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Volume 271, Number 19, Issue of May 10, 1996 pp. 11511-11517
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence Divergence Associated with Species-specific Splicing of the Nonmuscle -Tropomyosin Alternative Exon (*)

(Received for publication, December 29, 1995; and in revised form, February 22, 1996)

Anne-Marie Pret(§), (¶),  ,  Marc Y. Fiszman(¶)(**)

From the Department of Molecular Biology, Pasteur Institute, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.

INTRODUCTION

Splicing of pre-mRNA in eukaryotes requires the accurate pairing of 5` and 3` splice sites in order to obtain functional mRNA. The selection of splice donor and acceptor sites involves the interaction between loosely conserved sequence elements present at the exon/intron borders with small nuclear ribonucleoproteins and several auxiliary factors to form the functional spliceosome (reviewed in Refs. 1 and 2). However, it is now clear that such sequence elements alone are not sufficient to define exon/intron borders and that a variety of additional sequence and structural elements present in both exons and introns are important for efficient utilization of splice sites(3, 4, 5, 6, 7, 8) .

Alternative splicing of vertebrate beta-tropomyosin (beta-TM) (^1)pre-mRNA provides an interesting model system for the study of the mechanism of splice site selection. For the chicken beta-TM gene, we have shown that all of the sequences necessary to splice exon 6B specifically in skeletal muscle cells (quail differentiated myotubes) and exon 6A in all other cell types (quail myoblasts and fibroblasts) are present in a minigene consisting of the genomic sequences from exon 5 to exon 7(9, 10) . A complex combination of sequence and structural elements has been shown to be responsible for the repression of exon 6B in nonmuscle cells (11, 12, 13, 14, 15) . In quail myotubes, exon 6B is derepressed and we have shown that competition between the exon 6A and 6B splice sites for pairing with the exon 5 and 7 splice sites favors utilization of exon 6B(16) . Therefore, exon 6A, like other alternatively spliced exons, is associated with suboptimal splice sites that are sensitive to competition from flanking splice sites. Furthermore, in nonmuscle cells, efficient utilization of the splice sites flanking exon 6A requires an intronic splicing enhancer (S4), which consists of a 33-nucleotide polypyrimidine-rich tract located 37 nt downstream of exon 6A(12, 17) .

The structural organization and expression pattern of the vertebrate beta-TM genes is highly conserved, and parallel studies of the chicken and rat beta-TM genes indicate that the exon 6B repressor elements are conserved and that exon 6A splice sites in both species are inefficiently spliced relative to the consensus sequences(15, 18, 19, 20, 21) . However, when equivalent beta-TM minigene constructions from three different vertebrate species, namely chicken, rat, and Xenopus, are transfected into heterologous cell backgrounds, splicing of both exons 6A and 6B is deregulated while constitutive exons within the same constructions are accurately spliced(22) . In particular, exons 6A and 6B of the rat beta-TM gene are not recognized by the splicing machinery in quail fibroblasts and myogenic cells, whereas exons 6A of the Xenopus and chicken beta-TM genes become constitutive exons since they are included in mature transcripts in mouse myogenic cells, irrespective of the state of differentiation of the cells. These results are interesting since differences between the splicing machinery among vertebrates have not been reported previously. In contrast, sequence differences inhibiting splicing of vertebrate introns in invertebrate systems and vice versa have been characterized, including differences in the consensus splice site sequences and differences in the size and nucleotide composition of introns(23, 24, 25, 26, 27, 28) .

In order to determine whether sequence divergence between the chicken and rat beta-TM genes could account for the observed misregulation of splicing in heterologous systems, we generated hybrid constructions between the chicken and the rat beta-TM minigenes and transfected them into both quail and mouse cell lines. Here we demonstrate that sequence elements necessary for species-specific regulation of exon 6A splicing are present in the introns flanking exon 6A, while the exonic sequences of these two genes are interchangeable. Our experiments reveal, in particular, a significant disparity in the splicing efficiency of chicken and rat exon 6A, which can be attributed to nonconsensus nucleotide differences at the 5` and 3` splice sites flanking exon 6A and to a newly identified pyrimidine-rich splicing enhancer for exon 6A of the chicken beta-TM gene.

