing nPTB*, a codon-
optimized nPTB (data not shown).
Splicing of the del_a and del_c mutants was un-
affected by PTB ⁄ nPTB knockdown. In both cases,
complete exon skipping was maintained upon PTB
depletion, indicating that variations in PTB binding
are not the main reason for the skipping observed with
del_a and del_c, despite the strong increase in PTB

binding to del_a (Fig. 1C,D). This observation sug-
gests the presence of additional enhancer regulatory
sequences or proteins that could not be detected using
our affinity purification methods. Indeed, the presence
of additional enhancer ⁄ silencer elements acting on
pseudoexon inclusion in these regions is also evident
from the results of single-nucleotide mutagenesis
AB
CD
Fig. 1. Identification of pseudoexon splicing regulatory elements. (A) Pseudoexon sequence in upper case showing the del_a, del_b and
del_c deletions (highlighted and underlined). Intronic sequences are shown in lower case. The acceptor and donor site sequences of the
pseudoexon are in bold and underlined. (B) Transient transfection results for the hybrid minigenes carrying deletions. The + and ) signs
indicate pseudoexon inclusion and exclusion, respectively. (C) nuclear extract protein binding profile of WT, del_a and del_c RNAs following
Coomassie staining. The boxed area shows the protein bands that display the greatest change in binding profile. (D) Western blot probed for
PTB, hnRNP C, hnRNP A1 ⁄ A2, hnRNP DAZAP, hnRNP H, ASF2 ⁄ SF2, SRp55 and SC35.
M. Raponi et al. PTB pseudoexon repression
FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6103
analysis based on human ⁄ dog intronic sequence com-
parisons and ESEfinder in silico predictions. All dog ⁄
human and putative exonic splicing enhancer-inactivat-
ing substitutions are capable of completely repressing
pseudoexon inclusion (M. Raponi & D. Baralle,
unpublished observations). Studies are now being
performed to better characterize these additional
elements and their cognate binding factors.
Finally, it is interesting to note that PTB ⁄ nPTB
knockdown had no effect on the pseudoexon sequence
lacking the )279A>G mutation (Fig. 2E). This result
suggests that PTB binding sites could be maintained in
this pseudoexon sequence as a preventive measure

against 3¢ splice site-activating mutations.
Dissection of the PTB recognition elements
The preferred RNA binding sites of PTB ⁄ nPTB are
UCUU or CUCUCU in pyrimidine-rich contexts
[17,18], and we therefore focused on the role played by
such elements in the pseudoexon inclusion process.
Three UCUU motifs were identified in the pseudoexon
body itself and two were identified downstream of the
pseudoexon cryptic 5¢ splice site (Fig. 3A, referred to
as m1–m5), but no likely motifs for PTB binding were
observed upstream of the pseudoexon (a common
occurrence in several PTB-regulated exons). All these
sites were modified by site-directed mutagenesis in
order to inactivate putative PTB binding to each of
B
A C
D
E
Fig. 2. PTB and nPTB regulate pseudoexon definition. (A) Transient transfection results for the WT minigene in the presence of siRNAs
against PTB and nPTB. ‘Cont’, negative control siRNA; P1, siRNA against PTB; N1, siRNA against nPTB. The + and ) signs indicate pseudo-
exon inclusion and exclusion, respectively. The top panel shows the effect of PTB and PTB ⁄ nPTB knockdown on pseudoexon inclusion. The
middle and bottom panels show western blots probed for PTB, nPTB and ERK. The two bands observed in the PTB western blot corre-
spond to the PTB-1 (lower) and PTB-4 (upper) isoforms. (B) Effect of PTB overexpression on pseudoexon inclusion. From left to right,
increasing amounts (10, 100, 250 and 750 ng) of expression plasmid for PTB1 were cotransfected with the WT plasmid. Percentages of
pseudoexon inclusion are reported below the gel. (C) PTB overexpression can overcome the PTB ⁄ nPTB knockdown effect on pseudoexon
inclusion in the WT plasmid. (D) Knockdown of PTB and PTB ⁄ nPTB does not affect splicing in the artificial mutants del_a and del_c.
(E) Knockdown of PTB ⁄ nPTB does not affect splicing in the pseudoexon sequence lacking the )279a>g activating mutation (WT-279a).
PTB pseudoexon repression M. Raponi et al.
6104 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS
them, and the effects of the mutations were then tested

