INTRODUCTION

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Most eukaryotic primary RNA transcripts undergo posttranscriptional processing requiring splicing of introns. The best-appreciated regulatory outcome of posttranscriptional processing is alternatively spliced transcripts that differ in the coding exons or have distinct 3' or 5' untranslated ends (reviewed in reference 32). A second consequence of posttranscriptional regulation is the modulation of amounts of specific transcripts dependent on differential splicing efficiencies of different splice sites. It is generally thought that differences in cell-type-specific splicing machineries result in cell type-enriched or -specific alternative splicing (5, 19). In addition, efficiency of splicing could play a major role in gene regulation as primary transcripts that are not completely processed are generally not transported to the cytoplasm and are unlikely to code functional proteins (6, 21, 23). We decided to investigate the role of alternative and inefficient splicing in the regulation of the Drosophila erect wing (ewg) gene, as previous studies indicated a complex transcript profile, intron-containing cDNAs, as well as poly(A)⁺ transcripts with retained introns (9).

The Drosophila ewg gene provides a function that is vital in the nervous system and essential to the development of certain muscles (16). EWG protein contains an unusual DNA binding domain that is homologous to sea urchin P3A2 protein (4, 10), zebrafish Nrf (3), and mammalian transcription factors NRF-1 and initiation binding receptor (13, 18, 31). Our previous studies suggested that ewg primary transcript may be alternatively spliced, since the ewg gene has several introns and its Northern pattern shows multiple transcripts that are tissue and developmental stage modulated (15). However, at the protein level, only one major polypeptide, a 116-kDa, 733-amino-acid-long polypeptide encoded by the SC3 cDNA open reading frame (ORF), was observed in immunoblot analysis, although many other cross-reacting bands were also observed (9, 10). The translation start site of the SC3 ORF is an unconventional CTG codon, suggesting that translational regulation of ewg may be an important aspect of ewg regulation (10). Transgenes expressing the 116-kDa EWG protein provide compelling evidence that the 116-kDa protein is the major functional protein, as expression of 116-kDa protein in the neurons rescues lethality and general expression rescues both lethal and muscle phenotypes associated with ewg alleles (8, 10). An antibody generated against the 116-kDa EWG protein selectively labels all neurons in the embryonic and larval stages and certain migrating myoblasts in early pupae (8-10), suggesting a distinct tissue-specific expression of the protein and possibly transcript.

We investigated the splicing patterns of ewg RNA to address if ewg transcripts are indeed alternatively or inefficiently spliced and if the pattern of splicing shows tissue-specific differences. In this paper, we report the results on ewg splicing, using reverse transcription (RT)-PCR in head and body RNAs as representative of neuron-enriched and neuron-poor tissue, respectively. Our results show the following. (i) ewg is more widely transcribed than previously recognized, and total ewg RNA levels in heads and bodies are comparable. (ii) A subset of ewg introns are efficiently spliced, but another subset are inefficiently spliced and retained in poly(A)⁺ RNA. (iii) ewg RNA in bodies has a greater representation of unprocessed RNAs, and RNAs that include two exons that are not part of the SC3 ORF. One of these new exons is not included in ewg transcripts present in heads. (iv) SC3 ORF RNA is enriched in adult heads but low in the bodies. (v) Modest expression of the SC3-encoded ORF in the body can be lethal. Thus, ewg, which is widely transcribed, is primarily regulated by posttranscriptional mechanisms.

	MATERIALS AND METHODS

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Fly stocks and genetic crosses. Drosophila melanogaster flies were raised on standard media and at 25°C. The Canton-S strain was used as the wild type. EWG^HS and EWG^NS are two white⁺ marked transgenes that encode the 116-kDa EWG protein isoform under the control of heat shock hsp promoter and the neuron-specific elav promoter (8). ewg^l1 is a lethal, protein-null allele of the ewg gene (10). Df(1)cin-arth: uncovers several loci inclusive of cin, ewg, and y (14). Dp243: is a free duplication derived from Dp1187 which is y⁺ and also carries a P element marked with rosy (35).