MATERIALS AND METHODS

Constructions

The chicken wild-type beta-TM minigene plasmid pBS/SV-betaalt contains a genomic beta-TM fragment spanning exons 5-7 and has been described previously(10) . The rat wild-type genomic beta-TM fragment spanning exons 5-8 (29) was subcloned into the same SV40 expression vector used for the chicken beta-TM sequences(10) . To generate the chicken/rat hybrid beta-TM minigene constructions, a derivative of pBS/SV-betaalt, called Delta1, was used that contains a deletion of chicken beta-TM intron 5 (the intron between exons 5 and 6A, except for the first 10 bp), exon 6A, and the first 9 bp of intron 6A (the intron between exons 6A and 6B), which have all been replaced by a KpnI restriction enzyme site(17) . A reconstituted chicken wild-type beta-TM minigene (Ckpn) and the hybrid constructions P1-P7 (Fig. 3A) were all generated using the polymerase chain reaction (PCR, (30) ) with oligonucleotides that introduce KpnI restriction enzyme sites near the ends of the final PCR products for ligation into the Delta1 KpnI site. The newly introduced KpnI sites in Ckpn do not alter the splicing pattern of this construction with respect to pBS/SV-betaalt (data not shown). All PCR-generated plasmid constructions were sequenced using Sequenase (U. S. Biochemical Corp.) to verify that no nucleotide errors were introduced. To generate the P1 construction, rat beta-TM intron 5 (except the first 7 bp), rat exon 6A, and the first 17 bp of rat intron 6A were PCR-amplified from the rat wild-type minigene plasmid. To generate the P2 construction, the 5` half of intron 5 (143 bp starting 10 bp downstream of exon 5) of the wild-type chicken beta-TM minigene was fused to the 3` half of rat beta-TM intron 5 (the last 50 bp), followed by rat exon 6A and the first 17 bp of rat intron 6A, by overlap extension PCR mutagenesis(31) . For P3, the 3` half of chicken beta-TM intron 5 (the last 63 bp) was PCR-amplified from the chicken wild-type minigene with a 5` oligonucleotide primer, which also contained sequences from rat intron 5 including an EcoRII site, and a 3` primer, which also contained sequences from the 5` end of rat exon 6A including a PvuII site. In a separate PCR reaction, P1 sequences from exon 5 to 17 bp downstream of exon 6A were amplified and the amplification product was digested by EcoRII in rat intron 5 and PvuII in rat exon 6A. The 5` and 3` fragments released by this digestion were ligated to the 5` and 3` ends of the first PCR product, also digested with EcoRII and PvuII, such that chicken beta-TM exon 5 and the first 10 bp of chicken intron 5 are followed by the 5` half of rat intron 5 (the first 75 bp starting 7 bp downstream of rat exon 5), the 3` half of chicken intron 5, rat exon 6A, and the first 17 bp downstream of rat exon 6A. This ligation product was PCR amplified and cloned into the Delta1 KpnI site. For constructions P4-6, intron 5, exon 6A, and the 5` end of intron 6A of constructions P1, P3, and P2, respectively, were PCR amplified with oligonucleotide primers that replaced the first 17 bp of rat beta-TM intron 6A by the first 9 bp of chicken beta-TM intron 6A. For P7, chicken beta-TM exon 5 and intron 5 were fused to rat beta-TM exon 6A and the first 17 bp downstream of this exon by overlap extension PCR mutagenesis.

Figure 3: Transcript analysis of quail QT6 fibroblasts transfected with hybrid rat/chicken beta-TM constructions. A, schematic representation of beta-TM sequences present in hybrid rat/chicken minigene constructions and the results of the quantification of transcripts after stable transfection of these constructions in QT6 fibroblasts. For hybrid constructions P2-P7, only the region that has been modified with respect to P1 is shown. The relative position of the branchpoints upstream of exon 6A is marked with a dot. Otherwise, the figure is organized just as in Fig. 1A. B, RT-PCR analysis of transcripts produced upon stable transfection of QT6 cells with beta-TM constructions. The size and structure of the P1-P7 PCR products amplified with the same primer pair are indicated to the left of the figure. The radioactively end-labeled HaeIII-digested X174 DNA is included as a size marker.


