The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5 Splice Sites

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, Mar. 1997, p Vol. 17, No /97/$ Copyright 1997, American Society for Microbiology The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5 Splice Sites CHANG ZHU, JUN URANO, AND LESLIE R. BELL* Molecular Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, California Received 16 July 1996/Returned for modification 24 September 1996/Accepted 16 December 1996 The Sex-lethal (Sxl) early transcripts have a unique 5 exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5 splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron. Splicing reactions remove intron sequences from premrna and join exons together with exquisite precision. These reactions are carried out within the spliceosome, using several snrna molecules and many associated proteins. Accuracy in splicing requires that the appropriate pre-mrna splice sites, which may be separated by vast distances, be recognized and distinguished from among many closely related sequences. Three minimum sequence elements have been identified (33 35). At the 5 splice site, there is a short consensus sequence that is complementary to sequences of U1 snrna; the branch point sequence is recognized by U2 snrna; at the 3 splice site, the last two nucleotides of the intron are invariantly AG (31). In addition, in higher eukaryotic cells, a polypyrimidine tract is usually located between the branch point and 3 splice site; it is bound by the protein U2AF, which enhances the binding of U2 snrna to the branch point (56, 57). To some degree, the likelihood of choosing a particular splice site correlates with how closely it matches the consensus sequence. Nevertheless, these limited sequence requirements are not sufficient to explain the accuracy observed in splice site selection, as sequences matching the consensus are often found unused within introns. Additional features have also been identified as important for selection of the correct pair of 5 and 3 splice sites (5). Sequence context beyond the splice sites and branch point can influence splice site selection and splicing efficiency. For example, exon sequences have been known for some time to influence splice site selection, and several exonic purine-rich splicing enhancers have recently been characterized (12, 24, 28, 47, 53, 54). Some of these enhancers have been shown to be bound by members of the SR family of proteins, which as a class appear to be involved in joining 5 and 3 splice sites across exons as well as introns (14). While SR proteins are essential for splicing in general, they can also differentially influence the choice of splice sites in a concentration-dependent manner and therefore seem able to affect alternative splicing. In addition, there are specific splicing factors whose function is limited to the regulation of a few alternatively * Corresponding author. Phone: (213) Fax: (213) LBELL@mizar.usc.edu. spliced genes such as transformer, Sex-lethal (Sxl), and doublesex (35). These proteins act through specific intronic or exonic pre-mrna sequences to influence splice site selection and exert their effects by either recruiting or blocking components of the general splicing machinery (18, 25, 26, 29, 30, 44, 46, 49 52). Overall, both exon and intron sequences together with their associated binding proteins can participate in constitutive and/or alternative splice site selection. Beyond simple recognition and selection of a splice site, there is also the related problem of how each adjoining pair of splice sites becomes preferentially associated. Often exons appear largely interchangeable, and chimeric transcripts are readily spliced from exons of different genes (5). In some situations, more specific mechanisms for the pairing of particular exons seem important. In the case of the yeast gene RP51B, a region of secondary structure located within the intron near the 5 and 3 splice sites is important for preferential use of that pair of splice sites even when other usable splice sites are made available (16, 36). An argument for preferential pairings of splice sites, interpreted as a hierarchy of compatibilities between pairs of splice sites, has also been proposed for mammalian cells (15). Specifically, preference for two mutually exclusive exons of the myosin light chain 1/3 gene was found to depend on flanking exons, and their mutually exclusive nature was not intrinsic. In general, the data point to cooperation between particular 5 and 3 splice site regions in which certain pairs of splice sites are more likely to be chosen together, but a complete picture of how splice site compatibility might be achieved has not yet emerged. We have studied the regulation of splice site selection