(Received for publication, December 29, 1995; and in revised form, February 22, 1996)
From the
Alternative splicing of vertebrate -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
-tropomyosin alternative exons is not reproduced
in transfection experiments when heterologous myogenic cells are used.
By analyzing the splicing pattern of hybrid chicken/rat
-TM
constructions transfected into both quail and mouse cell lines, we
demonstrate that chicken
-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
-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.
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 -tropomyosin (
-TM) (
)pre-mRNA provides an interesting model system for the
study of the mechanism of splice site selection. For the chicken
-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 -TM genes is highly
conserved, and parallel studies of the chicken and rat
-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
-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
-TM gene are not recognized by the splicing machinery in quail
fibroblasts and myogenic cells, whereas exons 6A of the Xenopus and chicken
-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 -TM genes could account for the observed
misregulation of splicing in heterologous systems, we generated hybrid
constructions between the chicken and the rat
-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
-TM gene.
Figure 3:
Transcript analysis of quail QT6
fibroblasts transfected with hybrid rat/chicken -TM constructions. A, schematic representation of
-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
-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
-TM wild-type and hybrid constructions. A, schematic
representation of
-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
-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
-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
-TM and P1 constructions) and
to the right (rat wild-type
-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
-TM constructions. The figure is organized just as in B, except that the rat wild-type
-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 -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
-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 1. For the Rat+Pur and Rat+Pyr
constructions, a SacI site was first introduced into the
wild-type rat
-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
-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 -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
-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.
In mouse C2 myotubes, the rat
-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
-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
-TM minigene (<5% exon 6A inclusion; Fig. 1, A and C, lanes 3 and 4). Therefore,
the rat
-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
-TM minigene. This result
indicates that chicken
-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
-TM intronic sequences flanking exon 6A (chicken wild-type
minigene and RP1) confers exon 6A inclusion, while that of rat
-TM
intronic sequences flanking exon 6A (rat wild-type minigene and P1)
leads to exon 6A skipping.
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 -TM gene are associated with exclusion of exon
6A, while the analogous sequences of the chicken
-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
-TM gene.
Figure 2:
Sequence comparison of the splicing
elements flanking exon 6A of the chicken and rat -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.
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 -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
-TM intron
5 in HeLa nuclear extracts by replacing the central C
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
-TM genes, have a mixture of CU dinucleotides, as
well as stretches of both U and C nucleotides.
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
-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) .
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 -TM gene relies strongly on the presence of intronic
splicing enhancers. In the case of the rat
-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.