Figure 1: Transcript analysis of quail QT6 fibroblasts and mouse C2 myotubes transfected with rat and chicken beta-TM wild-type and hybrid constructions. A, schematic representation of beta-TM sequences present in rat and chicken wild-type and hybrid minigene constructions and the results of the quantification of transcripts after stable transfection of these constructions in QT6 and C2 cell lines. The chicken beta-TM sequences are indicated by white boxes and the rat sequences by black boxes. The exons are marked 5, 6A, 6B, and 7. The exons and introns are drawn only roughly to scale, and the relatively long intron between exons 6A and 6B is shown interrupted. The sizes of the relevant intronic regions are indicated. The quantification represents the percentage of the total transcripts that contain exon 6A. To avoid clonal variation, RT-PCR was carried out on total RNA isolated from a mixed population of 45-100 clones of stably transfected QT6 or C2 cells. B, RT-PCR analysis of transcripts produced upon stable transfection of QT6 cells with beta-TM constructions. The minigene transfected is indicated at the top of the gel. The size and structure of the products amplified with the same primer pairs are indicated to the left (chicken wild-type beta-TM and P1 constructions) and to the right (rat wild-type beta-TM and RP1 constructions) of the figure. The asterisk denotes the end of the PCR product that is radioactively labeled. Comparison of amplification products shown before(-) and after (+) digestion by NcoI reveals the composition of the co-migrating products. C, RT-PCR analysis of transcripts produced upon stable transfection of C2 cells with beta-TM constructions. The figure is organized just as in B, except that the rat wild-type beta-TM and RP1 amplification products were digested with PvuII.


In order to generate the RP1 construction, KpnI sites were introduced 6 bp downstream of exon 5 and 17 bp downstream of exon 6A of the rat wild-type beta-TM minigene plasmid by oligonucleotide-directed mutagenesis on single-stranded DNA (Rkpn)(17, 32) . The modifications in Rkpn do not alter the splicing pattern of this construction with respect to the rat wild-type beta-TM minigene (data not shown). Subsequently, the chicken intron 5 and rat exon 6A sequences of P7 were PCR-amplified with primers that introduced the chicken donor site sequence downstream of exon 6A and KpnI sites at either end of the PCR product in order to replace the KpnI fragment of Rkpn.

The P1don and P1env constructions were generated by PCR mutagenesis of the P1 construction with oligonucleotides containing the sequence modifications (see Fig. 4A) and KpnI sites for cloning into Delta1. For the Rat+Pur and Rat+Pyr constructions, a SacI site was first introduced into the wild-type rat beta-TM minigene 26 nt downstream of exon 5 by oligonucleotide-directed mutagenesis on single-stranded DNA and, subsequently, oligonucleotides containing the Pyr and complementary Pur sequences (see Fig. 4B) and SacI sites at the ends were annealed and ligated into the newly created SacI site of the rat beta-TM minigene.

Figure 4: A, RT-PCR analysis of transcripts produced upon transient transfection of QT6 cells with P1-derivative constructions containing modifications within the donor site region immediately downstream of exon 6A. The sequences at the 3` exon/intron border of exon 6A in these constructions, which represent the only differences between these constructions, is shown below the gel. The size and structure of the amplified products are indicated to the left of the gel. The quantification represents the percentage of total transcripts that contain exon 6A, as averaged from the results of several independent transient transfections showing a variation of less than 20%. B, RT-PCR analysis of transcripts produced upon transient transfection of QT6 cells with rat beta-TM-derivative constructions, which contain the insertion, 26 nt downstream of exon 5, of either the pyrimidine stretch (Rat+Pyr) present in intron 5 of the chicken beta-TM gene or the complementary sequence (Rat+Pur). The sequences inserted are indicated below the gel. The percentage inclusion of exon 6A was calculated as indicated in A.


