From the International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste 34012, Italy
Received for publication, December 17, 2002 , and in revised form, April 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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Skipping of constitutive exons may also occur for missense and silent
mutations. In this case, disruption of exonic splicing enhancer or the
creation of exonic splicing silencer has been considered an alternative
mechanism to explain the exon skipping phenotype
(2,
10,
14,
15). Some exonic splicing
enhancers in pre-mRNAs interact with serine/arginine-rich proteins (SR
proteins)1
(16). SR proteins are
essential splicing factors required for both constitutive and alternative
splicing, and changes in their relative concentrations with respect to
antagonistic splicing factors have been found to affect splice site selection
(17). Functional systematic
evolution of ligands by exponential enrichment (SELEX) strategies have
identified highly degenerate consensus sequences binding to SR proteins. These
sites are more represented in exons than in introns
(18,
19). In the BRCA1 and SMN
genes, changes in SR protein score motifs derived from these SELEX experiments
at exonic splicing enhancer sites have been shown to correlate with the
efficiency of splicing (15,
20). This has led to the
suggestion that SR protein score motif analysis might represent a useful tool
to identify general controlling elements of splicing efficiency. On the other
hand, exonic splicing silencer elements have always been classically separated
from enhancer sequences and may interact with negative regulators, which often
belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. In
particular, binding of hnRNPH at G-rich sequences has been recently found to
exert a strong inhibitory splicing effect in the rat -tropomyosin gene,
in the Rous sarcoma virus, and in human immunodeficiency virus-1
(2124).
The CFTR exon 9 alternative splicing represents an interesting model to evaluate the contribution of exonic sequences in normal and pathologic pre-mRNA processing. Exon skipping produces a nonfunctional CFTR protein, and alternative splicing of this exon has been associated with monosymptomatic forms of cystic fibrosis (CF). The cis-acting elements so far identified include the polymorphic region at the 3'-end of intron 8 and the intronic splicing silencer in intron 9. The polymorphic locus contains a variable number of dinucleotide TG (from 9 to 13) followed by a T repeat (T5, T7, or T9), and a high number of TG repeats and a low number of T tract induce exon skipping (2527). TDP43 binding to the UG polymorphic repeat reduces the proper recognition of the nearby 3' splice site and in association with the intronic splicing silencer element in intron 9 mediates exon skipping (27).
In this paper, we have analyzed the role of CFTR exon 9 sequences, taking advantage of several natural missense and nonsense mutations and extending them by extensive site-directed mutagenesis. Our results indicate that CFTR exon 9 splicing is extremely sensitive to small variations in its exonic sequence, suggesting that the entire sequence of the exon is important for exon recognition and processing, which occur independently from the maintenance of an open reading frame within the mRNA. We also identify at the 3' portion of the exon a composite regulatory element with juxtaposed enhancer and silencer properties.
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EXPERIMENTAL PROCEDURES |
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RNA Transcription, UV-cross-linking Assay, and Cross-linking of RNA to Adipic Dehydrazide-Agarose BeadsTo generate the WT and mutant RNAs, the different CFTR exon 9 hybrid minigenes were first amplified with the direct primer 5'-tacgtaatacgactcactataggatattaatttcaagatagaaagag-3', which contains a T7 polymerase sequence and the reverse primer 5'-ctaccttgcctgctccagtg-3' followed by in vitro T7 RNA polymerase transcription. The amplified region corresponds to the last 64 bp of CFTR exon 9. Transcription of labeled RNAs, nuclear extract preparation, and UV-cross-linking assay were performed as previously described (26, 27). Cold substrate RNAs for bead immobilization were synthesized by in vitro transcription using T7 RNA polymerase, and cross-linking of RNA to adipic dehydrazide-agarose beads was done essentially as previously described with the addition of heparin to a final concentration of 5 µg/µl (26, 27). Proteins were separated on a 10% SDS-PAGE gel and visualized by Coomassie Brilliant Blue staining or electroblotted onto a nitrocellulose membrane and probed with a rabbit polyclonal anti-hnRNPH antiserum. Immunoblottings were detected using the ECL chemiluminescence kit (Pierce).