by using the Sxl gene as a model system. Sxl has two types of transcripts, early and late, with different promoters and dramatically different splicing patterns. The Sxl early transcripts are activated transiently in early embryos by a female-specific promoter and have a unique 5 exon (E1) located between late exons 1 and 2 (diagrammed in Fig. 1). Exon E1 is spliced to exon 4, which is common to all Sxl transcripts, skipping both exons 2 and 3 (27). In contrast, the late Sxl transcripts derive from an essentially constitutive promoter but are spliced sex specifically (1). The male-specific exon, exon 3, is included by default in all male transcripts and contains in-frame nonsense codons that block Sxl protein production. In the presence of 1674

2 VOL. 17, 1997 Sex-lethal EARLY SPLICE 1675 FIG. 1. A reporter for the Sxl early splice and its deletions. Sxl early and late splicing patterns are shown at the top. The early transcript occurs only in female embryos; it begins at exon E1 and splices to exon 4. The late transcript begins at exon 1, is transcribed in both sexes, and is spliced sex specifically. Exon 3 is skipped exclusively in females under the control of Sxl protein. The minigene B/B consists of a 5.5-kb Sxl genomic fragment (BamHI-BspEI) which was placed under hsp83 control and fused to a T7 phage sequence as a marker. RT-PCR primers used in analysis of transcription patterns of transgenes and transfected cells are shown at the bottom. A summary of the observed splicing patterns is listed on the right, with E1-4 and E1-3 indicating splicing from exon E1 to exon 4 and exon E1 to exon 3. Downloaded from Sxl protein, the late transcripts skip exon 3 and splice in the female pattern. Studies by Horabin and Schedl (19, 20) and Sakamoto et al. (41) have demonstrated that the late splicing pattern can be reconstructed by using a minigene limited to those three exons, both in transformant flies and in Drosophila tissue culture cells. In this way, pre-mrna regions in the introns flanking exon 3 were defined as important for sex-specific splicing. The regions important for splicing of the early transcript are unknown. The function of the Sxl early transcripts, which are present only briefly, is to generate an initial pulse of Sxl protein that serves to direct splicing of the late Sxl transcript into the female mode (27). After the initial phase, Sxl female splicing and female development are maintained throughout life by Sxl protein generated from the late transcripts in a positive autoregulatory loop (1, 9, 27). It is essential that both early and late Sxl splicing be correctly regulated, because inappropriate levels of Sxl expression in either males or females will lead to lethality arising from inappropriate X-chromosome dosage compensation (2, 8, 17). Alternative promoters that produce different transcripts are relatively common in Drosophila melanogaster; however, the alternative first exons usually splice to a common downstream exon. In contrast, the Sxl early splicing pattern skips two exons that are included in the late transcripts. In this report, we compare how splice sites are chosen for these two Sxl transcripts. In particular, we ask what determinants are important for exclusion of exon 3 from the early transcripts but inclusion of exon 3 from the default (male) late transcripts. We demonstrate that even though the early transcripts are present briefly in early embryogenesis whereas the late transcripts are present virtually throughout life, no embryo-specific splicing factors are needed for the early splice. Neither are sex-specific factors required. Instead, the early splicing pattern is dependent on whether the 5 splice site region originates from exon E1 or exon 2. MATERIALS AND METHODS DNA constructs. The control plasmid B/B was constructed from a 5.5-kb BamHI-BspEI Sxl genomic fragment fused to a 300-bp phage T7 sequence. The T7 fragment (13) cloned into the BamHI and EcoRI sites of pgem4z (Promega) was a gift from W. L. Wong and M. Pellegrini. After destruction of the BamHI site by end filling and religation, and removal of the XmaI site by digestion with SacI and SmaI, blunt ending, and religation, the 300-bp T7 sequence was cloned as a PstI-EcoRI fragment into pbluescript KS( ) (Stratagene) to make pt7.bs. A Sxl genomic BamHI-StuI fragment containing sequences from early exon 1 to exon 5 was cloned into the vector pbs2n digested with BamHI and SmaI. pbs2n is a derivative of pbluescript KS( ) in which the KpnI site has been replaced with a NotI linker. The BamHI-NotI Sxl fragment was transferred to the same sites of pcasper-hs83 (19, 20). The SmaI (from the vector polylinker)-to-bspei Sxl fragment was then ligated into pt7.bs digested with ScaI and XmaI. This construct was named pb/b.bs. To make