Cells and Transfections

The culture conditions for the murine myogenic C2 cell line have been described(33) , as well as those for the avian fibroblastic cell line, QT6(34) . Transfections of these cell lines were carried out as described(22) .

cDNA PCR Analysis of the Transcripts

Total RNA was isolated from QT6 fibroblasts and C2 myotubes as described(35) . For analysis of the mature splicing products, specific cDNAs of the harvested transcripts were obtained with a first strand reverse transcription primer complementary to SV40 sequences 3` to the cloned beta-TM sequences as described previously(17) . PCR amplifications between exon 5 and the SV40 sequences in the 3` end of the various beta-TM minigene constructions were carried out with radioactively end-labeled oligonucleotide primers and the amplification products were digested with either NcoI or PvuII, which are present in exon 6A of both the chicken and rat beta-TM minigenes(17, 22) . A [-P]ATP end-labeled X174 HaeIII-digested DNA ladder (Promega, Madison, WI) was used as a size marker for all gels (not shown for all gels). Quantification of radioactive PCR products was performed on a Molecular Dynamics PhosphorImager. Sequencing of PCR products was achieved by replacing one of the PCR primers with a biotinylated oligonucleotide, which allowed subsequent single-stranded solid phase sequencing on magnetic beads coated with streptavidin as indicated by the manufacturer (Dynal, Oslo, Norway).

RESULTS

Exonic Chicken and Rat beta-TM sequences Are Interchangeable, while Intronic Sequences Are Responsible for Deregulated Splicing in a Heterologous Cell Context

In stably transfected quail fibroblasts (QT6 cell line), the chicken wild-type beta-TM minigene generates >95% spliced transcripts containing exons 5, 6A, and 7 (Fig. 1, A and B, lanes 1 and 2), while the rat wild-type beta-TM minigene produces mostly mature transcripts that skip directly from exon 5 to 7 (Fig. 1B, lanes 5 and 6) and only 8% of the mature transcripts contain exon 6A (Fig. 1, A and B, lanes 5 and 6, only observed with longer exposures). In order to identify which sequences in the rat beta-TM gene prevent utilization of rat exon 6A in this cellular context, we generated hybrid constructions between the chicken and rat beta-TM minigenes (RP1 and P1, Fig. 1A). In the RP1 construction, all beta-TM sequences derive from the rat minigene except for intronic sequences flanking exon 6A, which derive from the chicken minigene. In the P1 construction, all beta-TM sequences derive from the chicken minigene except for exon 6A and its flanking intronic sequences, which derive from the rat minigene. The RP1 construction is spliced like the chicken wild-type beta-TM minigene in quail fibroblasts, in that the majority of the transcripts contained exon 6A (>95%, Fig. 1, A and B, lanes 7 and 8). These results exclude all the exonic rat beta-TM sequences present in RP1, as well as all the splice sites associated with all the exons except exon 6A, from being responsible for rat exon 6A skipping in quail fibroblasts. Conversely, the high rate exon 6A inclusion for RP1 indicates that the minimum chicken beta-TM sequences required for efficient exon 6A splicing in quail cells are the intronic sequences immediately flanking this exon in RP1 (Fig. 1A). The P1 construction, on the other hand, behaves like the rat wild-type beta-TM minigene when transfected into quail fibroblasts: a minority (12%, Fig. 1A) of the mature transcripts contain exon 6A, while the majority of the transcripts skip from exon 5 directly to exon 7 (Fig. 1B, lanes 3 and 4). These results indicate that intronic rat beta-TM sequences flanking exon 6A in P1 (Fig. 1A) are not recognized by the splicing machinery in quail fibroblasts.

