©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Splicing Deficiency Induced by a C to T Mutation at Position 3 in the Intron 10 Acceptor Site of the Phenylalanine Hydroxylase Gene in a Patient with Phenylketonuria(*)

(Received for publication, March 21, 1995; and in revised form, June 25, 1995)

Jadwiga Jaruzelska (2)(§) Veronique Abadie (1) Yves d'Aubenton-Carafa (2) Edward Brody (3) Arnold Munnich (1) Joëlle Marie (2)(¶)

From the  (1)From theUnité 12 INSERM, Hôpital des Enfants Malades, 149 rue de Sèvres, 75015 Paris, France, the (2)Centre de Génétique Moléculaire du CNRS, Laboratoire Propre Associéà l'Université Pierre et Marie Curie, 91198 Gif-sur-Yvette, France, and the (3)State University of New York at Buffalo, Department of Biological Sciences, Cooke Hall, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A previous study has identified a C U mutation at position -3 in the 3` splice site of intron 10 of the phenylalanine hydroxylase pre-mRNA in a patient with phenylketonuria. In vivo, this mutation induces the skipping of the downstream exon. This result is puzzling because both CAG and UAG have been reported to function equally as 3` splice sites. In this report, we show that the C U mutation affects predominantly the first step of the splicing reaction and that it blocks spliceosome assembly at an early stage. The 3` region of the phenylalanine hydroxylase intron 10 has two unusual characteristic features: multiple potential branch sites and a series of four guanosine residues, which interrupt the polypyrimidine tract at positions -8 to -11 from the 3` splice site. We show that the mutation precludes the use of the proximal branch site, while having no effect on the remote one. We also show that in the UAG transcript, the four guanosine residues inhibit the splicing of intron 10. The substitution of these purine residues by one cytosine residue, regardless of the position, increases the splicing efficiency of the mutant UAG precursor while having no effect on the wild-type CAG precursor. Substituting the four purine residues by four pyrimidines relieves the inhibition and rescues the use of the proximal branch site. These results demonstrate that according to the context, the C and U nucleotides preceding the AG are not equivalent for the splicing reaction.


INTRODUCTION

The removal of intervening sequences from precursor mRNA involves the precise recognition of the 5` and 3` splice sites at the exon/intron boundaries. The splicing reaction proceeds by two transesterification steps(1, 2, 3) . In the first step, the precursor is cleaved at the 5` splice site to yield the upstream exon and the lariat intermediate. In the second step, the mature mRNA is formed and the intron is released as a lariat. The accuracy of the splicing process is due to the coordinated assembly of a large number of protein factors and small nuclear ribonucleoprotein particules (U1, U2, and U4/U5/U6 snRNPs) (^1)on the precursor to form the spliceosome(1, 2, 3) . The spliceosomal components interact with cis-acting sequences that include the 5` splice site, the 3` splice site (YAG), the branch site, and the pyrimidine tract downstream of the branch site.

The functional significance of these elements in the splicing reaction has been inferred from in vitro and in vivo site-directed mutational analysis in both yeast and mammalian systems (4, 5, 6, 7, 8) . Most of the mutations in the invariant 5` GU and 3` AG dinucleotides have a strong effect on the splicing process. Some mutations abolish or reduce mRNA production, whereas others induce exon skipping or utilization of cryptic sites. The effects of mutations in other positions are not so clear because of discrepancies between in vivo and in vitro results. However, the importance of nucleotides outside of the consensus sequences in the splicing process has been reported(4, 5, 9, 10) . Krawczak et al.(11) collected 101 cases of splicing mutations that are responsible for human genetic diseases. Although the most frequently observed mutations concerned the invariant 5`GU and 3`AG dinucleotides, some that occurred outside of these invariant sequences also dramatically altered the processing of pre-mRNA. For example, the mutation in position +5 of the 5` splice site, which in genetic disorders leads to abnormally spliced products, was shown recently to be a critical nucleotide for the accuracy of the splicing process by base pairing with U6 snRNP(12, 13, 14) .