RT-PCR. Total RNA was isolated using Trizol reagent (GIBCO-BRL) from heads and bodies of 2-day-old adults. After DNase I (GIBCO-BRL) treatment, RT of 1 µg of total RNA was primed with an oligo(dT) or gene-specific probe, using a Superscript II cDNA synthesis kit (GIBCO-BRL) according to the manufacturer's instructions except that the RNA was kept at 50°C for 5 min before initiation of the RT reaction. The manufacturer's instructions were followed to synthesize cDNAs primed by random hexamers. The RNase H step was omitted. Controls were done with no RNA and no reverse transcriptase. The sequence of the gene-specific probe is 5'-ACACTGTTCCATCGCTGTTCGT-3', which hybridizes to exon H. Cycle parameters for the PCRs were 30 s at 95°C, 40 s at 60°C, and 45 s at 72°C for 30 cycles, with an initial 2 min at 95°C and a final 8-min extension at 72°C. All PCRs were carried out in 50 µl, 8 µl of which was loaded on agarose gels. The Mg²⁺ concentration was optimized for each primer pair. Taq polymerase was from GIBCO-BRL, and PCR conditions were according to their instructions. Primers were used at a final concentration of 4 ng/µl. Primer positions are outlined in Fig. 1B, and their sequences are shown in Table 1. cDNA and genomic sequences were used for primer design. Primer sequences are shown in Table 1, and positions are outlined in Fig. 1B. Primer3, a web-based software program by Rozen and Skaletsky (27a), was used to assist in primer design. Identity of PCR bands was determined by restriction digests, internal primer PCRs, and/or direct sequencing. Direct sequencing was done with ABI automated sequencing equipment.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Schematic representation of ewg exons, cDNAs, and PCR primers. (A) Genomic map of the ewg locus and two cDNAs, MPA-1 and SC3, that share the SC3 ORF shown as filled boxes (adapted from reference 9). A map of all characterized ewg exons (A to J) and the nomenclature of alternative spliced introns is shown below the SC3 cDNA. Alternative splicing occurs only in introns 3 and 6. The new exons E and I are present within introns 3c and 6, respectively. (B) The primers are named according to the intron excision events that they were used to assess; for example, 2F and 2R amplify transcripts that span intron 2. Primers designated In were used to amplify intron-containing transcripts; all In primers with the exception of In1R and In3cF are within introns. Note that introns 1 and 6 are not drawn to scale. (C) Sequence of the 74-bp ewg exon. The underlined nucleotide T is a silent nucleotide polymorphism, as a C in this position was found by genomic sequencing. (D) Sequence of the body-enriched 38-bp exon I.

Sequencing of ewg^l1. ewg^l1 y w sn embryos were collected 24 h after egg laying and selected by the y marker after dechorionation. DNA was extracted by homogenizing ~50 embryos in 100 mM NaCl-10 mM Tris-HCl (pH 7.5)-1 mM EDTA. The homogenate was incubated in 1% sodium dodecyl sulfate (SDS)-1 mg of proteinase K per ml for 16 h at 55°C, phenol-chloroform extracted, and precipitated. A 2.4-kb genomic fragment spanning exons B to D was amplified by PCR with primers In1F and In3cR, using Pwo polymerase (Boehringer Mannheim). This fragment was then reamplified by using primers In1F/In2R and 2F1/e4R and sequenced on both strands. The sequence was compared to both cDNA and genomic DNA sequences, which match.

Protein expression. EWG protein was divided into three fragments for expression: exons B and C (EH1), exon D (EH2), and exons F to H containing J (EM3). Additionally, we made two fragments containing overlapping parts: exons B to D (EH4) and exons D to H containing J (EH5). Fragments were amplified from the SC3 cDNA, using the following primers containing an XbaI site in the return primer for cloning: 5'-CTGGCCACCACAAGCTATC-3' (e23F) and 5'-GCTCTAGATCAGTTATTGCTGTTGCCCGTC-3' (e23R) for EH1, 5'-CAACCGCAGCAGGTGAAT-3' (e4F) and 5'-GCTCTAGATCAATCAACATCGCTGAGCGTAA-3' (e4R) for EH2, and 5'-TACACCACGCAAACGGTC-3' (e6-8F) and 5'-GCTCTAGATCAGCTCCAGCTATTGTTCCAT-3' (e10R) for EM3. EM3 was cloned into pMal-c2 (New England Biolabs), using the XmnI and XbaI sites in pMal, yielding an N-terminal fusion to maltose binding protein (MBP). The remaining fragments were cloned into pSG05 via the SnaBI and NheI sites (17), yielding an N-terminal His tag, since we were unable to clone these fragments with the pMal system. Protein expression was done as described previously (17) for the EH fusions and according to the manufacturer's instructions for the EM fusions.