deletion O1, the BamHI-SpeI Sxl fragment was first cloned into those sites of pbs2n. The plasmid was linearized with SpeI and end filled, and a PvuII-StuI genomic Sxl fragment containing exons 3 through 5 was inserted. The resulting plasmid was digested with SmaI and BspEI and then ligated to pt7.bs digested with ScaI and XmaI. Deletion T5 was made by inserting the BamHI- SpeI Sxl fragment into pbs2n, and then the ApaLI-StuI Sxl genomic fragment was inserted into the end-filled SpeI site. The resulting construct was added to pt7.bs as described above. Deletions T1 through T4 and O2 were derivatives of pb/b.bs. For deletion T1, pb/b.bs was digested with SpeI and XbaI and religated. For deletion T2, an on July 22, 2018 by guest

3 1676 ZHU ET AL. MOL. CELL. BIOL. intermediate deletion plasmid was made by digestion with BclI (end filled), ApaLI (end filled), and EcoRI and then religation. That plasmid was then cut with SpeI and XbaI and religated. Deletion T3 was made by digestion with BclI (end filled), XbaI (end filled), and EcoRI and then religation. T4 was made by digestion with ApaLI (end filled), XbaI (end filled), and EcoRI and then religation. Deletion O2 was made by digestion with DraI, NdeI (end filled), and NcoI (in exon 3) and then religation to delete approximately 30 nucleotides (nt) around the exon 2 5 splice site. For deletion O3, pt5.bs was digested with DraI, NdeI (end filled), and NcoI (in exon 3) and then religated. The BamHI-EcoRI fragments from the constructs in pbluescript were then cloned into the BamHI and EcoRI sites of a P-element transformation vector modified from pcasper-hs83 (19, 20). The modification involved replacement of the polylinker sequences between the two PstI sites with a NotI linker and replacement of the polylinker XhoI site with a BamHI linker. B/B includes 204-bp sequences 5 of exon E1. Deletion O4 was made by modification of plasmid B/B. The 0.7-kb XbaI (end-filled)-bamhi fragment and the XhoI (end-filled)-bamhi fragment were joined. For deletion T6, the XhoI (end-filled)-ecori fragment containing the vector was ligated to the HpaI-EcoRI Sxl fragment. The R series was made by PCR (22). Four primers were used for each construct: a primer homologous to sequences at the 5 end of E1 with an introduced BamHI site (BE84; 5 -CCGGATCCATCTTCAGTCGAGTTC-3, BamHI site underlined), a primer homologous to sequences at the XhoI site (BE83; 5 -ATCGATAACAATCTCGAG-3, XhoI site underlined), and pairs of primers that generated the desired deletions. PCR-amplified fragments were either digested with BamHI and XhoI and cloned into pbluescript KS( ) digested with the same enzymes or directly ligated to a T vector as described elsewhere (32). The identities were confirmed by sequencing. The BamHI-XhoI fragments were then used to replace the BamHI-XhoI fragment in B/B. Specific primer pairs used for each deletion were as follows (the PstI site is underlined): R1, 5 -GTGACTCCCTGCAGCAACAATAATCCGGGTAG-3 and 5 -TTGT TGCTGCAGGGAGTCACAGTATC-3 ; R2, 5 -GATGGCGAGGTAAATTTT TAAACTTG-3 and 5 -GTTTAAAAATTTACCTCGCCATCTTAAAG-3 ; R3, 5 -CTAGCTGCAGAATGACAATCCCCAATC-3 and 5 -GGATTGTCATTC TGCAGCTAGTAAATAAGCTGCGC-3 ; R4, 5 -CCCTGGCTGCAGCCCAC ACATATAACACAC-3 and 5 -GTGGGCTGCAGCCAGGGTTGCCATACC -3 ; and R5, 5 -GCTTAGAAACTGCAGAAAATAGTTGCAGTCAACG-3 and 5 -CTATTTTCTGCAGTTTCTAAGCAGATCCCG-3. P-element transformation and transfection. P-element-mediated transformation was done as described by Rubin and Spradling (39). Sxl flies were of the genotype y w Sxl 7BO /Y (42). Schneider line 2 (SL-2) cells (45) were grown in Schneider s medium (Gibco) supplemented with 15% fetal bovine serum at 25 C as described by Di Nocera and Dawid (11). For transfections, cells at a late log phase were diluted to cells/ml, and 15-ml aliquots were seeded in 75-cm 2 T flasks overnight. The cells were transfected with 15 g of reporter plasmid and 45 g of pbluescript DNA per flask by the calcium phosphate technique described previously (11). Cells were harvested 48 to 65 h after transfection. RT-PCR. Total RNA was extracted from adult flies or transfected cells as modified from the method described by Sambrook et al. (43). For each RNA preparation, 50 flies were frozen in liquid nitrogen and homogenized in 500 lof lysis buffer (4 M guanidine thiocyanate, 0.1 M Tris [ph 7.4], 1% -mercaptoethanol). A 0.2 volume of 2 M sodium acetate (ph 4) was added. The homogenate was then extracted once with phenol-chloroform-isoamyl alcohol and precipitated with 1 volume of isopropanol. The RNA pellet was washed twice with 70% ethanol and resuspended in 40 l ofh 2 O. The sample was heated to 85 C for 5 min and cooled on ice, and insoluble debris was removed by centrifugation. Of the sample, 12.5 l was used for reverse transcription (RT) in a 20- l reaction. The