Concerning the position -3 at the 3` splice site, two mutations were reported in patients with beta thalassemia(15) . One was a C A substitution at the 3` splice site of intron 2 of the beta globin gene. The other was a U G substitution at the 3` splice site of intron 1. The resulting RNA splicing defects were consistent with what was previously observed by means of in vitro mutational studies. The replacement of 3` CAG by 3` GAG impaired the second step of the splicing reaction, whereas AAG or UAG had only a limited effect on the splicing process(16, 17, 18) . These studies were extended in a more recent paper; using the properties of AG-independent introns, which can support the first step of the splicing reaction in the absence of the dinucleotide AG, it was shown that the nucleotides preceding the AG are not equivalent, with CAG = UAG > AAG > GAG(19) . This finding can be related to the frequency with which wild-type 3` splice sites are represented in the human genome where CAG is the most frequent (75%), followed by UAG (23%), AAG (2%), and GAG (<1%)(20) . More recently, a C U transition at position -3 in intron 2 of the CFTR gene was reported in a patient with cystic fibrosis(21) . PCR sequencing analysis revealed that exon 3 was skipped during splicing of the mRNA.

In 1993, a CAG to UAG mutation was described in the 3` splice site of intron 10 of the phenylalanine hydroxylase pre-mRNA in a patient with phenylketonuria(22) . This mutation causes the skipping of exon 11 and the premature termination of RNA translation in the following exon. This was the first report showing that a pyrimidine to pyrimidine (C U) substitution at the -3 position in the 3` splice site affects the splicing process.

To get more insight into the molecular mechanism responsible for such a deleterious effect, we investigated the mutated precursor by an in vitro splicing assay. We have shown that the mutation markedly reduces the splicing efficiency and that it prevents the use of the branch site located at -22 nt upstream of the 3` splice site. In addition, the substitution of four contiguous purine residues, which interrupt the pyrimidine tract, by pyrimidine residues relieves the inhibition process on the mutant precursor. In contrast, a more limited effect is observed on the wild-type transcript. These results demonstrate that, according to the context, the C and U nucleotides preceding the AG are not equivalent for the splicing reaction.


EXPERIMENTAL PROCEDURES

Cloning and Site-directed Mutagenesis

An EcoRI DNA fragment derived from the wild-type and mutant phenylalanine hydroxylase gene containing exon 10 (the last 54 nt), the whole of intron 10 (556 nt), and exon 11 (81 nt from the 5` end) was cloned into the pSP72 vector using standard procedures(23) . Site-directed mutagenesis was performed as described (24) or using the USB T7-Gen kit after subcloning the EcoRI fragment into the pTZU19 phagemid. The G residues at positions -8 to -11 were substituted by C residues by using 24-mer degenerate oligonucleotides (5` CTTTTCACTT(G/C, G/C, G/C, G/C)CCTATAGTAC 3` for the wild-type construct and 5` CTTTTCACTT(G/C, G/C, G/C, G/C)CCTACAGTAC 3` for the mutant construct), where (G/C, G/C, G/C, G/C) indicates an equal mixture of G and C nucleotides at these positions.

Transcription and Splicing

The pSP72 and pTZU19 vectors were linearized by EcoRV and PvuII and transcribed by SP6 and T7 RNA polymerases, respectively. Uniformly labeled capped transcripts were synthesized in a 20-µl transcription reaction and purified by electrophoresis on a 5% acrylamide/7 M urea sequencing gel(18) . The specific activity of the pre-mRNA was between 10 10^6 and 20 10^6 cpm/pmol. HeLa cell nuclear extracts were purchased from Computer Cell Center (Mons, Belgium) and prepared as described (25, 26) . In vitro splicing reactions were performed at 30 °C for the indicated times. Splicing reactions were carried out in 50 µl containing 30 µl nuclear extract, 5 fmol of labeled precursor, 1 mM MgCl(2), 20 mM creatine phosphate, 0.5 mM ATP, 3% (w/v) polyvinyl alcohol, and 0.5 units/ml RNasin(5, 18) . Samples were phenol extracted, ethanol precipitated, and analyzed on a 5% denaturing polyacrylamide gel. Splicing complexes were separated on a 4% acrylamide gel(27) . Results were quantified using a PhosphorImager (Molecular Dynamics). The splicing efficiency was measured as a ratio of the amount of the final lariat over the amount of the final lariat plus the precursor. A correction factor of 1.7 was used for the final lariat to take into account the loss of radioactivity after cleavage of the downstream exon. The precursor corresponds to the amount of radioactivity that remains after the incubation in splicing condition. We choose to calculate the splicing efficiency from the final lariat and not from the spliced mRNA due to a high background below the precursor.