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RESULTS |
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To evaluate the effect of exonic substitutions in relation to the strength of the 3' splice site, we took advantage of the presence of the polymorphic TG and T repeats, which have been shown to modulate the efficiency of the exon recognition. The splicing efficiency of the missense and nonsense variations was analyzed not only in the TG11T5 construct but also in the TG11T9 and TG13T5 minigenes, two variants that cause a respective increase and decrease in the splicing efficiency. Each mutation showed a percentage of exon 9 inclusion to be higher in the presence of the TG11T9, intermediate in the TG11T5, and lower in the TG13T5 context (Fig. 1C). This indicates that both the splicing controlling elements affected by the missense and nonsense mutations along with the 3' splice site definition independently contribute to the overall efficiency of CFTR exon 9 splicing.
Reading Frame Contribution to CFTR Exon 9 Alternative SplicingNonsense mutations have been found in some cases to induce abnormal skipping of the exon. We have evaluated the possibility that some of the changes in the splicing pattern induced by the natural substitutions in exon 9, particularly the Q414X, might be related to nonsense mediated altered splicing. The two mechanisms suggested to be involved in the skipping of exons carrying stop codons are a putative nuclear open reading frame scanning process (31) and the contribution of exonic splicing regulatory elements (15). We evaluated the role of these two mechanisms in the CFTR exon 9 using our minigene system that provides a particular advantage, since alternative splicing produces one mRNA always in an open reading frame (the CFTR exon 9 form) and one mRNA that can contain stop codons according to the reading frame and to the mutation introduced (CFTR exon 9+ form). In the construct containing the normal exon 9 (hCF), the processed CFTR exon 9 mRNA is in frame; however, the CFTR exon 9+ mRNA contains numerous stop codons (Fig. 2A). This is due to the nonnatural context of the minigene, where the presence of globin-fibronectin exons results in a one-nucleotide shift of the reading frame. Transfection of this minigene showed about 65% of exon inclusion. To restore an open reading frame in the minigene that results in the elimination of the multiple stop codons, we prepared the F1 and F2 constructs (Fig. 2A). The open reading frame in both of the mRNAs produced by these minigenes was restored by a base deletion at the 5'-end (F1 contains the T16 deletion, and F2 contains an A23 deletion along with an A20G substitution) and a G insertion at the 3'-end (G164+) (for details, see "Experimental Procedures"). Cotransfection of these two constructs produces about 80% of exon inclusion (Fig. 2D, lanes F1 and F2, respectively). To exclude the possibility that this increase in exon recognition may be due to differential mRNA stability of one of the two alternatively spliced mRNAs (with or without exon 9), the cells were treated with cycloheximide or actinomycin D. Cycloheximide, impairing the efficiency of translation, has been shown to inhibit nonsense-mediated decay (3234). Analysis of the hCF, F1, and F2 constructs did not show any difference on the relative abundance of the two mRNA variants when treated with the translation inhibitor or actinomycin D (Fig. 2, B and C). This indicates that differential stability of the mRNAs in and out of frame cannot explain the different proportion of plus and minus forms observed.
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Thus, the increase in exon recognition of the F1 and F2 constructs compared with hCF may be explained by the interference of the base insertions, deletions, and substitutions with exonic regulatory elements. To fully investigate the putative role of the stop codons in nonsense altered splicing, we introduced in the three hybrid minigenes two A to T substitutions in the central part of the exon (positions 46 and 49 of the exon, respectively) (Fig 2A, Stop constructs). These nucleotide substitutions, according to the different reading frames of the hCF, F1, and F2 minigenes, do or do not generate stop codons. In the F1 and F2 contexts, these nucleotide changes generate two unique stop codons. The transfection experiments showed no changes in the splicing patterns with about 80% of exon inclusion (Fig. 2D, F1-Stop and F2-Stop). On the other hand, in the hCF minigene, the two A to T substitutions eliminated one stop codon, but again the splicing pattern was not affected, remaining at the initial 65% for this construct. This indicates that the two A to T substitutions, even if they produce nonsense codons, do not induce altered proportions of plus and minus forms, since they are not affecting splicing regulatory elements. We then tested, in the three different contexts, two of the natural substitutions with a splicing-inhibitory effect, the C31T and the C155A. In the hCF context, the C31T and the C155A do not introduce a new stop codon but reduce the 65% hCF exon inclusion to 48 and 16%, respectively (Fig. 2D, compare hCF with hCF-C31T and hCF-C155A). On the other hand, in the F1 and F2 minigenes, C31T creates a stop codon (Q414X), whereas C155A does not. The results shown in Fig. 2D indicate that these two mutations maintain a similar splicing-inhibitory effect both in F1 and F2 contexts. In fact, the 80% of exon inclusion in F1 and F2 is reduced in F1-C31T and F2-C31T to about 65%, and in F1-C155A and F2-C155A it is reduced to about 35% (Fig. 2D). These results indicate that the mutations have a negative effect on the exon recognition independently from the nonsense codon specification. In addition, we introduced on the F2 context two different stop codons in the same 5'-end position, with the creation of the F2-TAA and F2-TGA constructs. In one case, the stop codon (TAA) did not change the splicing pattern, whereas a 70% exon inclusion was observed for the TGA codon (Fig. 2D).