primer homologous to the T7 sequence, and therefore specific for the reporters, BE50, was used for RT. Total RNA from transfected cells was prepared essentially as described above except that 5 ml of cells was collected and washed twice with phosphate-buffered saline before lysis in 500 l of lysis buffer. Five microliters from the RT reaction was PCR amplified with primers specific for the T7 marker (BE50) and for exon E1 (BE7) in a 50- l reaction unless otherwise noted. The first PCR cycle was 95 C for 45 s, 58 C for 45 s, and 72 C for 10 min, followed by 25 cycles of 95 C for 45 s, 58 C for 45 s, and 72 C for 2 min). Then 1/500 from the first PCR was amplified for an additional 30 cycles (95 C for 45 s, 58 C for 45 s, and 72 C for 2 min) with primers as described in the figure legends. Primer sequences were as follows: BE7 (E1), 5 -GACAAGTCC AACTTGTGTTCAG-3 ; BE5 (E1), 5 -GTTCGACCATGTCGTCCTAC-3 ; BE6 (exon 4), 5 -CGGATGGCAGAGAATGGGAC-3 ; BE50 (T7), 5 -CTATT GGAAGTCGTTCCGTGG-3 ; and BE12 (exon 2), 5 -GTGGTTATCCCCCAT ATGGC-3. RNase protection. For RNase protection, total RNA was prepared from 15 ml of transfected cells, and half of the RNA was used for each RNase protection experiment. The RNase protection assay was done as described previously (1) except that hybridization was at 50 C. The probe plasmid was made by cloning the 600-bp HindIII-BspEI (end-filled) fragment from the early cdna clone (27) into the pgem4 vector (Promega) digested with XbaI (end filled) and HindIII. The Sxl fragment contains exon E1 and most of exon 4. The plasmid was linearized with BglII, and 32 P-labeled antisense probe was transcribed by using SP6 RNA polymerase. FIG. 2. Analysis of reporter gene splicing in adult flies. Total RNA was extracted from adult transformants and analyzed by RT-PCR. Lanes 1 and 2, the patterns observed with primers in exons E1 and 4. The early E1-4 splice is present as identical bands in males and females (M and F). Lanes 3 and 4, the patterns observed with primers in exons 2 and 4. The late splicing pattern is present and correctly regulated, with exon 3 present in males and absent from females. RESULTS Sxl early splicing does not require embryo-specific or sexspecific splicing factors. To test whether the early splicing pattern requires any specific splicing factor, we constructed a reporter gene, B/B, in which a Sxl genomic fragment extending from exon E1 through exon 4 was placed under the control of the hsp83 promoter (Fig. 1). The hsp83 promoter is constitutively expressed at a basal level without heat shock in most tissues and stages. A sequence from T7 phage was fused to exon 4 for use in distinguishing reporter-specific transcripts. B/B was transformed into Drosophila by P-element-mediated germ line transformation, and then its splicing pattern in adult male and female flies was analyzed by RT-PCR. A primer specific to the T7 marker sequence (BE50) was used in RT and used again to specifically PCR amplify the reporter gene along with a primer specific to exon E1 (BE7); reamplification was conducted with the exon E1 primer BE7 and an exon 4-specific primer, BE6 (diagrammed in Fig. 1). The E1- to-4 splice typical of the early transcript was present in both male and female adult flies, observed as a 203-bp band (Fig. 2, lanes 1 and 2). Its identity was confirmed by cloning and sequencing of the PCR product. The reporter therefore mimics the endogenous early transcript. No late spliced transcript was observed from the early reporter construct. However, because it would be much larger than the early transcript, we investigated whether any late pattern transcripts (2-3-4 or 2-4) could be observed when latespecific primers were used. PCRs were conducted with the exon 2-specific primer BE12 in place of the exon E1 primer BE7. The late pattern of splicing from exon 2 was indeed observed, and a larger product was observed in males than in females, as expected from the inclusion of exon 3 in males (Fig. 2, lanes 3 and 4). The small bands in lane 3 do not appear when analyzed on Southern blots probed with B/B (data not shown). These bands might derive from either E splicing or from incompletely spliced transcripts containing E1-intron-2-3-4, in the male situation. In either case, these bands are observed only when late-specific primers are used and are not observed as a second band when the early primers are used, suggesting that is a relatively low-level transcript. Absolute amounts cannot be compared between lanes in these nonquantitative PCRs. We note that the E1-to-2 splice is not normally present among the endogenous Sxl early transcripts. However, the results indicate that there is some potential for exon 2 splicing from the reporter. A comparison of all four lanes leads to the conclusion that while both early and late patterns are present, the correct spliced partners (E1-4 and 2-3 or 2-4) are always chosen. Sig-

