erect wing

REGULATION

Post-transcriptional Regulation

An unusual mode of tissue-enriched gene expression is documented that is primarily mediated by alternative and inefficient splicing. Posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles, has been analyzed. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. Thus, 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. 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. 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. 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, suggesting a distinct tissue-specific expression of the protein and possibly transcript. In contrast to the protein distribution, Erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. The analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. It is also shown that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, fly bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed Erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal (Koushika, 1999).

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, 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. In conclusion the results show that (1) ewg is more widely transcribed than previously recognized, and total EWG RNA levels in heads and bodies are comparable; (2) a subset of EWG introns are efficiently spliced, but another subset are inefficiently spliced and retained in poly(A)+ RNA; (3) EWG RNA in bodies has a greater representation of both 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); (4) SC3 ORF RNA is enriched in adult heads but low in the bodies and (5) 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 (Koushika, 1999).

Although the Drosophila erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa Ewg protein in neurons, while protein expression elsewhere (or of other isoforms) is below detection at this stage. This posttranscriptional control is important, since broad expression of Ewg can be lethal. Elav, a neuron-specific RNA binding protein, is necessary to regulate Ewg protein expression in Elav-null eye imaginal disc clones and Elav is sufficient for Ewg expression in wing disc imaginal tissue after ectopic expression. Analysis of Ewg expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in Elav-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and Elav-deficient eye imaginal discs and in wild-type and ectopic Elav-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to Elav levels. The data presented in this paper are consistent with a mechanism by which Elav increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism by which stability of specific splice forms is enhanced by Elav (Koushika, 2000).

The primary transcript of ewg, which has 10 exons, A to J, is alternatively spliced in two regions. Neuron-enriched heads and neuron-poor bodies have different EWG RNA splicing profiles. Heads show enrichment for a transcript encoding a 116-kDa protein, whereas bodies have lower amounts of the transcript that encodes the 116-kDa protein and greater amounts of unprocessed RNA. The head-enriched transcript encoding the 116-kDa protein results from inclusion of exon D and exclusion of exons E and I. Additionally, splicing of introns 3a, 3c, and 6 is inefficient, since these introns are retained in polyadenylated EWG RNA (Koushika, 2000).

Additionally, Elav promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. The neuron-specific arm transcript, n-arm, is generated by an alternative splice event that results from the exclusion of exon 6 of ubiquitous-arm (u-arm). The primer pair used amplifies both u-arm and n-arm transcripts; the 147-bp smaller band corresponds to n-arm, while the 244-bp band corresponds to u-arm. To test if Elav has a role in the formation of n-arm transcripts, RNA from wild-type and elav null allele (edr) eye discs, as well as from wild-type eye discs and wing discs ectopically expressing Elav were analyzed by RT-PCR. The amount of n-arm is reduced in Elav-deficient eye discs, and in the ectopically expressing wing discs expression of n-arm is clearly induced. No change was observed in the band representing u-arm splicing. In summary, the presence of n-arm is correlated with the presence of Elav in both neural and nonneural tissues, implying that arm transcripts are regulated by Elav. Similar data were obtained for splicing of exons VIIa and VIIb of Neuroglian transcripts (Koushika, 2000).

Elav ensures that the correct alternatively spliced protein isoforms of certain genes are generated in neurons. Currently three target genes, ewg, Nrg, and arm have been identified. Both Nrg and arm are vital genes and are broadly transcribed and ubiquitous protein isoforms are broadly expressed, but an additional isoform, encoded by an alternatively spliced transcript, is pan-neurally expressed. The significance of the neural Nrg (n-Nrg) isoform is not known, but the distinct cytoplasmic domain could be important in signaling. The n-Arm isoform differs from the ubiquitous Arm (u-Arm) isoform because it lacks the Wingless interacting domain; moreover, it preferentially interacts with DE-cadherins. Even with these differences in properties, the current evidence suggest that the u-Arm is sufficient to provide the n-Arm function. Perhaps a more detailed phenotypic analysis may reveal a specific role for n-Arm (Koushika, 2000).