Altogether, these results indicate that substitutions introduced in the exon may affect the exon recognition, but this is completely independent from the fact that the substitutions do or do not create a stop codon. In conclusion, these data exclude the presence of a nuclear reading frame mechanism regulating CFTR exon 9 splicing and suggest that exonic mutations, including nonsense substitutions, cause changes in the splicing efficiency by affecting exonic splicing-controlling elements.
Identification of Regulatory Elements of Splicing in CFTR Exon 9 Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides. This suggests that this short sequence at the 3' end of exon 9 may contain both enhancer and silencer functions, whose fine tuning on splicing could be missed if analyzed using multiple base deletions. To better characterize the mechanism by which some of the CFTR exon 9 mutations alter splicing, we performed a detailed investigation by site-directed mutagenesis of this short region (positions 144157). This analysis was further extended to other point mutations distributed in the entire length of the exon (Fig. 3).
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In the 15-bp sequence at the 3' portion of the exon, the substitutions of wild type C in position 155, which corresponds to the base affected by A4555E, to either A (the natural mutation), G, or T induced exon skipping (Fig. 4, A and B). Interestingly, the 155G and 155T produced a similar percentage of exon inclusion of about 5%, which was the lowest among the different mutants. A reduction in the percentage of exon 9 inclusion was also observed as a result of point substitutions in the adjacent positions at 154 and 156 and for one of the mutants at position 153 (153T). On the contrary, the 153C and the 157T (V456F) variants did not significantly affect the splicing pattern. Extension of the mutagenesis in the 5' direction, including the Q452P (146C) variant showed that mutants from position 145 to 149, with the notable exception of the 148G, induced exon inclusion (Fig. 4, A and B). The 148G substitution, in fact, contrary to the other nearby mutations, produced significant exon skipping. It is interesting to note that this particular mutation creates an overlapping and adjacent consensus sequence for 5' and 3' splice sites consisting of CAGGTG. Finally, substitutions of wild type A in position 144 caused significant exon skipping (144T) or mild reduction in the percentage of exon inclusion (144G). These results indicate the presence of two juxtaposed silencer and enhancer elements in a short region of 15 nucleotides.
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To study these elements in detail, we evaluated the effect on the splicing efficiency of double mutants. The 146C natural missense substitution (Q452P) with 95% of exon inclusion was analyzed in association with the nearby exon-skipping mutations in position 154 (C or T) and 155 (G or T). These two mutations produced a similar percentage of exon 9 inclusion of about 20% for 154C and 154T and 5% for 155G and 155T (Fig. 4C). Hybrid minigene experiments showed in three of the double mutants (146C-154C, 146C-154T, and 146C-155T) a percentage of exon 9 inclusion between 80 and 95% very similar to that found in 146C, indicating that the 146C enhancing mutation overrides the downstream silencing mutations. On the contrary, among the double mutants, only the 155G induced significant exon skipping when associated to the 146C enhancing variant (Fig. 4C). This indicates a strict functional interdependence of the juxtaposed silencer and enhancer elements related to the type of nucleotide substitutions. This points to a complex situation where these two functions may coexist in the same element as recently reported for the composite regulatory elements of splicing (CERES) in CFTR exon 12 (35).
We also analyzed another 14 mutants distributed in the rest of the exon.
The results showed that seven mutants induce changes in the splicing pattern
with either a positive or negative effect on the exon recognition (see
Table I for the percentage of
exon 9 inclusion). In particular, three nucleotide changes between positions
16 and 20 (T16, T18G, A20G) induce exon inclusion, indicating the
presence of a putative silencer element at the 5' portion of the exon,
thus explaining the increase in exon inclusion observed in the F1 and F2
minigenes. On the other hand, three mutations (G61A, C72G, and G164 ins)
induced exon skipping. These results are compatible with the idea that the
entire CFTR exon 9 is composed of several adjacent and functionally related
splicing regulatory elements with both enhancer and silencer functions.
