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, t
Trang 1splicing of an aberrant pseudoexon in NF1
Michela Raponi1, Emanuele Buratti2, Miriam Llorian3, Cristiana Stuani2, Christopher W J Smith3 and Diana Baralle1
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: D.Baralle@soton.ac.uk
(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 propro-tein 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.
Trang 2and 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-the omigra-ther hand, migra-the del_a mutant yielded three sequences: NP_114368 corresponding to PTB iso-form c, NP_114367 corresponding to PTB isoiso-form 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 (http://rulai.cshl.edu/tools/ESE/) and Splicing Rainbow (http://www.ebi.ac.uk/asd-srv/wb.cgi?method=8) 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
Trang 3negative 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
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.
Trang 4analysis 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 repressenhancer-inactivat-ing
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
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).
Trang 5them, 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
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.
Trang 6tution 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
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 patidiffer-ent’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
Trang 7skip-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
DELa forward 5¢-TTATAGTGGAGGAAAATAAGAC-3¢;
DELb reverse 5¢-AACAGTCCATTTTAGTCCTT-3¢ and
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· 105 Hela cells in serum-free medium with
8 mL Lipofectamine reagent (Invitrogen, Carlsbad, CA,
USA) Cells were grown overnight, washed with NaCl⁄ Pi,
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 4C 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 MgCl2, 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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