4 VOL. 17, 1997 Sex-lethal EARLY SPLICE 1677 FIG. 3. The splicing patterns of deletion constructs. The various deletions are diagrammed in Fig. 1. Total RNA was extracted from adult transformants and analyzed by RT-PCR, except that the O4 and T6 plasmids were transiently expressed in SL-2 cells. Southern blots were probed with the Sxl early cdna. In lanes 1 to 11, all constructs show the early pattern of splicing from E1 to 4, but O1 and O3 also show some E1-to-3 splicing. Lane 12 shows the O3 lane probed with an exon 3-specific probe. nificantly, aberrant E1-to-3 splicing was never observed (see also B/B splicing in Fig. 3 and Fig. 5A). In confirmation of these results, when the T7 sequences were replaced with SxlcF1 cdna sequences (1) and the sequences upstream of, and 50 nt into, exon E1 were deleted, identical splicing patterns were observed (data not shown). Because the early splice was observed in adult flies, while the endogenous early splice is normally present only in early female embryos, these results demonstrate that no stage-specific splicing factor is necessary for the E1-4 splice. It also implies that a specific concentration or composition of general splicing factors present in early embryos is not necessary. Finally, the observation of the early splice in both males and females indicates an absence of necessary sex-specific factors. It would seem that the early and late splicing patterns arise from an intrinsic or default mechanism for preferentially pairing splice sites. Deletions of many intron sequences fail to disrupt the early splice. Having ruled out the possibility of an embryo-specific splicing factor for exon E1-to-4 splicing, we searched for cisacting regulatory sequences. Various deletions of the B/B reporter were transformed into the Drosophila germ line, and their splicing patterns were assayed by RT-PCR of RNA from adults. To avoid potential complications of the endogenous Sxl gene, RT-PCR analyses were performed with male transformant flies carrying the Sxl deletion Sxl 7BO. Maps of deletion constructs are illustrated in Fig. 1. Two of the deletions, O4 and T6, were tested in Drosophila SL-2 cells, which seem male and splice Sxl in the male pattern (40). As with flies, the B/B reporter gene transiently expressed in SL-2 cells showed only exon E1-to-4 splicing (Fig. 5A and B, lanes B/B). The male and female Sxl late patterns have also been reproduced by using reporter genes in SL-2 cells (52). Most deletions of the intron between exons E1 and 2 failed to change the early splicing pattern and showed exclusively the E1-4 splice (Fig. 3, lanes 2 to 10). However, there were two exceptions in which E1-to-3 splicing was activated to create a novel splice that was never observed in either flies or the original B/B reporter gene. The first exception was the construct O1, in which nearly all sequences between exons E1 and 3 were removed, leaving approximately 75 nt of intron sequences adjacent to exon E1. Not only was E1-to-4 splicing observed, but two larger bands that correspond in size to products of E1-3-4 splicing ( 380 bp) and exon E1 unspliced to exon 3 then spliced to exon 4 ( 480 bp) were observed (Fig. 3, lane 1). The top two bands in lane 1 are of unknown identities but might be DNA or unspliced RNA. The second unusual result was obtained for the O3 deletion, in which two regions were removed: the intron sequences between exons E1 and 2 (extending from 71 nt downstream of exon E1 to 128 nt into exon 2, removing its 3 splice site), as well as the 5 splice site of exon 2 (20 nt of exon and 10 nt of intron). For deletion O3, both the normal E1-4 product and the aberrant E1-3-4 product were observed (Fig. 3, lanes 11 and 12). The E1-3 splice was confirmed by cloning and sequencing of the PCR fragment. Identical results were obtained for two other deletions that differed from O3 only at the exon 25 splice site, having 6 or 22 nt of exon and 19 nt of intron removed (data not shown). Thus, the two exceptions O1 and O3 that activate the E1-3 splice share the characteristic that in addition to large intronic deletions, both splice sites of exon 2 were also removed. It is noteworthy that when present separately, neither the large intron deletion extending into exon 2 (T5) nor the 5 splice site deletion (O2) was sufficient to activate E1-to-3 splicing (Fig. 3, lanes 2 and 8). The 800-bp band observed for T5 (Fig. 3, lane 8) was the product of splicing, which could not splice upstream of exon 2 because the 3 splice site had been deleted. It would seem that in removing the 3 splice site of exon 2, and in simultaneously altering the E1 splice site, a competition between 5 splice sites