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Correlation between SR Protein Score Matrices and CFTR Exon 9 SplicingIt has been suggested that exonic splicing controlling elements affected by point mutations contain consensus sequences derived from SR-specific score matrices that mediate exon splicing efficiency (15, 1820). We have determined the contribution of SR protein binding sites using the four available SF2/ASF, SC35, SRp40, and SRp55 motif-scoring matrices (15) to investigate wild type and mutant CFTR exon 9. The wild type CFTR exon 9 contains 15 multiple high score motifs (three for SF2/ASF, five for SC35, seven for SRp40, and no matches for SRp55) (Fig. 3). All of the mutants tested in the hybrid minigene experiments were analyzed according to the changes of the SR protein matrix scores (Table I). None of the natural missense mutations disrupts a preexisting SR protein site of this group, even if some do induce exon skipping. Interestingly, the majority of the mutations (10 of 15) inducing exon inclusion creates one or multiple new high score motifs, suggesting that the creation of new sites could be responsible for the increased exon usage. In the same manner, the majority of the mutants (10 of 15) that do not change the splicing pattern did not modify the SR protein scores. On the contrary, no correlation can be observed when the mutants induce exon skipping; in fact, among the 17 mutants inducing exon skipping, none disrupts an SR protein site. These results suggest that the currently available SR protein high score motif can explain only partially the complex regulation of CFTR exon 9 and that in this context any individual change in SR motif score cannot predict the splicing efficiency.
To directly evaluate the effect of one SR protein SF2/ASF, we cotransfected the WT exon 9 and mutant minigenes with SF2/ASF coding plasmid. The CFTR exon 9 is negatively regulated by SF2/ASF, and this inhibition is mediated by the interaction of the splicing factor with the intronic splicing silencer element (26). We reasoned that the creation or disruption of an SF2/ASF binding site in the exon should modify the response to SF2/ASF. For this reason, we studied the effect of SF2/ASF on several exonic mutants. The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I). In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig. 4 and Table I).
Cotransfection of SF2/ASF with the wild type CFTR exon 9 resulted in a decrease in exon 9+ transcripts as previously reported (26). In this case, the exon 9+ form goes from 65% to about 25% (Fig. 5). Cotransfection of SF2/ASF with each of the site-directed mutants showed in all cases a similar inhibitory effect of the splicing factor, with a significant reduction in the percentage of exon 9 inclusion (Fig. 5). In conclusion, cotransfection of SF2/ASF induced a significant reduction in the percentage of exon 9 inclusion not related to the SF2/ASF score motif variability (Fig. 5).
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The Silencing Effect of the 155G Mutant Correlates with Specific hnRNPH
Binding at the CFTR Exon 9 To identify specific changes in binding
property or affinity of trans-acting factors at the juxtaposed
enhancer and silencer elements that can explain the effect of the mutants, we
performed UV-cross-linking experiments and pull-down assay on the 3'
portion of CFTR exon 9. In particular, we focused our attention on the two
substitutions 155G and 155T, which, even if located at the same nucleotide
position, were observed to induce exon skipping with different mechanisms.
When alone, both caused severe exon skipping defects, but only 155G maintained
a strong splicing inhibition when associated to the enhancing substitution
146C (Fig. 4C).
UV-cross-linking experiments were done on mutants at position 155, on the 146C
enhancing variant, on the 148A and 148G mutants, and on the double mutants
146C-155G and 146C-155T (Fig.
6). These mutant sequences and the WT were transcribed in
vitro in the presence of [-32P]UTP and equal quantities
of labeled transcripts were then used in a UV-cross-linking assay with HeLa
nuclear extracts. Among the numerous proteins that could be cross-linked to
the labeled RNAs, a 55-kDa approximate molecular weight band could be observed
only in the 155G and 146C-155G substitutions. No significant changes in the
UV-cross-linked pattern compared with normal exon 9 were observed for the
other mutants, which can be possibly ascribed to the fact that this
experimental assay can detect predominant changes in binding properties of
abundant splicing factors. To identify the 55-kDa splicing factor binding at
155G, we performed pull-down experiments with WT exon 9, 155G, and 155A
variants (Fig. 7A).
Again, in the 155G mutant, a protein of about 55 kDa was evident from the
proteins eluted from the beads that could not be pulled down by the WT or 155A
sequence. Internal sequence analysis by mass spectrometry of the excised
55-kDa band yielded four different peptides and a search in the
DDBJ/EMBL/GenBankTM data base revealed that their sequences were
identical to residues 5068, 99114, 151167, and
300316 of hnRNPH. hnRNPH is an abundant splicing factor that in certain
contexts binds to G-rich sequences, inducing splicing inhibition when the G
residues are exonic
(2124).