Identification of a Lariat Structure and Mapping of a Branch Point

The band migrating more slowly than the precursor was cut out of 5% denaturing acrylamide gel after large-scale splicing reactions. RNA was eluted, ethanol precipitated, and subjected to debranching(28) . The branch point was identified by primer extension analysis using a 15-mer oligonucleotide complementary to positions -15 to -1 of either the wild-type or the mutant intron 10. Primer extension was performed for 45 min at 50 °C, and the extended products were analyzed on an 5% acrylamide denaturing gel(18) . The sequencing was performed by the chain termination method on pre-mRNA in the same conditions as described above with the addition of the corresponding ddNTP (50 µM). Quantification was done by using a PhosphorImager.


RESULTS

The Mutation C U in the -3 Position Decreases Splicing Efficiency

In this study we attempted to find out why a C U substitution at position -3 within the intron 10 acceptor site of the phenylalanine hydroxylase gene induces skipping of exon 11 in a patient with phenylketonuria. For that purpose, we constructed a mini-gene that contains 54 nucleotides of the 3` end of exon 10, the whole of intron 10 (556 nt), and 81 nucleotides of the 5` part of exon 11 cloned in the pSP72 vector. The mutant and the wild-type transcripts of these constructs were subsequently tested by in vitro splicing experiments using HeLa cell nuclear extracts. Fig. 1shows that the wild-type precursor is efficiently spliced, generating the correct intermediates and final mRNA product. The identification of the intermediate and final lariat was done by the debranching reaction (data not shown). In contrast, the mutant precursor shows a dramatic decrease of the splicing efficiency with barely detectable products. The mutation C U seems to predominantly affect the first step of the splicing reaction (see also Fig. 4and 5). Depending on the nuclear preparation used, the splicing efficiency of the mutant transcript was 10-30% of that of the wild type. To further investigate the mechanism by which the C U mutation inhibited the splicing reaction, we looked at spliceosomal assembly using non denaturing gel electrophoresis (Fig. 2). Clearly, the pattern of complex formation is different between the wild-type and the mutant precursor. Retarded complexes (Fig. 2, SP), which migrate more slowly than the nonspecific complex (Fig. 2, H), are visualized with the wild-type transcript. These complexes appear after 5 min of incubation in splicing conditions and accumulate with increasing incubation times. The faint band, which migrates just above complex H, most probably corresponds to part of the nonspecific complex (this band does not change during the incubation). Due to the length of the transcript (772 nt), the distinction between the prespliceosome (Fig. 2, A) and the spliceosome (Fig. 2, B) was rather difficult (for comparison, complexes formed on adenovirus (Ad) precursor (362 nt) are shown on the right in Fig. 2). Similar observations have been already reported for long precursors(29) . In the case of the mutant precursor, the specific complexes are barely detectable even after 1 h of incubation in splicing conditions. This suggests that the splicing defect occurs at an early stage of spliceosome assembly.


Figure 1: In vitro splicing reaction of the wild-type 3`CAG and the mutant 3`UAG precursors. Splicing reactions were incubated for 0 (lanes 0), 30 (lanes 1), 60 (lanes 2), or 120 min (lanes 3). Mut and WT designate the mutant and the wild-type precursor, respectively. M designates P-labeled HpaII fragments of PBR 322. Due to the cloning procedure, 5` exon migrates at 120 nt (exon 10, 54 nt plus 67 nt from the polylinker).




Figure 4: The effect of one G C mutation within the tetraguanosine sequence on splicing of the 3` CAG and 3` UAG precursors. The tetraguanosine sequence and the G C mutation introduced at each position are indicated at the top of the figure. Splicing on the 3` UAG (Mut) and 3`CAG (WT) were analyzed for each mutation for 0 (lane 0), 30 (lane 1), 60 (lane 2), or 120 min (lane 3).