ewg, also a vital gene, is broadly transcribed, but the protein product, a likely transcriptional regulator, is almost exclusively neural. In the case of ewg, it is clear that the expression of the 116-kDa protein isoform is essential for viability in the nervous system and that, when expressed in nonneural tissues, it can be lethal. These Elav-regulated genes provide insight into the regulatory role of Elav in neurons. Experiments reported here demonstrate for the first time that the prevalence of neuron-specific ewg, nrg, and arm transcripts positively correlates with Elav levels, and these results are achieved through the increased use of specific splice sites (Koushika, 2000).

ELAV inhibits 3'-end processing to promote neural splicing of ewg pre-mRNA

ELAV is a gene-specific regulator of alternative pre-mRNA processing in neurons of Drosophila. A functional in vivo binding site for ELAV in neurons is described through the development of a reporter gene system in transgenic animals in combination with in vitro binding assays. ELAV binds to erect wing (ewg) RNA 3' of a polyadenylation site in the terminal intron 6. At this polyadenylation site, ELAV inhibits 3'-end processing in vitro in a dose-dependent and sequence-specific manner, and ELAV binding is necessary in vivo to promote splicing of ewg intron 6. Further, the AAUAAA poly(A) complex recognition sequence, together with ELAV, is required to regulate neural 3' splice site choice in vivo. In addition, the use of segmentally labeled RNA substrates in UV cross-linking assays suggest that ELAV does not inhibit or redirect binding of cleavage factor dCstF64 at the regulated polyadenylation site on ewg RNA. These data indicate that binding of 3'-end processing factors, together with ELAV, can regulate alternative splicing (Soller, 2003).

Although the ewg gene is ubiquitously transcribed, a salient feature is the unusual posttranscriptional regulation of this transcription factor. The last intron 6 is only spliced in the presence of ELAV, as in neurons, or when ELAV is provided ectopically. This, in turn, leads to the expression of the major Ewg protein isoform sufficient for full rescue of viability and neuronal function. A rescue reporter transgene, tcgER, has been developed that recapitulates ELAV-mediated regulation of ewg transcripts in neurons of developing and adult Drosophila flies. ELAV binds directly to ewg RNA close to an intronic pA site and inhibits 3'-end formation at this site to promote neuronal splicing of ewg intron 6 (Soller, 2003).

Several lines of in vitro and in vivo evidence converge to identify the AU_4-6 motifs 3' of pA2 in ewg intron 6 as a functional ELAV-binding site. Deletions introduced in tcgER reporter transgenes show that only ~25% of intron 6 is sufficient for ELAV-dependent regulation. Within the remaining RNA, ELAV UV cross-links in neuronal nuclear extracts to AU_4-6 motif containing region pA-I, but not to the flanking sequence or the 3' UTR. In addition, EMSAs show that recombinant ELAV binds with nanomolar affinity to ewg RNA pA-I. Mutational analysis further substantiates ELAV's binding to AU_4-6 motifs in vitro; U-to-C substitutions considerably reduce ELAV binding in UV cross-linking assays as well as in EMSAs. Moreover, AU_4-6 motifs are necessary to inhibit cleavage of substrate RNA in in vitro cleavage/pA assays with neuronal nuclear extracts or when recombinant ELAV is added to nonneuronal extract. Finally, tcgER reporter transgenes with mutated AU_4-6 motifs fail to show the neuronal processing mode of ewg intron 6, demonstrating the importance of these motifs to ELAV regulation in vivo (Soller, 2003).

The ELAV-binding site on ewg RNA consists of several AU_4-6 motifs, consistent with previously reported binding preferences of ELAV/Hu proteins to AU-rich sequences. Within this site individual tandem AU_4-6 motifs contribute to ELAV binding, indicating that several ELAV molecules bind to ewg RNA. Recently, Hu proteins were found to interact with each other in yeast two-hybrid assays and coimmunopreciptations, and could thus potentially form a complex on binding to target RNA. This is consistent with the current observations and might indicate that ELAV/Hu proteins associate cooperatively on target RNA to form a complex (Soller, 2003).