To investigate the binding properties toward hnRNPH of mutant RNAs, we
performed Western blotting of the proteins eluted from the pull-down
experiments. hnRNPH-specific immunoreactive material was present in the 155G
mutant and in the double mutant 146C-155G, but not in 155A, 155T, and
146C-155T (Fig. 7B). A
specific band for hnRNPH was also evident in the 164G+ silencing mutation,
where a run of three G residues is formed by the insertion
(Fig. 3), indicating that
binding of these splicing factors is associated to the silencing effect. In
conclusion, the 155G variant creates a new exonic splicing silencer element
composed of five G residues, and presumably it is the specific binding of the
hnRNPH inhibitory splicing factor that makes it dominant over the nearby
enhancing mutations. These data suggest that the complex binding properties of
the two juxtaposed elements modulates the splicing efficiency.
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DISCUSSION |
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To study the contribution of exonic sequences, we evaluated several naturally occurring missense and nonsense mutations and extended these studies by new variations introduced by site-directed mutagenesis. Our results show that single nucleotide substitutions may have a profound effect on the splicing efficiency inducing both exon inclusion and skipping. The large unexpected sensitivity to splicing variations (35 substitutions of 47 cause changes in the splicing pattern) (Table I) may be related to the weak definition of the CFTR exon 9. In fact, the changes in the splicing pattern were modulated by the composition of the polymorphic TG-T locus in intron 8. This indicates the strict relationship between the definition of the exon mediated by the strength of the 3' splice site and the exonic regulatory structures affected by the natural and site-directed mutants.
The existence of a putative nuclear scanning mechanism for nonsense codons that directly affects the splicing process, causing defective exon recognition, is controversial (1, 59, 15, 3638). Using several hybrid minigene constructs, in which we systematically create and/or disrupt nonsense codons and reading frames, we showed that the creation of nonsense codons in exon 9 is not responsible per se in changes in splicing efficiency. In fact, exonic nucleotide substitutions may or may not modify the splicing pattern independently from the reading frame (Fig. 2). In addition, the treatment with protein synthesis or transcription inhibitors showed that the presence of multiple stop codons in exon 9 did not selectively affect the relative amount of one of the two mRNAs with or without exon 9. This indicates that selective degradation of the two spliced variants with stop codons is not responsible for changes in the measurement of splicing efficiency. Even if gene- and exon-specific determinants might be responsible for a selective exon splicing defect caused by nonsense codons, our results do not support the idea that the efficiency of exon recognition can be regulated by a putative nuclear reading frame scanning for nonsense codons. On the contrary, the fact that changes in splicing efficiency can be caused by a large number of point substitutions in CFTR exon 9 strongly indicates that nucleotide variations induce changes in splicing efficiency by affecting exonic cis-acting regulatory elements. Our results are consistent with studies where the exon skipping phenotype has been shown to be reproduced also in in vitro splicing assay independent from the reading frame disruption (15, 20).
A significant result of this study is the lack of the predictive capacity of SR protein score matrices in CFTR exon 9 (Table I). These matrices have been previously found to correlate with the splicing phenotype in other gene systems (15, 20). However, this is the first study to our knowledge that addresses the role of SR protein score matrices by extensive site-directed mutagenesis. Our analysis does not support a general predictive value of the SR matrices for the identification of CFTR exon 9 cis-acting elements. Thus, their weight in splicing regulation cannot be taken without a proper evaluation that will necessarily include appropriate functional splicing assay in each specific system. In addition, the SR protein score matrices have been selected for the identification of "pure" exonic splicing enhancers binding SR proteins. It is also possible that such enhancers may not be represented in the CFTR exon 9.