Immunoblot analysis. Drosophila protein extracts were prepared and resolved as previously described (30). Drosophila embryos were 14 to 18 h old. Bacterial extracts were prepared by pelleting the bacteria and dissolving them in 2× sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis at 12.5% and 8% SDS for the bacterial extract and Drosophila extracts respectively. Anti-EWG antibody (10) was used 1:5,000 for immunoblot analysis of bacterial extracts and 1:1,500 for Drosophila extracts. A peroxidase-conjugated goat anti-rabbit secondary antibody (Amersham) was used at a dilution of 1:2,000, and blots were developed by chemiluminescence (LumiGLO; Kirkegaard & Perry).

Nucleotide sequence accession numbers. The cDNA sequence (accession no. L11345) and genomic sequence (accession no. AF135590) have been submitted to GenBank.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Splicing of known ewg exons differs in adult heads and bodies. Figure 1A represents our current understanding of the exon/intron structure of the ewg gene. This map is based on sequences of ewg genomic DNA (15), two cDNAs, SC3 and MPA-1 (9, 10), that shared a common ORF, referred to as the SC3 ORF, consisting of exons B, C, D, F, G, H, and J, and the RT-PCR analysis presented in this paper (see below). The SC3 cDNA differed from MPA-1 in that it contained part of intron 1 and lacked the noncoding exon A (Fig. 1A).

(i) Introns 2, 4, and 5. Analysis of RT-PCR products using exon-specific primers revealed that introns 2, 4, and 5 are excised efficiently, as in each case a single band representing the spliced product was observed for head or body RNA. Moreover, for each primer set the band densities in both head and body lanes were comparable (Fig. 2, lanes 3, 4, 19, 20, 12, and 13). The efficient splicing of these introns, which are all small (Table 4), is to be expected since small introns are known to be spliced efficiently in Drosophila (29). However, the observation that the PCR bands for both body and head RNAs were comparable was unexpected, as it implied that nonneural tissues expressed ewg RNA to a greater extent than previously thought (see below and Fig. 3). In these RNA samples, levels of a control ribosomal protein transcript, rp49, are similar in the two tissues (Fig. 2, lanes 17 and 18).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Characterization and comparison of ewg splicing in wild-type adult tissues. All RT-PCR assays were carried out with DNase I-treated total RNA isolated from 2-day-old heads (H) or bodies (B). The italicized letters below each pair of lanes represent the specific splice events as outlined in Fig. 1B, e.g., primers 3aF and 3aR for intron 3a splicing. These data are summarized in Table 2, which also lists the lengths of PCR products. rp49 transcripts were used as a control. Molecular size markers (GIBCO-BRL) are shown in lanes 11 and 16. Note that splicing of introns 3a, 3c, and 6 in heads is mostly in the mode of the SC3 cDNA.

(ii) Intron 1. Intron 1 is more efficiently spliced in body than in head RNA (Fig. 2, lanes 1 and 2). This inefficient splicing in heads perhaps explains the SC3 cDNA, which contains part of intron 1 (Fig. 1A).

(iii) Introns 3a, 3b, and 3c. Intron 3a is inefficiently spliced in both heads and bodies, as both spliced (150-bp lower band) and unspliced (449-bp upper band) products are observed in RT-PCRs using primers 3aF and 3aR with poly(A)⁺ head and body RNAs (Fig. 2, lanes 5 and 6). The excision of intron 3c (154-bp lower band) occurs more efficiently in adult heads than in bodies (lanes 9 and 10), as the body lane shows the unspliced product (highest, 961-bp band in lane 9), while it is undetectable in heads under these assay conditions (lanes 9 and 10). Two unexplained bands were present predominantly in the body lane in RT-PCRs using primers 3cF and 3cR (lanes 9 and 10), representing a new exon (see below). Thus, both introns 3a and 3c are retained in a fraction of body transcripts, while 3a is also retained in a fraction of head RNAs.

(iv) Intron 6. Using primer set 6F/6R and head RNA, only one band (278 bp) expected from splicing of intron 6 was seen. However, in the body RNA lane, two relatively faint bands of equal intensities were observed; the lower band represents the splicing of intron 6, while a slightly larger and unexpected band represents a second new exon (Fig. 2, lanes 14 and 15; see below). The low level of spliced product in the body lane suggests that intron 6 is inefficiently spliced and/or that some transcripts terminate within it.