ELAV inhibits cleavage of ewg substrate RNA in in vitro cleavage assays in a sequence-specific and concentration-dependent manner. The inhibitory activity of ELAV resides in its ability to bind RNA. Thus, ELAV is not inhibitory via titrating any essential component. This is of particular importance, since ELAV was also found to interact with dCstF64 in nuclear extracts. Although it is not yet know if the interaction of ELAV and dCstF64 is direct, inhibition of pA by ELAV cannot be explained by sequestering pA factors (e.g., dCstF64) from binding to ewg RNA in vitro. Rather, specificity in the substrate RNA and assembly of ELAV and CstF64 on ewg RNA might play a critical role in inhibiting 3'-end processing. The results, however, argue against a role of ELAV in competing with pA site recognition by Cleavage and polyadenylation specificity factor (CPSF) and Cleavage stimulation factor 64 kilodalton subunit (CstF) (Soller, 2003).

In neurons, splicing of ewg intron 6 is achieved through inhibition of intronic 3'-end formation at pA2 and distal 3' splice site selection. By what mechanism does ELAV inhibit 3'-end processing to allow splicing? ELAV's binding in the proximity of the cleavage site could slow the recruitment of cleavage factors (CF I and CF II) and/or poly(A) polymerase (PAP) resulting in a delay of the cleavage reaction. Alternatively, execution of the cleavage reaction could involve a structural rearrangement that is affected by ELAV binding. In either case, this intermediate pA complex consisting of at least CPSF and CstF, together with ELAV, alters the timing of 3'-end processing to allow for the assembly of the splicosome to the neuronal 3' splice site of intron 6 and for splicing to proceed (Soller, 2003).

Transcription and RNA processing are coupled through the C-terminal domain of the largest subunit of RNA polymerase II (pol II). Low processivity of RNA pol II could occlude the availability of a 3' splice site and thus favor intronic 3'-end processing. The short distance of only 164 nt from the AAUAAA sequence to the 3' splice site of exon I, however, makes this an unlikely scenario. Furthermore, ELAV's ability to inhibit cleavage in vitro in a concentration- and sequence-dependent manner argues against a role in stimulating RNA pol II processivity to make the neural 3' splice site available for splicing before 3'-end processing occurred (Soller, 2003).

What drives the choice of the neural splice site in ewg intron 6? An interesting alliance between ELAV and components of the pA complex in choosing the neural 3' splice site was revealed when analyzing mutations of the AAUAAA pA complex recognition sequence (DeltapA2). In DeltapA2, inclusion of exon I can occur even in the presence of ELAV, whereas in the absence of ELAV, inclusion of exon I is the major splice product. Thus, the ability of the pA site to initiate the assembly of pA factors in the presence of ELAV is key to the tight regulation of usage of the distal 3' splice site in neurons. As a consequence, exon I is not included in wild-type neurons. In nonneuronal tissue, inclusion of exon I is observed at low frequency, because the few transcripts that escape 3'-end formation at pA2 are spliced to exon I. Thus, ELAV and factors bound to the pA2 site together block the 3' splice site of exon I (Soller, 2003).

In summary, this study shows that the RNA-binding protein ELAV can inhibit 3'-end formation without affecting recognition of the pA site by CPSF and CstF64. ELAV and components of the pA complex then direct exclusive use of the distal 3' splice site to promote the neural processing mode. Because bona fide pA sites are frequently found in introns, binding of pA complex components could contribute to localize splice sites, and, as shown in this study, can regulate alternative splicing (Soller, 2003).