of exons E1 and 2 has been revealed. If the E1 splice site is altered, and presumably weakened, the exon 2 splice site becomes active. In comparison, the E1 splice site apparently remains functionally intact in deletions T2 and T4, which have smaller intronic deletions than T5. Although the product of E splicing is only very slightly larger than the E1/2-3-4 splice observed for T5, it is not observed in any of the constructs. We conclude that, as with the endogenous Sxl transcripts, the E1-2 splice is not used. Only by removing the 3 splice site of exon 2 have we set up an observable cis competition between exons E1 and 2. It remains possible that there is some level of E1-intron transcript in constructs with intact exon 2, but that it is not observed due to its comparatively large size and the preferential amplification of smaller transcripts by PCR. These data suggest two points. First, because E1-to-4 splicing was not abolished in any deletion, at least some of the sequences essential for the early splice must lie beyond the sequences so far deleted. Second, the E1-to-3 splice was activated in deletion O3, which removes both the intron and the exon 2 5 splice site, but was not activated in the separate component deletions T5 and O2. This result indicates that splicing to exon 3 could be observed only in the absence of the presumably competing 5 splice site of exon 2. Consistent with those results, the E1-to-3 splice was also activated in the deletion O1, which contains the same amount of intron sequences from the E1 5 splice site region as deletion O3. The early or late 5 splice site region determines the early or late splicing pattern. The foregoing experiments suggested that the cis-acting RNA sequences essential for the early splice might include exon E1 and its adjacent intron sequences to within 240 nt, as determined by the position of the XbaI site used in deletions O4. To further define the important sequences, we constructed a series of deletions in which there was a single 5 splice site upstream of exon 3. These constituted replacements between corresponding regions in and near exons E1 and 2 (Fig. 4). The reporter plasmids were transiently expressed in Drosophila SL-2 tissue culture cells and assayed by RT-PCR. Southern blot analysis of RT-PCR products probed with an antisense RNA derived from the early Sxl cdna is shown in Fig. 5A. Total RNA from transfected cells was reverse transcribed, and PCR amplified as before except that reamplifica-

5 1678 ZHU ET AL. MOL. CELL. BIOL. Downloaded from FIG. 4. Replacements between the late exon 2 and early exon E1 alternative 5 splice site regions. (A) Schematic of deletions R1 to R5. The numbers denote the distances from either 5 splice site. (B) Sequences around the 5 splice sites of exons E1 and 2. The open boxes denote exons. Pairs of downward and upward arrows with the same designation indicate the boundaries of each deletion. tion used primers BE12 and BE6 for deletion R1 and BE5 and BE6 for deletions R2 through R5 (primer locations are diagrammed in Fig. 1). The early E1-to-4 splicing pattern generated a band of 80 bp, and the late male splicing pattern generated bands of 260 bp. In R1, exons E1 and 2 were fused so that the 5 splice site derived only from late exon 2, with about 70 nt of its surrounding exon and all of its downstream intron sequences. All splicing from R1 includes exon 3, like the late transcript (Fig. 5A, lane R1). Therefore, R1 must contain all necessary sequences for efficient splicing in the late pattern. In R2, the 5 splice site is a chimera consisting of exon sequences from E1 and intron sequences from downstream of exon 2. The R2 RNA was spliced equally to exons 3 and 4 in both late and early patterns (Fig. 5A). For R3, addition of 75 nt of E1 intron sequences was sufficient to switch splicing to a predominantly early pattern (Fig. 5A). This splicing pattern is similar to that observed in O3 (Fig. 3A), in which there are 71 nt of intron sequences and the exon 2 5 splice site is deleted. For R4, with 130 nt of intron sequences, splicing is similar to that for R3 (Fig. 5A). Complete exclusion of exon 3 was achieved only in R5 splicing (Fig. 5A), showing that intron sequences up to 233 nt are important for efficient early splicing. The bulk of sequences from the exon portions of E1 and 2 seem unimportant for selection of exon 3. However, 70 nt of exon 2 (present in R1) and 40 nt of exon E1 (absent from R1 but present in R2 through R5) acted to improve splicing to their respective normal downstream partners. Nevertheless, these exon regions are not sufficient, and in fact the presence of the entire exon E1 (as in R2) is not sufficient to promote efficient splicing to exon 4. Overall, there was a switch in splicing pattern from complete