Characterization of new ewg exons E and I. The novel bands were isolated and directly sequenced on both strands with the primers that had been used for their amplification to determine if they resulted from additional exons in the ewg gene. The sequence of the 230- to 250-bp product detected in the 3aF/3cR (Fig. 2, lane 7) and 3cF/3cR (lane 9) PCRs revealed the presence of a 74-bp exon in intron 3c. This new exon, E in Fig. 1A, codes for 24 amino acids and alters the translational frame. Further RT-PCR analysis of exon E revealed high enrichment in female abdomens (data not shown).

ewg RNA is abundant in heads and bodies. Comparison of levels of efficiently spliced introns 2, 4, and 5 (Fig. 2A, lanes 3, 4, 12, 13, 19, and 20) suggests that ewg RNA is present in heads and bodies at comparable levels. To verify that ewg RNA was indeed present at high levels in bodies, either random hexamers or a gene-specific primer in exon H were used for RT, allowing the amplification of all splice isoforms of ewg regardless of their state of polyadenylation in subsequent PCR. For both reactions, primers 4F and 5R yielded very similar signals with head and body RNAs (Fig. 3A, lanes 1, 3, 6, and 8). Primers 3cF and 5R revealed that both tissues contain ewg RNAs that either retain or excise intron 3c (lanes 2, 4, 7, and 9). Exon E-containing transcripts were detected mostly in bodies (lanes 2 and 7). Also, some intron 3c-1 retention in body RNA is observed (lanes 2 and 7). Thus, ewg RNAs appear to be efficiently polyadenylated, as the ewg splicing profiles are similar for cDNAs primed with oligo(dT), gene-specific, and random hexamer primers.

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Abundance of ewg transcripts is independent of polyadenylation and is equal in heads (H) and bodies (B). (A) RT-PCR using random-primed or gene-specific-primed cDNAs. The RT reaction shown in lanes 1 to 4 was primed with a primer in exon H, an exon common to all ewg transcripts. The RT reaction shown in lanes 6 to 9 was primed with random hexamers. The italicized letters below each pair of lanes represent the specific splice events assayed as outlined in Fig. 1B. The uppermost bands in lanes 2, 4, 7, and 9 show ewg transcripts containing intron 3c. The 408-bp band in lanes 2 and 7 contains exon E. Molecular size markers (GIBCO-BRL) are shown in lane 5. (B and C) Cycle titration of PCRs using primers 4F and 5R to amplify parts of ewg transcripts common to all ewg transcripts. cDNAs were synthesized with a gene-specific primer in exon H. Aliquots were removed from the PCR beginning at cycle 18 and continuing until cycle 28. Quantitation of bands reveals that PCR is in the linear range (data not shown). Note that the intensities of bands in both heads and bodies are similar at all cycles.

Alternative splicing of ewg exons. The RT-PCR studies suggested that both head and body RNAs have populations of alternatively spliced transcripts that include or exclude exon D and that body-specific transcripts are enriched in transcripts that include exon I. Further, the splicing of introns 3a and 3c and splicing of intron 6 in bodies appear to be independent of each other since ewg transcripts containing exon I were found with or without exon D (436 bp in Fig. 4A, lane 6; 451 bp in lane 7; summarized in Table 3).

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Alternative splicing of ewg transcripts is restricted to introns 3 and 6, alternative splice events are independent of each other, and splicing of introns 1, 3, and 6 is inefficient. Comparison was done between head (H) and body (B) poly(A)+ RNAs. (A) PCR products spanning introns 3 and 6 reveal all combinations of alternatively spliced introns. The italicized letters below each pair of lanes show the amplified section of ewg transcripts. Table 3 provides a complete listing of PCR products and summarizes the data. The forward primer was 3aF or 3cF, the return primer was RV, 6R, or 38R. Molecular size markers (GIBCO-BRL) are shown in lanes 5 and 11. In lane 8, a lambda hindIII digest was used as a marker. Note that heads and bodies show differences in the ewg transcript population and abundance due to the body-enriched usage of exons E and I, while increased inclusion of exon D occurs in heads. (B and C) Primer pairs spanning several introns reveal no additional alternative splice events. The italicized letters below each pair of lanes show the amplified section of ewg transcripts. Table 3 provides a complete listing of PCR products and summarizes the data. Molecular size markers (GIBCO-BRL) are shown in lanes 9 and 18 of panel B and lanes 1 and 8 of panel C. In lane 15 of panel C, a lambda hindIII digest was used as a marker. (D) Introns 1, 3a, 3c, and 6 are retained in polyadenylated ewg transcripts. A listing of PCR products and summary of the data are shown in Table 2. Note that differences between heads and bodies are mainly detected in the retention of introns 1 and 6 but not 3. Molecular size markers (GIBCO-BRL) are shown in lanes 5 and 13.