ELAV multimerizes on conserved AU^4-6 motifs important for ewg splicing regulation

ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. A combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes was used to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU_4-6 motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU_4-6 motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes it has been demonstrated in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU_4-6 motifs and that introduction of spacer sequence between conserved AU_4-6 motifs has a minimal effect on splicing. Collectively, these results suggest that ELAV multimerization and binding to multiple AU_4-6 motifs contribute to target RNA recognition and processing in a complex cellular environment (Soller, 2005).

Based on several lines of evidence, a model is proposed for a multimeric ELAV complex consisting of 12 ELAV molecules that associate with ewg RNA between a functional poly(A) site (pA2) and exon I in intron 6 in vitro. (1) ELAV assembles with RNA into a defined and saturable RNA-protein complex when assayed by EMSA. This association occurs in an RNA substrate-specific manner, since some RNAs do not form an ELAV-RNA complex even at a concentration of 3.2 microM, which thus clearly distinguishes the ELAV-RNA complex from an unspecific aggregation. (2) Two substrate RNAs of different size form two separable complexes, demonstrating that only one RNA is present in the final ELAV complex. (3) In size exclusion chromatography experiments under physiological salt conditions, ELAV bound to ewg RNA pA2-I results in a defined complex of about 700 kDa, and the smaller RBD60 protein yields an RNA-protein complex of appropriately reduced size of about 500 kDa, suggesting assembly of a complex in the range of 12 protein molecules. (4) In stoichiometry EMSAs the final ELAV complex forms at around a ratio of one RNA per 12 ELAV molecules. (5) Titration of ELAV against RBD60 (an N-terminal truncation mutant of ELAV) at complex-forming concentrations in EMSAs reveals 13 bands, as expected for a dodecameric complex. (6) Reducing the length of the substrate RNA does not result in ELAV complexes of intermediate size, indicating that binding of ELAV as dodecameric complex is an intrinsic property of ELAV to associate with target RNA. Although the tools to demonstrate an in vivo assembly of a dodecameric ELAV complex with target RNA in fly neurons are currently not available, circumstantial evidence supports the presence of large ELAV-RNA complexes in vivo. In the nucleus, ELAV has been found to sublocalize to sites of higher concentration in discrete dots and webs, indicating that complex formation with ewg pA2-I RNA at around 350 nM in vitro could meet in vivo conditions. Further, Hu proteins have also been shown to be present in large particles in cells and neurites (Soller, 2005).

Although ELAV shares the tetramer configuration characteristics with general heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins, binding to RNA induces a rearrangement into dimers. In addition, the ELAV complex forms on 43 to 135 nucleotides of RNA pA2-I, while a tetramer unit of general hnRNPs isolated from 40S particles binds to 200 to 240 nt, and the RNA present in the 40S particle is about 500 to 800 nt in length. The length differences of the RNAs present in the ELAV complex and general hnRNP complexes likely reflect a different packaging mode. Models for hnRNP C binding to RNA have favored a loose wrapping around the tetramer. For an ELAV-RNA complex, however, a different model might apply, since the two RRMs of Sex-lethal (Sxl) and the first two RRMs of HuD, both closely related to ELAV, were shown to cover 11 nt in cocrystallization experiments. A linear assembly of 12 ELAVs with RNA is therefore unlikely, particularly with RNAs as short as 43 nt, unless not all RRMs are in contact with RNA in such a complex. Moreover, phylogenetically conserved AU^4-6 motifs contain only 5 to 7 nucleotides. A possible alternate model for the assembly of the ELAV complex might therefore include that ELAV surrounds the RNA upon binding, similar to the core of Sm proteins bound to snRNA (Soller, 2005).