Figure 2: Spliceosome assembly on the wild-type 3`CAG and 3`UAG mutant pre-mRNAS. Splicing reactions were incubated under splicing conditions for 0 (lanes 0), 5 (lanes 1), 10 (lanes 2), 15 (lanes 3), 30 (lanes 4), or 60 min (lanes 5), treated with heparin (2 mg/ml), and loaded onto native polyacrylamide gels. H, nonspecific complexes; SP, splicing complexes; Mut, mutant (C U) precursor; WT, wild-type precursor; Ad, adenovirus precursor used as a control; A, pre-splicing complex; B, spliceosome.



The Mutation C U Prevents the Utilization of the Major Branch Site

These results raised a question: Why is the UAG 3` splice site being efficiently used in introns of other mammalian genes (particularly in six out of the 13 phenylalanine hydroxylase introns) because it provokes a reduced in vitro splicing efficiency in this particular case(20, 30) ? Examination of the sequence upstream of the 3` splice site reveals multiple potential branch sites that match the mammalian consensus YURAC sequence (Table 1). Previous reports have shown that inactivation of normally used branch site sequence leads to a decrease of in vitro splicing efficiency. The reduced rate in the splicing process has been explained by the activation of inefficient cryptic branch points(28, 31, 32) . Furthermore, it has been reported that in vivo, a single base substitution within the branch point sequence altered 3` splice site recognition(31) . From these observations, it was important to assess whether the mutation could affect branch site selection. Final lariats were isolated from large-scale splicing reactions and extended with an oligonucleotide complementary to the 3` end of the wild-type or the mutant intron. The amount of the final lariat isolated is 4-fold less than the wild-type amount, which means that the splicing efficiency was 25% relative to the wild type. Wild-type RNA yields three stops (Fig. 3). The major stop maps to the adenosine residue at position -22 upstream of the acceptor site within the sequence UAAUAAC (where A is the stop). In six out of seven positions it closely matches the yeast consensus branch site sequence. The second stop corresponds to the A residue at position -23, just 5` to this branch point A. This finding is consistent with previous reports showing that two adjacent adenosine residues can be alternatively used as a nucleophile(33) . The third maps to the adenosine residue at position -34 upstream of the 3` splice site within the sequence UUUAAC. The relative usage of these sites is from the proximal (closer to the 3` splice site) to the distal, 62, 12, and 26% respectively. In the case of the mutant transcript, only 23% of the stops correspond to a termination of transcription on the A residues at position -22 and -23 nt upstream of the 3` splice site, the distal one(-34) becoming the predominant stop (77%). To further investigate how the mutation affects the use of branch sites, the mutant/wild type ratio for each branch site usage has been measured. The mutant/wild type ratio for the use of the distal branch site(-34) was 0.78, whereas it was 0.13 for that of the proximal one(-22). Whatever the splicing efficiency, the ratio for the distal site was always very close to 1, that of the proximal site being between 0.08 and 0.29. From these results we can conclude that i) the mutation C U predominantly prevents the use of the proximal branch sites and ii) the recognition of the branch site located at -34 nt upstream of the 3` splice site is only slightly affected. One must speculate that in vivo, the inefficient recognition of the UAG as a 3` splice site allows the use of the downstream 3` splice site preceding exon 12.




Figure 3: Localization of the branch point by primer extension analysis. In order to get enough material for branch site determination, the amount of mutant pre-mRNA was increased 2-fold. Wild-type (Wt) and mutant final lariats were subjected to primer extension analysis using a 15-mer oligonucleotide complementary to positions -1 to -15 from the 3` splice site in the presence (lanes A, C, G, and T) or in the absence (lane E) of dideoxynucleotides. The nucleotide sequence of the 3` region of intron 10 of both wild-type and mutant precursors are also shown. The arrows show the branch sites used in the splicing process.