In mouse C2 myotubes, the rat beta-TM minigene generates mostly mRNA spliced directly from exon 5 to exon 6B, while exon 6A is included in approximately 10% of the mRNA (Fig. 1, A and C, lanes 5 and 6). In contrast, inclusion of chicken beta-TM exon 6A is high (80%) in these cells (Fig. 1, A and C, lanes 1 and 2). Analysis of C2 stable transfectants containing the P1 hybrid construction showed that P1 is regulated like the rat beta-TM minigene (<5% exon 6A inclusion; Fig. 1, A and C, lanes 3 and 4). Therefore, the rat beta-TM intronic sequences flanking exon 6A in P1 are sufficient to obtain proper down-regulation of exon 6A in mouse C2 myotubes. In contrast, the RP1 hybrid construction exhibits high (95%) exon 6A inclusion (Fig. 1, A and C, lanes 7 and 8) like the chicken beta-TM minigene. This result indicates that chicken beta-TM intronic sequences immediately flanking exon 6A in RP1 are responsible for deregulated splicing of chicken exon 6A in mouse myogenic cells. Taken together (see Fig. 1A), our results show that in both cellular contexts, quail fibroblasts and mouse myotubes, the presence of chicken beta-TM intronic sequences flanking exon 6A (chicken wild-type minigene and RP1) confers exon 6A inclusion, while that of rat beta-TM intronic sequences flanking exon 6A (rat wild-type minigene and P1) leads to exon 6A skipping.

Regulation of Exon 6A Utilization Requires Three Independent Intronic Sequence Elements: the Donor and Acceptor Site Regions Flanking Exon 6A and Sequences Upstream of the Intron 5 Branchpoint

The rat beta-TM intronic sequences, present in the P1 construction and responsible for exclusion of exon 6A in quail fibroblasts and mouse myotubes, can be divided into three separate regions (Fig. 1A and 2): 1) the donor site and donor site context immediately downstream of exon 6A, 2) the acceptor site region immediately upstream of exon 6A, and 3) the intron 5 sequences between the donor site and the branchpoint(s). We tested each of these three rat beta-TM regions independently for their contribution to rat exon 6A skipping in quail fibroblasts and mouse myotubes. To do so, we exchanged each one of these regions within the P1 construction with the corresponding sequences from the chicken beta-TM minigene (P2, P3, and P4, Fig. 3A). Analysis of the mature transcripts produced in QT6 stable transfectants containing the P2, P3, and P4 constructions showed significant activation of exon 6A splicing for all three constructions (55%, 85%, and 60%, respectively) relative to the P1 construction (8%) (Fig. 3, A and B, lanes 1-8). These results demonstrate that each of the three rat intronic elements, namely the donor region, the acceptor region, and the intron 5 distal region, contributes to the poor recognition of rat exon 6A in this cellular background. In the P5, P6, and P7 hybrid constructions, different combinations of two of the three rat intronic elements in P1 were exchanged with the corresponding chicken beta-TM sequences (Fig. 3A). With these constructions, even higher rat exon 6A inclusion rates are obtained (95%, 90%, and 90%, respectively; Fig. 3, A and B, lanes 9-12). These rates are close to those observed for the chicken wild-type beta-TM minigene (>95%, Fig. 1A).

As in quail fibroblasts, transfection of the P1-P7 hybrid constructions into mouse C2 myotubes shows that the same three intronic regions flanking exon 6A of the rat beta-TM gene are associated with exclusion of exon 6A, while the analogous sequences of the chicken beta-TM gene activate splicing of this exon (data not shown). Taken together, our experiments demonstrate the presence of independent exon 6A splicing regulatory signals, not only in the donor and acceptor site regions flanking exon 6A, but also in the region upstream of the branchpoint of intron 5 of the chicken and rat beta-TM gene.

Nonconsensus Nucleotides within the Chicken Exon 6A Donor Site Confer Rat Exon 6A Utilization in Quail Cells

Our results clearly demonstrate that the intronic sequences flanking the chicken and rat beta-TM exon 6A are not functionally equivalent, since exchanging these sequences does not allow proper splicing regulation of this exon in quail nonmuscle cells and mouse myotubes, respectively. The P1 and P4 constructions only differ in the presence of the rat and chicken exon 6A donor site within their own sequence contexts, respectively ( Fig. 2and Fig. 3A). The chicken and rat exon 6A donor sites differ at positions +4 and +6 relative to the exon/intron border, neither site containing the consensus nucleotide at these positions. The exon 6A donor site context in P1 and P4 also differs since the sequences following the rat and chicken exon 6A donor sites have diverged (Fig. 2). In order to determine which differences in the chicken and rat exon 6A donor site and/or donor site context are responsible for the difference in utilization of chicken and rat exon 6A, we generated P1-derivative constructions that pair either the chicken donor site with the context of the rat donor site (P1don, Fig. 4A) or the rat donor site with the context of the chicken donor site (Plenv, Fig. 4A).