The assembly of a dodecameric ELAV complex on RNA pA2-I suggests that an array of repetitive cis elements might mediate complex formation. Results from various approaches show that a series of AU^4-6 motifs present between pA2 and exon I in the last ewg intron are important for ELAV complex binding. (1) Using RNA substrates with mutations in AU^4-6 motif element m1, m2, or m3 demonstrated that all elements spread over 135 nt contribute to ELAV binding in vitro in UV-cross-linking assays and EMSAs. (2) UV-cross-linking assays with segmentally labeled substrate RNAs using RNA pA2-ivs further demonstrate that the ELAV complex binding site extends over about 135 nt. (3) Phylogenetic analysis of the ELAV binding site reveals evolutionary conservation of six AU^4-6 motifs, suggesting that an ELAV dimer might bind per AU^4-6 motif. (4) Functional importance in vivo of AU^4-6 motifs is further shown in ELAV-mediated splicing of the last ewg intron, using Drosophila transgenes. Although an array of AU^4-6 motifs is important for ELAV complex formation, not all AU^4-6 motifs contribute equally. In particular, AU^4-6 motifs in the m3 element and the polypyrimidine tract have a much higher impact on high-affinity binding than the m1 and m2 elements, both in vitro and in vivo. A similar situation has been observed in the hnRNP A1 binding site in intron 3 of human immunodeficiency virus tat transcripts, and the following model has been proposed. A few hnRNP A1 molecules bind first to the high-affinity portion of the binding site and then recruit further hnRNP A1 molecules to nucleate to a higher-order complex. A similar model might also apply to ELAV complex formation. Here, high-affinity binding of few ELAV molecules to the 3' part of the complex binding site could lead to recruitment of more ELAVs that will enhance complex formation in the presence of additional AU^4-6 motifs. Alternatively, clustering of binding motifs might enhance cooperative interactions among ELAVs that then trigger formation of a stable complex upon reaching local concentrations close to the stoichiometry of the final ELAV complex at a specific target site, thereby contributing to gene-specific recognition of target RNAs (Soller, 2005).

The ewg ELAV complex binding site from the melanogaster species subgroup harbors three tandem AU^4-6 motifs (in m1, m2, and m3 elements) that can be aligned. Collectively, the results presented here argue against tandem AU^4-6 motifs in element m1, m2, or m3 either as individual tetramer binding sites or as overlapping binding sites for two tetramers. (1) ELAV assembles as dimers in stoichiometry EMSAs. (2) Deleting tandem AU^4-6 motifs does not result in ELAV complexes of intermediate size, since no one- or two-tetramer complexes are detected as the main product in EMSAs. Rather, ELAV complex formation and its affinity for a specific substrate RNA depend on length and poly(U) content of the substrate RNA. (3) Tandem AU^4-6 motifs in the m1 and m2 elements are not sufficient for complex formation in EMSAs. (4) Only six AU^4-6 motifs (m1r, m2r, m3l, m3r, and two in the polypyrimidine tract) are evolutionarily conserved. The additional AU^4-6 motifs present in the melanogaster species subgroup might therefore represent redundancy. This is also indicated by the minimal difference in affinity between RNA pA2-I, Delta1, and Delta12 in EMSAs, as the remaining AU^4-6 motifs still suffice for almost optimal positioning of the ELAV complex in this assay (Soller, 2005).

Besides increasing binding specificity, multimerization of RNA binding proteins might also provide a mechanism to locate distant RNA-processing signals by looping out intronic sequence to bring splice sites into proximity. The organization of the ELAV complex binding site illustrates flexibility in positioning of AU^4-6 motifs relative to each other, since they are not strictly conserved between different Drosophila species and introducing spacer sequences only marginally affects processing of the last ewg intron 6. Forming a complex with distant recognition sequences also offers an appealing explanation for ELAV-mediated regulation of pre-mRNA processing in the more complex situation of nrg, where a distal terminal exon is chosen in the presence of ELAV. UV-cross-linking studies of the whole nrg-regulated intron reveal four areas of binding in the vicinity of splice sites and poly(A) signals. An ELAV complex bound to these extensively spaced binding sites would exclude nonneuronal RNA-processing signals from recognition. A similar model has been proposed for autoregulation of hnRNP A1 alternative splicing. Here, hnRNP A1 binds sequences flanking both sides of the regulated exon, leading to skipping of this exon (Soller, 2005).