Increasing the Pyrimidine Content in the 3` Region Increases the Splicing Efficiency of the Mutant Precursor but Not That of the Wild Type

Examination of the sequence between the branch point and the 3` splice site revealed another particularity. The sequence corresponding to the polypyrimidine stretch was short and was interrupted by four guanosine residues at positions -8 to -11 upstream of the 3` splice site. Numerous studies have demonstrated that the pyrimidine tract is an important cis-acting element for the splicing reaction (reviewed in (3) ). We hypothesized that the four G residues situated at a very short distance from the 3` splice site subtantially contributed to making this splicing site relatively weak. It would be in this context that the C to U mutation at position -3 would block usage of this 3` splice site. This hypothesis was supported by a recent report of Dworniczak et al.(34) , who identified a G to A mutation within intron 10 from the phenylalanine hydroxylase gene at position -11 upstream of the 3` splice site. This mutation creates a UAG acceptor site upstream of the guanosine residues. The consequence of the mutation results in an in-frame insertion of nine nucleotides between exon 10 and exon 11, which signify that this UAG site is preferentially used in vivo over the wild-type 3` splice site. These results demonstrate that there is no inherent difficulty for a UAG to act as a 3` splice site in intron 10 and suggest that the conjunction of the four G residues together with the C U mutation at position -3 could be responsible for the defect. If our hypothesis is correct, the substitution of four guanine residues by pyrimidines would allow the mutant C U transcript to become efficiently spliced.

To test if this was the case, we constructed plasmids replacing the guanine residues at each position by one cytosine residue. Two or four cytosine substitutions were also tested. The results of splicing experiments with the CAG and UAG pre-mRNAs containing a single guanine to cytosine substitution at each of four positions are shown in Fig. 4. The results demonstrate that a single cytosine substitution has a significant stimulating effect on the mutant pre-mRNA splicing. Depending on the HeLa cell nuclear extract used, the level of stimulation with one G C substitution was 2-3-fold, regardless of the position (see Fig. 6). These results demonstrate that there is no positional effect and indicate the importance for the nucleotide to be a pyrimidine in all four positions. The subsequent splicing experiments with pre-mRNAs containing two or four guanines substituted with cytosines indicated that the effect of splicing stimulation was additive; increasing the number of pyrimidines proportionately increases the splicing efficiency (Fig. 5). The level of stimulation with four G C substitutions was about 8-10-fold. The substitution of all guanine residues by cytosines roughly restores the same splicing efficiency as the wild-type precursor, which is correlated with the predominant use of the proximal branch site (data not shown). In contrast, the single substitutions have no effect on the wild-type precursor. Only the substitution by four cytosine residues leads to an increase of the splicing activity by about 2-fold (Fig. 6).


Figure 6: Splicing efficiency of the 3`CAG and 3`UAG precursors after substituting cytosine residues within the tetraguanosine sequence. Quantification was performed on the final lariat after 2 h of incubation under splicing conditions. The results were from five independent experiments that were carried out under the same experimental conditions. The splicing efficiency was calculated for each precursors with or without G to C substitution as described under ``Experimental Procedures'' (y axis). They are expressed as percentages of the splicing efficiency of the wild-type 3`CAG precursor, where 100% is that of the wild-type transcript. The x axis corresponds to pre-mRNAs. 4G, wild-type tetrapurine sequence; 3G+1C, one substitution G C independent of the position; 2G+2C, two substitutions by cytosine residues in the tetrapurine sequence at positions -10 and -11; 4C, four substitutions by cytosine residues. Because substitution of the tetrapurine sequence by one pyrimidine residue has the same effect on the splicing efficiency regardless of the position, we present them by only one bar graph. The shaded bars designate the 3`CAG precursor, and the hatched bars designate the 3`UAG precursor.




Figure 5: The effect of double and quadruple G C substitutions within the tetraguanosine sequence on splicing of the 3` CAG and 3` UAG precursors. The splicing reaction was analyzed on the 3` UAG (Mut) and 3`CAG (WT) after mutation of the four guanosine residues by either two cytosine residues at positions -10 and -11 or four cytosine residues. The incubation times were 0 (lane 0), 30 (lane 1), 60 (lane 2), or 120 min (lane 3).



From these results, we propose that the G residues within the pyrimidine tract are negative elements for the splicing of the UAG precursor. The substitution by cytosine residues markedly increases the splicing efficiency and has a greater effect on the UAG precursor than on the CAG (Fig. 7). However, even after restoration of an uninterrupted polypyrimidine tract, the CAG and UAG transcripts behave differently. The splicing efficiency of the UAG transcript remains lower than its CAG counterpart.