Figure 2: Sequence comparison of the splicing elements flanking exon 6A of the chicken and rat beta-TM genes. The donor, acceptor, and branch site sequences are also compared to the vertebrate consensus sequences. The exon/intron borders are indicated by a slash. The donor and acceptor site positions that diverge from the consensus sequence are boxed and underlined, respectively. The sequences immediately downstream of the donor site that comprise the donor site context are shown in lowercase letters. Intronic sequences immediately upstream of exon 6A are shown in the section ``pyrimidine tracts,'' and two of the mapped branchpoints upstream of rat exon 6A are underlined(20) , as well as the first potential branchpoint upstream of chicken exon 6A. The distal portion of intron 5 refers to the sequences between the donor site and branchpoint(s).


In transiently transfected QT6 fibroblasts, the P1 construction shows almost no splicing of exon 6A (5% of mature transcripts, Fig. 4A), while exchanging the rat donor site and donor site context in P1 by that of the chicken (P4) leads to 37% splicing of exon 6A (Fig. 4A). QT6 transient transfection experiments with the P1don construction indicated that 26% of the mature transcripts contain exon 6A with this construction (Fig. 4A), while the P1env construction shows no activation of exon 6A splicing with respect to P1 (data not shown). These results indicate that the two-nucleotide difference between rat and chicken exon 6A donor sites present in the P1 and P1don constructions, respectively, but not the differences in the exon 6A donor site context, are responsible for the poor recognition of the rat donor site in quail cells.

A Pyrimidine-rich Sequence in Chicken Intron 5 Activates Utilization of Rat Exon 6A Splice Sites in Quail Cells

Exchanging only the 5` half of rat beta-TM intron 5 in P1 with the corresponding region of the chicken beta-TM gene (P2, Fig. 3A) leads to an increased splicing efficiency of the downstream rat exon 6A in both quail fibroblasts and mouse myotubes (Fig. 3B and data not shown). These regions of chicken and rat intron 5 differ in size (approximately 143 and 95 nt, respectively) and in the striking presence, only in the chicken beta-TM gene, of a stretch of 33 nt comprising 85% pyrimidines and located 25 nt downstream of exon 5. Since we have previously identified a pyrimidine-rich splicing enhancer downstream of chicken beta-TM exon 6A, we tested here the possibility that this upstream pyrimidine-rich sequence may also act as a splicing enhancer. The chicken intron 5 33-nt pyrimidine-rich sequence was inserted into the rat beta-TM minigene, 26 nt downstream of exon 5 (Rat+Pyr, Fig. 4B), and, for comparison, the complementary purine-rich sequence was inserted at the same position (Rat+Pur. Fig. 4B). In transiently transfected QT6 cells, the rat wild-type beta-TM minigene exhibits very low levels of exon 6A splicing (5%, Fig. 4B). In the presence of a purine tract in intron 5, there is no change in the utilization of rat beta-TM exon 6A compared to the rat wild-type minigene (Fig. 4B). In contrast, the addition of the pyrimidine stretch leads to 18% inclusion of rat exon 6A in quail cells (Fig. 4B). Therefore, the pyrimidine tract upstream of the branchpoint of chicken intron 5 functions as an exon 6A splicing enhancer when placed within the rat beta-TM minigene.