Functional importance of multimerization has also been indicated for hnRNP A1-regulated alternative splicing of intron 3 from human immunodeficiency virus tat transcripts. Here, multimerization of hnRNP A1 in the context of RNA secondary structure on intronic and exonic splicing silencer sequences competes with other factors for exon recognition. In the case of PTB-regulated splicing of a neural exon in c-src, a neuronal form of PTB, nPTB, is postulated to interfere with multimerization of PTB and release the blocked exon for inclusion in neurons. nPTB has also been found to interact with Nova-1 and to inhibit Nova-1-stimulated GlyRalpha2 E3A alternative splicing. In Drosophila, overexpression of the ELAV family member FNE can regulate expression of ELAV, similar to autoregulation by ELAV at endogenous levels. Since ELAV family members interact in yeast two-hybrid assays, they likely can also form heteromultimeric complexes in vivo, and multimeric binding of HuB at the c-myc 3' UTR has been indicated. In addition, many other RNA binding proteins have also been shown to engage in homo- and hetero-philic interactions (Soller, 2005).

In conclusion, multimerization of RNA binding proteins into macromolecular complexes likely is an important feature of this abundant class of proteins to localize pre-mRNA processing signals in a complex cellular environment in constitutive splicing and affect use of alternative splice and polyadenylation sites. In addition, multimerization of RNA binding proteins might also provide the combinatorial setup to posttranscriptionally coordinate the expression of functionally related genes in higher eukaryotes (Soller, 2005).

DEVELOPMENTAL BIOLOGY

Embryonic

The protein is present after 6 hours of embryogenesis. The level of EWG protein increases during embryogenesis and then appears to drop dramatically in third-instar larvae. A comparision of head and body extracts shows an enrichment in head preparations (DeSimone, 1993). The protein is nuclear and does nor appear in neuropil regions, axons or neuronal cytoplasm. EWG protein is first detected in the developing CNS shortly after the onset of germ band shortening (early stage 12) [Images]. Staining is barely visible at this stage and becomes progressively stronger during germ band retraction; it does not appear to change after full germ band retraction. EWG is also detected in cells of the dorsal, lateral and ventral peripheral nervous system clusters and in the head PNS structures, as well as in the brain (Fleming, 1989). It is unlikely that EWG is present in neuroblasts, since the protein is not detected until neuroblast segregation from the epidermis has been completed. The spatial and temporal distributions of EWG are remarkable similar to those of ELAV, but EWG appears later than ELAV after the birth of a neuron (DeSimone, 1993).

Larval

The six Dorsal Longitudinal flight muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult muscle fibers. In response to experimental ablation, myoblasts that would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles (DVMs), which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. These results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts; in so doing, they regulate formation of the correct number of DLM fibers (Fernandes, 1996).

Effects of Mutation or Deletion

Mutants of ewg exhibit both neural and muscle phenotypes; these include breaks in the central nervous system commissures and longitudinal tracts, aberrant intersegmental axonal projections pathways in the embryo, aberrant giant fiber position in the adult fly, missing or reduced indirect flight muscles associated with erect wing posture, and adult hypoactivity. ewg is also associated with neural defects in the retina and optic ganglia. Certain ewg mutations cause embryonic lethality (DeSimone, 1993 and references). Studies of gynandromorphs indicates that there is no compelling evidence of a strictly neural origin for the muscle defect in ewg mutants (de la Pompa, 1989).

Analysis of mutants of two gene pairs stripe and erect wing, and erect wing and vertical wings reveals that these loci exhibit a synergism. In addition, a dosage effect is apparent between ewg and sr. The ewg phenotype is similar to that of stripe These interactions suggest the existence of a functional relationship between the three loci (de la Pompa, 1989).

Most ewg lethal alleles lead to either a late embryonic or early larval lethal pase, indicating that the ewg gene product is necessary for the development of more than the dorsal longitudinal flight muscles (Fleming, 1983).

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date revised: 20 January 2007 

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