inclusion of exon 3 in R1 to its complete exclusion in R5. This change in exon 3 inclusion can be seen when an exon 3-specific probe was used, as shown in Fig. 5B. The increase in early splicing from R2 to R5 was independently validated by RNase protection (Fig. 5C), and phosphorimager measurements from a similar analysis showed that E1-to-4 splicing increased approximately ninefold from R2 to R3. These experiments demonstrate that when only one 5 splice site is available upstream of exon 3, its identity as either exon E1 or exon 2 determines whether there will be splicing to exon 3. The sequences sufficient for efficient early splicing were narrowed to the 5 splice site of exon E1, including a short region of exon sequences and 233 nt of adjacent intron sequences. DISCUSSION To determine the mechanism that regulates the seemingly paradoxical splicing pattern of the Sxl early transcripts, we on July 22, 2018 by guest

6 VOL. 17, 1997 Sex-lethal EARLY SPLICE 1679 FIG. 5. Progressively replacing the 5 splice site region of late exon 2 with that of early exon E1 changes the splicing pattern from late to early, as determined by analysis of transcripts from the deletions R1 to R5 diagrammed in Fig. 4. (A) Southern blot analysis of RT-PCR products probed with an antisense RNA containing derived from the early Sxl cdna. Constructs were assayed by transient expression in SL-2 cells. (B) The Southern blot in panel A probed with an exon 3-specific sequence. (C) RNase protection with an E1-4 antisense RNA probe shows the increase in exon E1-to-4 splicing in constructs R2 through R5. constructed a reporter gene that mimics the early pre-mrna. Although these early transcripts are normally present only transiently during female embryogenesis, when the reporter gene is transcribed ectopically in adult flies of both sexes and in Drosophila tissue culture cells, it correctly reproduces the early splicing pattern. This finding demonstrates that no embryo-specific splicing factor or combination of general splicing factors is required for the early splice; neither are sex-specific factors required. Instead, the early splicing pattern must represent the default state for a transcript that begins with early exon E1. Thus, the apparently compatible interactions between pairs of splice sites must be generated by the default splicing machinery and would be expected to involve preferences of certain general factors for one splice site over another as well as preferred interactions between such proteins. Instead of a requirement for stage-specific factors, the critical aspect in the choice of early or late splicing pattern is whether the 5 splice site from early exon E1 or late exon 2 is used. This could most easily be seen in reporter genes that contained a single exon upstream of male-specific exon 3, created by replacements between the early and late exons (E1 and 2 respectively). When the entire 5 splice site of exon 2 replaced a similar region of exon E1 (deletion R1), all splicing was to exon 3 in the late pattern. In contrast, when exon E1 and 230 bases of adjacent intron were present, replacing all such regions of exon 2 (deletion R5), all splicing was to exon 4 in the early pattern. Intermediates derived from progressively more early exon E1 and less late exon 2 showed a transition toward more early splicing. These substitutions also suggest that more than one region near the 5 splice site may be important for exon 3 selection, because incremental increases in the early splicing pattern were observed when successive E1 regions were sequentially added. Addition of comparable regions from exon 2 increased the likelihood of splicing to exon 3 rather than exon 4. The importance of E1 intron sequences in enhancing the E1-4 splice suggests that this region might possess an intronic splicing enhancer. Exonic enhancers that increase use of nearby splice sites of both constitutive and regulated splicing have been reported for several genes (12, 24, 28, 47, 50, 53, 54). Positively acting intronic regulatory elements have also been identified (3, 4, 6, 10, 23). Several of these elements have been shown to act in a heterologous context. Most of these enhancers are purine-rich sequences bound by SR proteins. Candidate repeats in the E1 intron region are nine copies of TAT(A/G) and five copies of TTAAG. The putative enhancer might improve absolute splice site strength or might create a specific compatibility between the E1 and 4 exons. On the one hand, the E1 5 splice site may be very strong, causing the E1-4 splice to be very efficient; when the E1 splice site is altered and weakened, the intrinsically less favored spliced pattern becomes possible. This might occur if there is robust recognition of exon 4 followed by scanning upstream for