DISCUSSION

Species Specificity of beta-TM Exon 6A Splicing Is the Result of a Disparate Splicing Efficiency

Splicing of the nonmuscle exon 6A of the chicken and rat beta-TM gene is strictly regulated when minigene constructions are transfected into quail and mouse myogenic cells, respectively, but not when transfected into a heterologous cell background(22) . Our results demonstrate that the exon sequences of the chicken and rat beta-TM genes are entirely interchangeable (exon 6A in these two species differs at 13 out of 75 nt). In contrast, the intronic sequences flanking chicken and rat exon 6A have diverged significantly, so that these sequences are not functionally equivalent. In both quail QT6 fibroblasts and mouse C2 myotubes, we show that the rat beta-TM exon 6A donor and acceptor sites flanking exon 6A, as well as the intron 5 sequences upstream of the branchpoints, are not efficiently recognized by the splicing machinery, while the cognate chicken beta-TM sequences ensure efficient utilization of exon 6A in these cells. The misregulation of exon 6A splicing presented here can therefore be interpreted as being due to a significantly greater splicing efficiency for chicken versus rat exon 6A splice signals. In order to obtain proper splicing regulation of this exon then, a specific adaptation is needed between the intrinsic strength of the regulated splice sites and the ``strength'' that the nuclear environment can provide in the way of specific splicing factors and/or the balance of general splicing factors. In particular, from our results we can infer that the relatively weaker rat beta-TM exon 6A splice sites require at least one potentiating factor for efficient splicing in mammalian nonmuscle cells. A screen for such a factor(s) is under way.

The Disparate Splicing Efficiency between Exon 6A of the Chicken and Rat beta-TM Genes Is Associated with Nonconsensus Nucleotides in the Flanking Splice Sites

The donor splice site adjacent to beta-TM exon 6A in both chicken and rat is degenerate with respect to the consensus sequence at the same intronic positions (Fig. 2). Both splice sites contain a U instead of a G at the highly conserved position +5 relative to the exon/intron border (present in 80-90% of vertebrate donor sites; (36) ). In addition, neither donor site contains the consensus nucleotides at the two positions immediately flanking the U (A and Y are found at these two positions in approximately 70% and 60% of vertebrate donor sites, respectively). The sequence context immediately downstream of these donor sites also differs between the two species (Fig. 2). We showed that the two nucleotidic sequence differences at positions +4 and +6 of the donor site alone, and not the donor site context, is responsible for the disparate splicing efficiency of the chicken and rat donor sites in quail cells. We have shown previously that exon 6A splicing in beta-TM minigene constructions derived from the owl, Otus scops, and from Xenopus, like that of chicken beta-TM exon 6A, is inappropriately activated in mouse C2 myotubes(22) . The contribution of the exon 6A donor site to this misregulation of splicing is supported by the fact that both the owl and Xenopus beta-TM genes contain the same exon 6A donor site sequence as the homologous chicken gene. (^2)

Recognition of the intronic portion of the donor site sequence is thought to occur sequentially by base pairing first with U1 snRNA, and, subsequently, with U6 snRNA over the course of the splicing reaction (37, 38, 39, 40, 41, 42, 43, 44) . The G at intronic position +5 of the donor site is thought to be important for both of these events. Given that little is known about donor site recognition, there are several possibilities for the significantly different efficiency of splicing at the chicken and rat exon 6A donor sites. The repetition GUAGUA in the rat exon 6A donor site may present two possible binding sites for U1 snRNA which could interfere with splicing at both sites. Alternatively, there may be more favorable alternative sites for U6 snRNA base pairing for the chicken than for the rat exon 6A donor site. Since vertebrate donor sites can be highly divergent relative to the consensus sequence, it is likely that additional RNA-RNA and RNA-protein interactions are needed to specify the donor site in these organisms. This is probably the case for the donor sites that do not contain a G at position +5, as is the case for the exon 6A donor sites. It is possible that snRNA variants and/or splicing factors not yet characterized are involved in recognition of these divergent splice sites.