Figure 7: The substitution of the purine residues by cytosines has a greater effect on the splicing reaction of the UAG precursor than of the CAG. The results shown in Fig. 6have been expressed as the ratio of the splicing efficiency of the CAG and UAG transcripts containing the G to C substitutions over their respective nonsubstituted transcripts.




DISCUSSION

In this paper we show that in vitro, the mutation C U at position -3 of the 3` splice site of phenylalanine hydroxylase intron 10 severely impairs both the spliceosomal assembly and the catalytic steps of the splicing reaction. This mutation was identified in a patient with phenylketonuria. In vivo, the effect of this mutation was to induce the skipping of the downstream exon, leading to a truncated protein terminated in exon 12(22) . This is the first report showing that in position -3 the C and U pyrimidines are not equivalent for the splicing process. The results we obtained were surprising for several reasons. First, in over 98% of human 3` splice sites a pyrimidine residue precedes the AG consensus dinucleotide. Although the CAG acceptor site is more common (75%), the UAG is still quite frequently used (23%; 20). This was particularly relevant for the phenylalanine hydroxylase gene, where in six out of the 13 introns the UAG is present at the 3` splice site(30) . Second, recent results have shown that when two closely spaced AG dinucleotides are in competition, the U and C at the -3 position are equally recognized by the splicing apparatus(19) . Third, as in other mammalian introns, a UAG can be used to remove intron 10 from the phenylalanine hydroxylase gene. This has been observed in a patient with phenylketonuria in which a G to A substitution at the position -11 creates a UAG 3` splice site (34) . This last observation is of particular interest because it suggests that the elements upstream of the four G residues function perfectly well in a UAG context. It also points to the role of the four G residues in the inhibitory process in a UAG context.

Our in vitro experiments show that the C U mutation predominantly blocks the first step of the splicing process. This is correlated with a dramatic reduction of splicing complexes (Fig. 2). The mechanism by which this inhibition occurs remains unknown. However, we can postulate that the C U mutation disrupts an early interaction needed for the 3` splice site to be efficiently recognized by the splicing machinery.

The pyrimidine tract is known to be an important element involved in this process. In the case of AG-dependent introns, in which the pyrimidine tract is short, decreasing the number of pyrimidines downstream of the branch site or upstream of the 3`AG was shown to strongly impair the first step of the splicing reaction(16) . Recently, Roscigno et al.(35) reported that reducing consecutive uracil residues in the pyrimidine tract by inserting purines affected spliceosomal assembly before A complex formation. Moreover, they showed that there is both a positional and a compositional effect. The substitution by G residues has a more pronounced effect on the splicing efficiency than that by A residues; they totally block the splicing reaction. All these findings point to the fact that the length of the pyrimidine tract, its composition, and the distance between the branch site and the pyrimidines can affect the splicing efficiency(16, 35, 36) . Examination of the pyrimidine tract in intron 10 of the phenylalanine hydroxylase gene revealed several characteristic features. It is short and interrupted by four guanine residues very close to the 3` splice site. Indeed, substituting these purine residues by pyrimidines increases the splicing efficiency of the mutant pre-mRNA (see Fig. 6). Individually each substitution is able to increase the splicing reaction. Replacement of the four purines by four cytosine residues restores roughly the same splicing activity as is found in the wild-type precursor. These results confirm all the previous reports concerning the role of the pyrimidine tract in the splicing reaction and support the finding that the 3` region is recognized at an early step of complex formation(37) .