by transient transfection.
As shown in Fig. 3B, the m1 and m5 mutations had
negligible effects upon pseudoexon splicing. However,
the pseudoexon inclusion efficiency (48%) was moder-
ately increased to 62% and 72%, respectively, follow-
ing introduction of the m2 and m3 mutations.
Strikingly, mutating the UCUU motif m4 immediately
downstream of the pseudoexon 5¢ splice site induced
almost complete pseudoexon exclusion. However,
given that the m4 motif is immediately adjacent to the
5¢ splice site, it is very likely that this mutation is
involved in recognition of this sequence either directly
or through interaction with a positive trans-acting
factor.
Taken together, these results suggest that the m2
and m3 are the sequences that are principally responsi-
ble for mediating the repressive activity of PTB.
Consistent with this, pulldown and western blot anal-
ysis showed decreased PTB binding to pseudoexon
sequences carrying mutations m2 and ⁄ or m3 compared
with the WT pseudoexon sequence [Fig. 3C, normal-
ized using the uniformly binding deleted in Azoosper-
mia associated protein (DAZAP) protein]. Moreoever,
although PTB overexpression in HeLa cells induced a
fourfold decrease in WT pseudoexon inclusion, this
effect was reduced to threefold for the individual m2
and m3 mutants (Fig. 3D) and to 1.7-fold for the
double mutant m2 + m3. In conclusion, these results
suggest that the effects of PTB on pseudoexon
exclusion are mediated by the two central UCUUCUU

(m2) and UCUU (m3) sequences.
Discussion
Introns frequently embed potential exonic sequences
[19], and their activation through the creation or acti-
vation of a cryptic splice site is a common cause of
genetic disease. We recently reported the example of a
deep intronic mutation c.31-279A>G in the NF1 gene
of a patient with a severe form of neurofibromatosis
type 1, in whom this mutation was associated with a 3¢
A
BD
C
Fig. 3. Role played by UCUU-type motifs in pseudoexon splicing. (A) Pseudoexon (upper case) and partial downstream intron (lower case)
nucleotide composition. The acceptor and donor site sequences of the pseudoexon are in bold and underlined. m1–m5 indicate the nucleo-
tide substitutions analysed. (B) RT-PCR products from transfection experiments using minigenes carrying each substitution. The + and )
signs indicate pseudoexon inclusion and exclusion, respectively. The percentages of pseudoexon inclusion are also shown. The nucleotide
substitutions analysed are indicated above each lane and labelled m1–m5. (C) Western blot probed for PTB and DAZAP. Comparison
between the PTB binding capacity of the WT pseudoexon and mutant pseudoexons m2, m3 and m2 + m3. (D) Effect of PTB overexpression
on mutants with various m2 and m3 combinations; 750 ng of expression plasmid for PTB1 was cotransfected in each case.
M. Raponi et al. PTB pseudoexon repression
FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6105
splice site activation event [15]. In this work, we have
focused on identifying the internal pseudoexon
sequences and trans-acting factors capable of affecting
the levels of inclusion of the pseudoexon. The fact that
such sequences ⁄ factors are likely to exist is based on a
previous comparison [15] with the dog genome, where
the presence of a naturally occurring )279A>G substi-
tution does not lead to any exonization event.
In line with this hypothesis, we provide experimental

evidence that altering the human pseudoexon sequence
can heavily affect its recognition. In particular, our
findings demonstrate that PTB and nPTB are major
repressors of pseudoexon splicing, with a role in regu-
lating inclusion of the pseudoexon. These functional
effects are in line with the view that PTB ⁄ nPTB might
act as general repressors of weak exons, including
pseudoexons [20], although PTB may also act as a
positive splicing regulator [21]. We were able to detect
increased or decreased binding of PTB to the pseudo-
exon when the 5¢ (del_a) or 3¢ (del_c) thirds of the
pseudoexon, respectively, were deleted. In line with
this, we show that mutation of putative PTB binding
motif m1 (which is deleted in del_a) had no influence
on pseudoexon inclusion rates but mutation of m3
(which is deleted in del_c) had a strong effect on
pseudoexon exclusion.
The two deletions leading to strong repression of
pseudoexon inclusion may have also damaged splice
site recognition as well as altered binding sites for
exonic splicing enhancer regulatory elements. In parti-
cular, changes in secondary structure could explain
why PTB binds more strongly to the deletion mutant
del_a. Furthermore, involvement of positive regulatory
elements is suggested by the strong repression of
pseudoexon inclusion in mutant m4. This effect could
be due to disruption of a T-cell intracellular antigen 1
(TIA-1) binding site immediately downstream of the 5¢
splice site, as this splicing factor has been shown to
bind at pyrimidine tracts, competing with PTB [22]. In