ewg is inefficiently spliced. To determine if introns other than 3a and 3c were present in poly(A)⁺ ewg RNA, PCR was done with intron-specific primers. Introns 1, 3a, 3c, and 6 (Fig. 4D) are present in poly(A)⁺ ewg transcripts of heads and bodies. From these introns, only intron 1 is differentially retained in body RNA compared to in head RNA. All of these introns are larger than 81 bp and are classified as large Drosophila introns (26). Among these introns, the 5' and 3' splice sites of 3c and the 3' splice site of 3a diverge significantly from the Drosophila consensus (Table 4) (26). Introns 2, 4, and 5 were not detected in poly(A)⁺ ewg transcripts, confirming that they were efficiently spliced (Fig. 4D, lanes 3, 4, 10, 11, 12, and 14; Table 3). The overall levels of unspliced ewg transcripts were assessed in assays using primer 3cF and return primer RV in intron 6. This analysis indicated the presence of greater amounts of unprocessed ewg transcripts containing both intron 3c and intron 6 in bodies than in heads (Fig. 4A, lanes 9 and 10; data summarized in Table 3).

Expression of 116-kDa protein is able to rescue ewg-null phenotypes. We previously demonstrated that the 116-kDa protein is able to rescue the three well-characterized phenotypes associated with ewg mutations: embryonic lethality, erect wings, and formation of dorsal longitudinal muscles (8, 10). Expression from two cDNAs expressing ewg minigenes, EWG^NS (neuron specific) and EWG^HS (basal level expression), rescued viability of ewg^l1, an ethyl methanesulfonate-induced lethal allele, which was thought to be genetic null and 116-kDa protein null (10). The possibility that the ewg locus was formally able to generate several other isoforms made us wonder if ewg^l1 was a true null for all possible EWG proteins. Sequencing of genomic DNA from exons B to D revealed a C-to-T base pair change in exon B that resulted in the termination of the ORF at amino acid 187. Since exon B is part of all putative EWG isoforms, the ewg^l1 allele is a functional null allele for all possible EWG proteins; moreover, it lacks the DNA binding domain.

Putative isoforms encoded by the ewg locus. ewg can potentially encode several polypeptides in addition to the 116-kDa EWG protein that includes exon D. Figure 5 depicts the conceptual ewg-generated isoforms. The presence or absence of exon D, which encodes 154 amino acids, does not interrupt the translational frame of the ewg ORF, while inclusion of exon E or I alters the translational frame, leading to a premature stop compared to that of the SC3 ORF (Fig. 5). Additional protein isoforms can also be generated by the ewg transcripts that retain either intron 3a, 3c, or 6. All intron 3a-, 3c-, and 6-containing RNAs result in a premature stop in the SC3 ORF, encoding 408, 574, and 840 amino acids, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   ORFs of ewg splice isoforms deduced by RT-PCR analysis. All deduced ewg RNA isoforms are outlined from data presented in Table 2. The size of each ORF is given in amino acids (aa). Transcripts including exon E and I are not shown since the ORF terminates in exon F. Also, ORFs resulting from intron-retaining transcripts are not shown.