From the results we present here, we can suggest that increasing the pyrimidine content in the UAG precursor allows efficient recognition of the pyrimidine tract/3` splice site. But if this is so, why is the wild-type precursor so different for its requirement for the pyrimidine stretch? In fact, we have observed that increasing the number of pyrimidines in the wild-type transcript has no effect on the splicing reaction except when the four G residues are replaced by four C residues. In that case, the splicing efficiency increased by 2-fold. These results seem to indicate that the wild type intron partially escapes the need for a very strong pyrimidine tract. In apparent contradiction of this assertion, these results also suggest that the information content of the pyrimidine tract is limited. It was previously reported that the C and U residues at position -3 are equally efficient for 3` splice site recognition(19) . In contrast to these findings, our results clearly indicate that in the phenylalanine hydroxylase intron 10, the pyrimidines adjacent to the 3`AG are not equivalent. This is also illustrated by the fact that even after restoration of an uninterrupted pyrimidine tract, the splicing efficiency of the UAG precursor remains lower than the CAG counterpart. From these results, we propose that the C residue at position -3 increases the strength of the 3` splice site and is one of the determinants that compensates for the relative weakness in the pyrimidine tract. This assumption might explain why in mammalian introns CAG 3` splice sites are more abundant than UAG sites.

The recognition of the 3` splice site involves collaborative interactions between the branch site and the pyrimidine tract/3` AG (31, 38) . We have shown that splicing of the wild-type precursor involves the use of several branch sites, localized at -22, -23, and -34 nt upstream of the 3` splice site. The proximal one lies in the UAAUAAC sequence, which, in six out of seven positions, closely resembles the yeast branch site sequence. The results show that it is preferentially used for the splicing of the wild-type transcript. This is consistent with previous work from Zhuang et al.(38) showing that the UACUAAC sequence is the most efficient branch site for mammalian mRNA splicing. In the previous section, we suggested that the information content of the pyrimidine tract is weak and that a cytosine residue at position -3 contributes to increase the strength of the 3` splice site. Another element that could compensate for the weak pyrimidine tract would be the presence of a strong branch site. In agreement with this hypothesis, it has been reported that in vitro a strong branch point sequence can partially overcome a weak polypyrimidine tract and vice versa(35) . In the course of branch site determination, we noticed that a small percentage of the lariats have branch sites on A residues at positions -25, -26, and -39. Why multiple branch sites are used in the splicing of intron 10 is presently unknown. The existence of multiple branch sites has already been reported and is particularly well illustrated in alternative splicing(39, 40, 41, 42) . In those cases, it was proposed that the presence of multiple branch sites provides a number of binding sites for splicing factors. In a similar way, we can suggest that the presence of several branch sites could increase the recruitment of splicing factors, allowing the stabilization of U2 snRNP on the selected branch site.

The results presented here show that the splicing defect occurs at an early stage of spliceosome assembly (Fig. 2). We can suggest that the mutation interferes with some steps required for the formation of the A complex. Among the numerous proteins required for stable U2 snRNP interaction on the branch site is the splicing factor U2AF(43) . It is an abundant component of E complex whose binding to the pre-mRNA is strongly dependent on the 3` splice site and the integrity of the pyrimidine tract(37, 44) . SF3a and SF3b splicing activities are also involved in the U2 snRNP binding process(45, 46) . Recently, a p80 protein, which associates with the branch site sequence prior to A complex formation, has been identified, and it has been proposed that this protein could be involved in the communication between U1 snRNP and the branch region(47) . One possibility could be that the mutation C U at the 3` splice site blocks the interaction of one or several of these proteins, thus preventing the recognition of the branch site by the U2 snRNP particle. Currently experiments are in progress to understand the molecular mechanism by which the mutation C U at position -3 upstream of the 3` splice site prevents the splicing of intron 10.


FOOTNOTES

*
This work was supported by Grant 881002 from the CNRS, by funds from the INSERM, the Association Française contre les Myopathies, the Association pour la Recherche sur le Cancer, and the Ligue Nationale Contre le Cancer, and by Grant 405209101 from the Committee for Polish Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Inst. of Human Genetics Polish Academy of Sciences, 32 Strzeszynska, 60-479 Poznan, Poland.

To whom correspondence should be addressed. Tel.: 33-69-82-38-00; Fax: 33-69-82-33-54.

(^1)
The abbreviations used are: U snRNP, U-rich small nuclear ribonucleoprotein particule; nt, nucleotide(s).


ACKNOWLEDGEMENTS

We thank members of our laboratory and particularly M. Gallego, P. Sirand Pugnet, and A. Expert Bezançon for helpful discussions and critically reading the manuscript. We are grateful to P. Durosay and R. Matuszak for technical support. Special thanks to K. Tanner for carefully reading the manuscript.


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