general, however, PTB cannot be considered the sole
determinant of pseudoexon splicing, which is evidently
controlled by more complex processes that are
currently under investigation. This complexity may well
be important in explaining the severe phenotype
observed in the patient, where the requirement and
balance of antagonistic splicing factors involved in
pseudoexon definition defines the degree of aberrant
NF1 intron 30 exonization in various tissues.
The functional effect of PTB on intervening
sequence 30 (IVS30) pseudoexon inclusion appears to
be mediated by cooperative binding sites within the
pseudoexon. In particular, the central m2 and m3
elements have the strongest effect on pseudoexon
inclusion and can function as an exon splicing silencer.
The requirement for multiple PTB binding sites for
optimal repression has already been highlighted in
other model pre-mRNAs. Recent research on other
genes such as c-src and Fas has shown that PTB can
repress exon inclusion by interfering with several steps
of intron ⁄ exon definition (reviewed in [23]).
Most importantly, we have established the physio-
logical importance of these results by using an
expression vector to increase PTB and nPTB protein
levels in living cells, and shown that this results in
efficient repression of pseudoexon aberrant splicing
both with and without PTB ⁄ nPTB knockdown. Our
observation that different PTB ⁄ nPTB expression levels
can successfully alter pseudoexon inclusion suggests
that quantitative differences in PTB ⁄ nPTB expression

may be responsible for cell-type-specific restrictions
(upregulation) in pseudoexon splicing. This has
considerable importance when considering potential
methods for the control of aberrant splicing (see
below). Indeed, PTB ⁄ nPTB expression levels in differ-
ent tissues may be the cause of the patient’s particu-
larly severe spinal NF1 phenotype described
previously [15]. It is important to note that the effect
of nPTB in regulating aberrant pseudoexon exclusion
in neurons may be weak due to translational repres-
sion. It has been recently shown that nPTB is
expressed in vivo at a lower level than PTB or codon-
optimized nPTB* [24].
Finally, our findings open the way to development
of novel therapeutic strategies aimed at rescuing splic-
ing inhibition in patient cells. In general, pseudoexon
inclusion in pathological situations can be targeted
through use of antisense oligonucleotides or modified
U7 snRNA molecules against the cryptic 5¢ and 3¢
splice sites, as recently described for PCCA, PCCB,
PTCH1 and BRCA1 [25,26]. Although these strategies
may represent viable therapeutic approaches for
repression of the NF1 pseudoexon, the results pre-
sented in this work have expanded the list of poten-
tial options. The use of bifunctional oligonucleotides
{targeted oligonucleotide enhancer of splicing (TOES)/
targeted oligonucleotide silencing of splicing (TOSS)
methodology reviewed by Garcia-Blanco et al. [27]}
that carry a binding domain and an effector domain
with binding sites for known splicing factors has been

recently described for the successful splicing recovery
of spinal muscular atrophy disease gene (SMN)
exon 7. In our case, we hypothesize that the use of
such reagents will increase recruitment of additional
PTB molecules and in this way achieve downregula-
tion of pseudoexon inclusion. This type of strategy
would also have the advantage that successful skip-
PTB pseudoexon repression M. Raponi et al.
6106 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS
ping of the pseudoexon would remove the bifunctional
oligonucleotide from the rescued mRNA and would
not eventually interfere with subsequent steps of the
mRNA life cycle such as transport to the cytoplasm
and ⁄ or its translation.
Experimental procedures
Site-directed mutagenesis and deletion
Site-directed mutagenesis was performed by the overlap
extension method [28] using previously described
pNF1c.31-279A>G as a template and NF31-F and
NF31-R as flanking primers [15]. Deletions were introduced
using the same method with overlapping primers designed
according to the portion to be deleted as follows: DELa
reverse 5¢-TCCTCCACTATAAAAGGAAATG-3¢ and
DELa forward 5¢-TTATAGTGGAGGAAAATAAGAC-3¢;
DELb reverse 5¢-AACAGTCCATTTTAGTCCTT-3¢ and
DELb forward 5¢-AAAATGGACTGTTCTTTCTT-3¢;
DELc reverse 5¢-TACCTAGAAGAAAGAACAGT-3¢ and
DELc forward 5¢-TCTTTCTTCTAGGTAATAGT-3¢.
Transient transfection assay and pre-mRNA
splicing analysis