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Characterization of anti-EWG antibody and immunoblot analysis of EWG proteins in Drosophila embryos and heads. (A) Anti-EWG antibody recognizes epitopes all along the EWG protein. Different parts of EWG protein were expressed in E. coli and tagged with either six histidines (6His) or MBP at the N terminus. As controls either bacterial extracts expressing only MBP or bacterial extracts from uninduced E. coli (lanes ) were loaded. Note that the part encoded by exons B and C has a calculated molecular mass of 43 kDa but runs at ~70 kDa. Molecular mass markers are shown on the left in kilodaltons. (B) Anti-EWG antibody recognizes one major and several minor proteins in wild-type embryos (+; lane 1), which are absent in ewgl1 embryos (lane 2). Proteins marked with arrows are degradation products of the 116-kDa EWG protein, since they are also present in ewgl1; EWGNS4 embryos (NS4; lane 3). EWGNS4 is a rescue construct where a cDNA for the 116-kDa EWG protein is fused to a elav promoter fragment restricting expression to the nervous system. Molecular mass markers are shown on the left in kilodaltons. (C) Basal expression of the 116-kDa EWG protein under a heat shock promoter is comparable to EWG levels in the wild type. Head extracts from wild-type females (Tb [+/+; +/+; +/Tb]; lane 1) are compared to head extracts from females with either one copy (HS1 [ewgl1/ewgl1; +/+; EWGHS1/+] [lane 2] and HS7, Tb [ewgl1/ewgl1; EWGHS7/+; +/Tb] [lane 3]) or two copies (HS1, HS7 [ewgl1/ewgl1; EWGHS7/+; EWGHS1/Tb] [lane 4]). Note that the amount of EWG protein is about half of the wild-type amount with only one copy. Molecular mass markers are shown on the left in kilodaltons. (D) The 40-kDa proteins recognized by the anti-EWG antibody in head extracts are not EWG isoforms. Head extracts from the wild type (+; lane 1) are compared to head extracts from an ewgl1; EWGNS4 mutant (NS4; lane 2). Molecular mass markers are shown on the left in kilodaltons.

Overexpression of 116-kDa EWG is lethal. The splicing regulation of ewg guarantees that the 116-kDa protein is generated in the head but down regulated in the body. To test if this regulation is biologically important, the effect of overexpression of 116-kDa protein in nonneural tissues was tested. Overall overexpression was achieved by using EWG^HS1 and EWG^HS7, two independent insert lines for a transgene in which the SC3 cDNA is driven with the heat shock promoter. The expression from the EWG^HS transgene at 25°C mimics endogenous transcription, likely because of the inclusion of regulatory sequences upstream of the ORF (SC3 cDNA in Fig. 1A). Both of these transgenes lead to lethality when homozygous. We determined the viability of flies with different doses of the wild-type ewg allele that also carry EWG^HS1 and EWG^HS7 (Table 5). Indeed, flies carrying two doses of heat shock transgenes are less viable, and the males have a lower viability index than females. Males of genotype ewg⁺/Y; EWG^HS7/Tb or ewg⁺/Y; EWG^HS1/+ have viability indices of 0.76 and 1.00, respectively, but the viability index of ewg ⁺/Y; EWG^HS7/Tb; EWG^HS1/+ drops to 0.005. Female survival is better than male survival, but the viability indices are still low: 0.10 and 0.12 for females carrying both transgenes and one or two doses of wild-type ewg, respectively (Table 5).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

EWG protein provides a vital function in the nervous system, as exclusive neural tissue-specific expression of the 116-kDa EWG protein rescues the lethality caused by the ewg-null alleles. This neural role of ewg is underscored by a robust expression of the 116-kDa protein in neurons at all stages. In fact, the high expression of EWG seen with anti-EWG antibody and the functional information had led us to assume that the weak signal seen outside the nervous system (with the exception of myoblast expression) with the antibody staining in larvae and adults was due to high background (9). Thus, it is surprising and puzzling that despite the broad distribution of the transcript, the two known functions of ewg are associated with specific cell types: neurons and myoblasts. The studies described in this paper show that although the gene is broadly transcribed, the functional transcript encoding the 116-kDa protein is highly enriched in neuron-rich head tissue compared to that in neuron-poor bodies. The neurons in the body must contribute to the functional transcript observed in the body RNA; however, our data do not address if cell types other than neurons are also capable of low-level production of the functional transcript.

Generally, transcriptional controls ensure that specific proteins are synthesized in specific tissues. In the case of ewg, however, posttranscriptional mechanisms are critical to ensure that the 116-kDa protein is present at high levels in neurons. Few examples that show broad tissue distribution of transcript but restricted protein expression are known. In Drosophila, the gene encoding P transposase is transcribed broadly but the productive splice is made only in the germ line cells (1). A second example is the Drosophila Sex-lethal gene (Sxl), which is transcribed in both males and females but generates the functional Sxl protein only in females (22). Similar to the case of ewg, in both of these instances alternative splicing is involved; in the case of Sxl, a large number of intron-containing transcripts are also present (28).