Several lines of evidence suggest that the two juxtaposed enhancer and silencer elements in CFTR exon 9 cannot be considered "pure" enhancer and silencer but that they concur to the creation of a composite element with both functions overlapping. For example, all of the substitutions at position 155 that induce exon skipping may potentially inactivate an exonic splicing enhancer or create a new exonic splicing silencer. We show that this latter case occurs for one of the substitutions, the 155G, that creates a binding site for the hnRNPH inhibitory splicing factor. Binding of hnRNPH to exonic G-rich sequences mediates silencing in several exons (2124), and hnRNPH binding to the 164G+ mutant (that also creates a G triplet) induces exon skipping (Fig. 7B). On the other hand, the splicing inhibition induced by the 155A and 155T substitutions has a different molecular basis than the 155G. In fact, 155A and 155T do not bind to hnRNPH, and in the double mutants, their splicing-inhibitory effect is overcome by the nearby enhancing mutations (Fig. 4). This indicates that point mutations at the same position (155) can be mechanistically different and functionally modulated by the context. This aspect is difficult to predict from the currently available predictive algorithms such as the SR score matrices (15) and the enhancer identification programs (39). Another example of elements having a juxtaposed effect is given by analysis of the position 148. As demonstrated in Fig. 4, this position is placed among the enhancing sequences at the 5' portion of the element (from position 145 to 149); however, 148G induces significant exon skipping. Finally, cloning of the wild type and mutant regulatory sequences in a heterologous exonic context did not induce consistent alterations in the splicing pattern of the host exon (data not shown). These different observations suggest that the juxtaposed enhancer and silencer sequences may indeed represent a CERES with overlapping silencer and enhancer functions that are context-dependent, as recently found in the CFTR exon 12. In this case, analysis by extensive site-directed mutagenesis showed an even more complex splicing effect of mutations at the same and nearby positions (35).
Our results indicate that a single mechanism cannot explain the effect of single nucleotide substitutions at the juxtaposed enhancer and silencer elements in CFTR exon 9. The determination of the precise underlying mechanism for every nucleotide substitution in these regulatory sequences will require a deep investigation that will necessarily take into account several possibilities. Furthermore, each mutation may cause subtle changes of the binding specificity of trans-acting factors, but the limit of detection may be below that of UV-cross-linking and pull-down assays used in this paper. Alternatively, each mutation may modulate the cooperative propagation of splicing factors along the exon as previously suggested for hnRNPA1 in the human immunodeficiency virus-1 tat exon 3 (40). It is interesting to note that 144T, 155T, and most significantly 148G cause a variable degree of exon skipping and create an AG-GT sequence, which might represent a consensus for both 5' and 3' splice sites. In some cases, sequences with inhibitory activity have been shown to function as pseudosplice sites, which sequester the splicing machinery into a nonproductive complex (4143). Inappropriate exon skipping may also result from the creation of U1 small nuclear ribonucleoprotein binding sites (30). Not only creation, deletion, or changes in binding affinity of binding sites for splicing factors but also changes in secondary structure, having an indirect effect on such interactions, might participate in the regulation of the splicing efficiency, modulating the proper display of splicing factor binding sequences (44).
Point mutations in exonic splicing regulatory elements may have pathological consequences. CFTR exon 9 skipping has been associated with CF phenotypes of different severity and produces a nonfunctional CFTR protein (45). Some of the missense mutations analyzed in this study may exert part of their phenotypic expression and disease variability affecting the splicing pattern, in particular when associated to a unfavorable polymorphic variant near the 3' splice site. For example, it appears that A455E can achieve adequate levels of chloride conduction at the cell surface (46, 47), causing only a partial CFTR protein processing defect (48). Since it also increases exon skipping, the penetrance of its pathological effects may be better related to the deletion of the amino acids encoded by the entire exon 9. Furthermore, the modulation by the concentration of splicing factors, which have an inhibitory effect on the CFTR exon 9 (26, 49) and a specific and possibly individual variation distribution, can provide an explanation for the phenotypic and tissue-specific variability in CF patients, particularly in those carrying the A455E substitution. It is suggestive that as many as five natural mutants in the CFTR exon 9 modify the splicing pattern with either an increase or decrease in the splicing efficiency (Fig. 1A). It is also interesting to note that mutants inducing aberrant exon skipping may also occur at the third position of the codon usage and do not change the amino acid code. When these mutations are found in genomic screening, their effect on splicing may not be taken into account, a fact that would lead to an erroneous molecular diagnosis. Thus, medical geneticists should be aware that silent mutations cannot be ignored as a potential cause of disease. The extreme sensitivity of exonic sequences to splicing derangement is of great relevance to human pathology, since missense and even translation silent variations may cause human disease by affecting the extent and accuracy of pre-mRNA splicing.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 39-040-3757337; Fax:
39-040-3757361; E-mail:
baralle{at}icgeb.org.
1 The abbreviations used are: SR protein, serine/arginine-rich protein;
CERES, composite exonic regulatory element of splicing; CF, cystic fibrosis;
hCF, human CF; CFTR, cystic fibrosis transmembrane regulator; SF2/ASF,
splicing factor 2; hnRNP, heterogeneous ribonucleoprotein; hnRNPH,
heterogeneous ribonucleoprotein H; SELEX, systematic evolution of ligands by
exponential enrichment; RT, reverse transcription; WT, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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