The rat and chicken exon 6A acceptor site regions (Fig. 2) both contain branch site sequences with 6/6 matches to the consensus sequence, CAG/C cleavage sites that diverge from the consensus (CAG/G) at exonic position +1, which is a purine in 80% of vertebrate acceptor sites (36) , and a suboptimal pyrimidine tract interspersed with purines. Roscigno et al.(45) demonstrated that the presence of consecutive uracils substituting for cytosines in the polypyrimidine tract of an adenovirus 2 intron leads to improved splicing. The rat beta-TM intron 5 polypyrimidine tract, which is also particularly poor in uracil and contains instead three C stretches (Fig. 2), could account for the low efficiency of splicing of rat exon 6A splice sites. Significantly, Tsukahara et al.(46) , enhanced splicing of rat beta-TM intron 5 in HeLa nuclear extracts by replacing the central C(5) stretch and the flanking two G nucleotides by a stretch of 7 U nucleotides. It is not possible to determine from their experiments whether the increased splicing efficiency is due to the conversion of the C stretch or of the two purines or both. In contrast, the chicken intron 5 polypyrimidine tract, as well as that of the owl and Xenopus beta-TM genes, have a mixture of CU dinucleotides, as well as stretches of both U and C nucleotides.^2 From the experiments presented here, we demonstrate how subtle differences in the sequence composition of the acceptor site region, probably at the level of the pyrimidine tract, can induce large variations in the splicing efficiency of exon 6A of the vertebrate beta-TM genes.

Our results indicate that present definitions of splice site consensus sequences do not contain all the information necessary to determine splice site strength, probably due to a bias in the genes that have been characterized to date. Surveys of different subsets of splice sites, for example those associated with alternative exons expressed in neuronal cells, define consensus splice site sequences that differ from those of the general consensus splice sites(47) .

Recognition of Chicken beta-TM Exon 6A Involves Several Intronic Splicing Enhancers

The introduction of a 33-nt stretch made up of 85% pyrimidines, present 25 nt downstream of chicken but not rat exon 5, into the same position in intron 5 of the rat beta-TM minigene, leads to significant activation of rat exon 6A splicing in quail cells. These results indicate the presence of a splicing activating sequence in chicken intron 5, rather than the presence of negative elements in rat intron 5. Furthermore, the addition of the complementary purine-rich sequence at the same position of rat beta-TM intron 5, which has no effect on exon 6A splicing, indicates that the relatively smaller size of rat intron 5 probably does not contribute to the poor splicing efficiency of this intron in quail cells. In addition, deletion of the 33-nt pyrimidine-rich sequence from the chicken wild-type beta-TM minigene leads to a decrease in exon 6A utilization.^2 Significantly, the owl beta-TM intron 5 contains a sequence with 16/20 nt identity to part of the chicken intron 5 pyrimidine-rich splicing enhancer at approximately the same position in intron 5, and Xenopus beta-TM intron 5 also has pyrimidine-rich elements upstream of the potential branchpoint.^2

There are not many other examples of intronic sequences upstream of a branchpoint contributing to splicing regulation, and the mechanism by which these sequences function is unknown(48, 49, 50) . We have shown previously that splicing of the chicken exon 6A depends on the presence of a similar pyrimidine-rich intronic enhancer sequence (S4) located 30 nt downstream of exon 6A(12, 17) . In the present work, we provide additional evidence that splicing of exon 6A of the chicken beta-TM gene relies strongly on the presence of intronic splicing enhancers. In the case of the rat beta-TM gene, where neither of these enhancers exist (our results and (18) ), a different mechanism for the recognition of exon 6A has to be involved.

FOOTNOTES

*
The work was supported in part by grants from INSERM, the Association des Myopathes de France, CNRS, Association de Recherche sur le Cancer, the Ligue Française contre le Cancer, and the Fondation pour la Recherche Médicale Française. 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.

§
Supported by the Ligue Française contre le Cancer, the Société des Amis des Sciences, and the Association des Myopathes de France.

Present address: Unité INSERM 153, Pavillon Rambuteau, Groupe Hospitalier Pitié-Salpétrière, 47, blvd. de L'Hôpital, 75651 Paris Cedex 13, France.

**
To whom correspondence should be addressed. Tel.: 33-1-42-17-68-06; Fax: 33-1-42-17-68-11.

(^1)
The abbreviations used are: beta-TM, beta-tropomyosin; nt, nucleotide(s); bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; snRNA, small nuclear RNA.

(^2)
A.-M. Pret, unpublished observations.

ACKNOWLEDGEMENTS

We thank D. Helfman for the rat beta-TM minigene construction. We also thank Laurent Balvay, Maria Gallego, Hend Farza, and Laurent Théodore for helpful discussions and critical reviews of the manuscript.

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