Efficiency of alternative splice events that result in SC3-like transcript is higher in head RNA. Splice events that are crucial for the production of the functional SC3 transcript are inclusion of exons D and J and exclusion of exons I and E. Inclusion of exon D requires splicing of introns 3a and 3c instead of 3b. The 3' splice sites of both 3a and 3c and the 5' splice site of 3c diverge from the Drosophila consensus (Table 4) (26), making them likely targets for splicing regulation. Intron 3a is inefficiently spliced in both head and body RNAs, whereas 3c is inefficiently spliced in body RNA only. Since exclusion of D resulting from exon skipping is seen in both body and head RNAs, it is likely to be the default mode, with inclusion of D requiring a positive regulatory step. To what extent the inefficient splicing of 3a and 3c affects this regulation is difficult to assess. SC3 transcript also requires the appropriate choice of a 5' splice site, resulting in the excision of intron 6. The body RNA shows inefficient splicing in the intron 6 region and, among the spliced products, about equal amounts of exon I inclusion and intron 6 excision. It is likely that RNAs that retain intron 6 become polyadenylated as consensus sequences for polyadenylation exist in intron 6; in case these are used, transcripts that encode different C termini will be generated.

Splicing of introns 1 (~4.5 kb), 3a (299 bp), 3c (807 bp), and 6 (1,722 bp) is inefficient; all these are large introns. Some examples of intron retention in Drosophila include transcripts of Dopa decarboxylase (2), Suppresser-of-white-apricot (34), and Sxl (28). Whether intron-containing ewg transcripts exit the nucleus is not known, although the presence of 5' and 3' splice sites in a transcript is often sufficient to retain most intron-containing RNAs in the nucleus (23). However, the presence of intron-containing messages in the cytoplasm has been reported; examples include insulin pre-mRNA (33) and bovine growth hormone pre-mRNA (11). Intron retention can be a powerful means of gene regulation; the Drosophila Transformer-2 protein self-regulates the retention of intron M1 in germ line tissue (25). This intron retention in tra-2 mRNA has important phenotypic consequences, and it is thought to help maintain appropriate levels of Tra-2 protein in germ line tissues (25). Intron retention can also be a means to generate alternate protein isoforms, examples of which include C-CAM3 and rVDR1 (7, 12).

Alternative splicing is a widespread mechanism to regulate functional properties of DNA binding proteins to modulate DNA binding activity and dimerization properties (reviewed in reference 24). Since all conceptual alternative EWG isoforms contain the DNA binding domain encoded by exon B, these proteins are likely to differ in either dimerization or activation properties. The activation domain of NRF-1, a protein that has homology to EWG, has several glutamine-containing hydrophobic clusters (20). There are five such clusters in head-enriched exon D and two in exon H. Thus, the head-enriched 116-kDa protein may have stronger activation properties than proteins that lack exon D. The dimerization region for EWG has not been characterized.

Are any of the putative isoforms synthesized, do they have a function, or will they provide ewg function? The putative alternate protein isoforms have no essential role in the nervous system, as the 116-kDa EWG protein rescues viability of null ewg alleles, although they may still provide subtle functions in vivo or substitute for the 116-kDa function. Whether the alternative proteins are synthesized at low levels is not known.

The studies with heat shock transgenes show that overexpression of 116-kDa protein in nonneural tissues can be lethal, yet it is the expression of the 116-kDa protein in neurons that provides the viability function associated with ewg. With two doses of the EWG^HS transgenes, both ewg^l1 and ewg^l1/+ flies were about equally lethal, suggesting a formally neomorphic effect. The lethality could result from the expression of 116-kDa protein in nonneural tissue or be due to general overexpression. An alternative possibility of the transgene generating a toxic novel protein cannot be discounted but is unlikely because the transgene was constructed by using a cDNA and not genomic DNA. Since the 116-kDa protein levels in the head with two doses of EWG^HS are comparable to the wild-type levels, the protein is unlikely to attain a higher than normal level in the neural tissue. Moreover, higher than normal levels of 116-kDa protein generated through two doses of EWG^NS transgenes, which are expressed only in the nervous system, is not lethal (our unpublished observations). Therefore, expression outside the nervous system is likely to be the primary cause of lethality. Thus, given that ewg is broadly transcribed, its posttranscriptional regulation is crucial to the fulfillment of its function.