the best 5 splice site. However, it is difficult to explain the pattern of the endogenous gene with only the view that the E1 5 splice site is exceptionally strong and is always chosen over exons 2 and 3. Even if exon E1 is strong, it is not clear why exon 4 should be preferred over both exons 2 and 3, when all three are default exons in the late transcript. Perhaps the most likely explanation is a splicing enhancer that acts with a preferential effect on E1-4 splicing and forms the basis for splice site compatibility. By this model, one would expect to find compatible enhancing sequences associated with exon 4. Likewise, there appear to be preferential interactions between exons 2 and 3 (see below). Whether E1-4 or 2-3 compatibility has the predominant influence or whether both are important remains to be determined. Simple deletions of the reporter gene suggest that competition for substrate splice sites may nevertheless also play some role. As the E1 intron was shortened to 75 nt E1-to-3 splicing was observed, but only in the absence of the exon 2 5 splice site (O3) and not in its presence (T5). However, as long as the early exon E1 5 splice site and 230 nt of adjacent intron were intact, pre-mrna was spliced exclusively in the early E1-to-4 pattern, regardless of the state of exon 2. This finding suggests that competition for exon 3 by exon 2 decreases the likelihood of abnormal splicing from E1 to 3, and enhances the E1 to 4 splice, but exon 2 is not normally necessary for the E1-to-4 splice. It seems likely that both competition and splice site compatibility may be used to reinforce one another. Also important for a functional early transcript is the exclusion of exon 2 along with exon 3. This cannot easily be attributed to the length of the potential intron ( 525 nt), but it could be explained by a direct preferential interaction between exons E1 and 4. In addition, exon 2 is a relatively large exon of 449 or 497 nt (depending on an alternative 5 splice site), so it might be recognized differently from other internal exons. For example, the usual mechanisms of exon definition might not apply to it (37, 38, 48). On the other hand, exon E1 is a first exon that must have special features for exon recognition, and lacking a 3 splice site, it would naturally be excluded from the late transcripts. A functional comparison of late exon 1, which splices to exon 2, and early exon E1, which skips exon 2, might be revealing. Experimental data from Horabin and Schedl (19) indicate that exons 2 and 3 in the late splice show a preferential relationship. When exon 3 and its flanking sequences were inserted into the intron of the white or ftz gene, this exon was predominantly skipped in male flies, suggesting that appropriate flanking intron and exon sequences are required for default male splicing and that exon 3 is relatively weak when placed in a foreign context. It seems unlikely that uniform inclusion of

7 1680 ZHU ET AL. MOL. CELL. BIOL. exon 3, an event essential for male viability, could occur efficiently if exon 3 is always inherently very weak. It would be more plausible to postulate that specific sequences, missing from heterologous exons, are involved in compatibility between exons 2 and 3 and that these improve utilization of exon 3. By analogy, the early splicing pattern essentially places upstream of exon 3 a heterologous exon (E1) which lacks the sequences necessary for preferential splicing to exon 3. Exon E1 in turn seems to have its own preference for exon 4. Recent results using a different reporter for the Sxl early splice in adult flies are consistent with our observation that the E1-4 splice is a default pattern (21). In addition, it was shown that mutations in genes [snf, vir, and fl(2)d] required for the Sxl-dependent 2-4 splice have no effect on the early E1-4 splice in the presence or absence of Sxl. This finding argues for differences in the early and late mechanisms and is consistent with the model that the early and late splicing patterns arise from differences in intrinsic splice site compatibility. The regions important for the early splicing pattern are likely to involve interactions at the 5 splice site consensus sequences as well as more distal elements, because several regions at and near the splice site, including up to 230 nt of intron sequences, seem to be important for exon 3 exclusion. Although recent in vitro experiments have demonstrated that a 5 splice site with as few as seven intron nucleotides can trans splice to a 3 splice site with a downstream splicing enhancer or 5 splice site (7), the broader sequence context of the E1 5 splice site contributes to its character and is important for preferential splice site determination. 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