HeLa cells were grown in Dulbecco’s modified Eagle’s
medium (supplemented with 10% fetal calf serum,
450 mgÆL
)1
glucose, 110 mgÆL
)1
sodium pyruvate, 2 mm
l-glutamine and 50 mgÆmL
)1
penicillin ⁄ streptomycin) on
35 mm plates. Each minigene plasmid (0.8 lg) was trans-
fected into 3 · 10
5
Hela cells in serum-free medium with
8 mL Lipofectamine reagent (Invitrogen, Carlsbad, CA,
USA). Cells were grown overnight, washed with NaCl ⁄ P
i
,
and fresh medium with 10% fetal calf serum was added.
Cells were grown for an additional 24 h followed by
RNA extraction. siRNA transfection of HeLa cells was
carried out using 10 pmol of P1 and N1 siRNA accord-
ing to a 7-day, two-hit protocol as described previously
[16,20], where the target genes for knockdown were PTB
and nPTB, respectively. For the add-back experiments,
increasing amounts of PTB1 expression plasmid (10, 100,
250 and 750 ng) were cotransfected on the 5th day of the
knockdown protocol together with 250 ng of reporter
plasmid using 4 lL of Lipofectamine (Invitrogen). Total
RNA was extracted using an RNeasy mini kit (Qiagen,

Valencia, CA, USA) according to the manufacturer’s
instructions. Total RNA (3 lg) was reverse-transcribed
using random hexamer primers, and cDNA was then
amplified by PCR in a total volume of 50 lL using prim-
ers specifically designed to amplify processed transcripts
derived from the minigene. Each transfection experiment
was perfomed at least three times, and representative gels
are shown in each case.
Pulldown assay
Pulldown assays were performed essentially as described
previously [29]. Briefly, 500 pmol of the target RNA
(approximately 15 lg of a 100-mer RNA) were placed in a
400 lL reaction mixture containing 100 mm NaOAC pH
5.0 and 5 mm sodium m-periodate (Sigma, St Louis, MO,
USA), incubated for 1 h in the dark at room temperature,
ethanol-precipitated, and resuspended in 100 lL of 0.1 m
NaOAC, pH 5.0. To this RNA, 300 lL of an adipic acid
dehydrazide agarose bead 50% slurry (Sigma) equilibrated
in 100 mm NaOAC pH 5.0 were added, and the mix was
incubated for 12 h at 4 °C on a rotator. The beads with the
bound RNA were then pelleted, washed (5 min) three times
with 1 mL of 2 m NaCl, and equilibrated in washing buffer
(5 mm HEPES pH 7.9, 1 mm MgCl
2
, 0.8 mm magnesium
acetate). They were then incubated on a rotator with
approximately 1 mg of HeLa cell nuclear extract for 30 min
at room temperature in 1 mL final volume. Heparin was
added to a final concentration of 5 mgÆmL
)1

. The beads
were then pelleted by centrifugation at 3000 g for 3 min
and washed for 5 min, four times with 1.5 mL of washing
buffer, before addition of SDS sample buffer and loading
onto a 10% SDS–PAGE gel.
Western blot
Pulldown samples were electroblotted onto a Hybond-C
Extra membrane (Amersham, Chalfont St Giles, UK), and
antibody recognition was then performed using several
in-house antibodies against PTB, hnRNP A1 ⁄ A2 ⁄ C and H
proteins and commercial antibodies against ASF ⁄ SF2
(Zymed, Carlsbad, CA, USA), SC35 (Sigma) and SRp55
(1H4 antibody, Zymed). Protein bands were detected using
an enhanced chemiluminescence kit (Pierce, Rockford, IL,
USA) according to the manufacturer’s instructions.
Acknowledgements
E.B. and C.S. are supported by the Telethon Onlus
Foundation (GGP06147), Fondo per l’Investimento
sulla Ricerca di Base (FIRB) (RBNE01W9PM), and by
EC grant EURASNET-LSHG-CT-2005-518238. R.M.
and B.D. are supported by Action Medical Research
(grant SP4175) and EURASNET. C.W.J.S. and M.L.
are supported by a programme grant from the Well-
come Trust (077877) and EURASNET.
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