The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein

Abstract

Although the Drosophila melanogaster 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, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, 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 in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

FIG. 1

(A) Maps of ewg gene and ewg transgenes. ewgR19 is a genomic EcoRI fragment that includes ewg SC3 cDNA. elav-ewg is a transgene in which a genomic EcoRI-PstI fragment is driven by the neuron-specific elav promoter. elav-EWG is a minitransgene in which the elav promoter drives the SC3 cDNA. The black boxes show exons, and the grey boxes indicate 5′ and 3′ UTR sequences present in the SC3 cDNA. All constructs provide full rescue of ewg^l1, a protein-null allele. The transcriptional start site of ewgR19 is not known, but its general location is indicated with an asterisk. Exons E and I are not present in the SC3 cDNA. Retention of exons E and I prematurely terminates the 116-kDa ORF. On the right, levels of EWG expression in the third-instar ELAV-depleted edr eye imaginal discs shown in panel D are summarized for the different transgenes. Note that only the genomic fragments show a reduced expression. R and P denote restriction sites EcoRI and PstI. (B) Expression of EWG protein in ELAV-deficient eye discs. Third-instar larval eye discs of the wild type (Ba, c, and e) or an edr mutant (Bb, d, and f) were stained with either anti-ELAV (Ba and b), anti-EWG (Bc and d), or anti-APPL (Be and f) antibodies. Note that both ELAV and EWG levels are reduced compared to that of the control APPL antigen. Bar, 50 μm. (C) ELAV-null clone double labeled for ELAV and EWG. An eye disc of the genotype elav^e5/Y; elav^DMORF2/Ki p^p(Δ2-3ry⁺) depicting an ELAV-null patch simultaneously immunoreacted with anti-ELAV (Ca) and anti-EWG antibodies (Cb) and a merged image (Cc). Note that the ELAV and EWG signals overlap and that the EWG-null patch coincides with the ELAV-null patch. Bar, 5 μm. (D) EWG expression elicited from ewg transgenes in wild-type and ELAV-deficient eye discs. Different ewg transgenes were crossed into the EWG-null (ewg^l1) and ELAV-deficient (edr) genetic backgrounds to assess EWG levels by immunohistochemistry. Eye discs of genotype ewg^l1/Y; ewg transgene (Da, c, and e) and edr/Y; ewg transgene (Db, d, and f) were immunoprocessed; the transgene designation in each case is noted on the right edge. Note that in ewg^l1, expression is only from the transgene, while in edr, EWG levels reflect transgene expression superimposed on the endogenous (reduced) EWG expression. (Da and b) elav-EWG transgene expression. Note the similar level of EWG immunoreactivity in both genotypes. (Dc and d) ewgR19 transgene expression in ewg^l1 and edr. Note that the expression of EWG evident in panel Dd is reduced compared to that evident in panels Da, b, and c. (De and f) elav-ewg transgene expression in ewg^l1 and elav^edr. Note that the expression evident in panel Df is reduced compared to that evident in panels Da, b, c, and e. Bar, 50 μm.

FIG. 2

(A) Alternative splicing of ewg transcripts and PCR primer map. The top diagram shows ewg introns and exons except exon A. The splicing pattern for the neurally enriched SC3 ORF is drawn below the exons and the predominantly nonneuronal splicing pattern is shown above. The lower diagram depicts the positions of the primers used for RT-PCR. The primer sequences are as described in reference . (B and C) Splicing profile of ewg mRNA in wild-type and ELAV-deficient eye discs (B) and wild-type wing discs and wing discs ectopically expressing ELAV (C). Quantitative RT-PCR assays were carried out using DNase I-treated total RNA isolated from eye discs of third-instar wandering larvae. The primer sets used for the four reactions shown in each row of images are listed in the leftmost column. The rightmost columns depicts diagrams of the amplified fragments, and the black dots indicate the fragments expected of 116-kDa ORF-encoding transcripts. Each rectangle shows the results of four successive PCR cycles in the presence of [α-³²P]dCTP (cycles 18, 20, 22, and 24 for primer pairs 3cF-3cR and 3aF-3cR and cycles 20, 22, 24, and 26 for primer pairs 4F-5R, 3aF-3aR, 6F-6R, 6F-RV, and FV-6R). Sizes of bands are as listed in Table 1. As shown in panel B, ewg splicing was assessed in wild-type (cs) and edr eye discs. Note the threefold reduction of intron 3c splicing in 3cF-3cR (marked with an arrow) and in 3aF-3cR reactions (marked with an arrow) in edr. Additionally, intron 6 splicing efficiency decreases more than 10-fold in edr as revealed with primer pair 6F-6R (marked with an arrow). As shown in panel C, ewg splicing was assessed in wild-type (cs) and c309/UAS-ELAV^2e2; UAS-ELAV^3e1 (ectopic ELAV) wing discs (expression pattern shown in Fig. 3). ewg mRNA, but no protein, is expressed in wild-type wing imaginal discs (14). Note the two- to threefold increase of intron 3a splicing in the 3aF-3aR reaction (marked with an arrow) and the 10-fold increase of intron 3c splicing in 3cF-3cR (marked with an arrow) and 3aF-3cR reactions (marked with an arrow) upon ectopic ELAV expression. Additionally, intron 6 splicing efficiency increases by orders of magnitude after ectopic expression of ELAV, as revealed with primer pair 6F-6R (marked with an arrow).

FIG. 3

(A) Ectopic EWG expression levels correspond to ELAV levels. (Aa to Ad) EWG levels were monitored in wing discs ectopically expressing ELAV. Wing discs were stained with anti-ELAV (Aa) or anti-EWG (Ab to d) antibodies. Note that neither ELAV (Aa) nor EWG (Ab) is expressed at this stage in wild-type wing discs. Wing discs of the genotypes c309/UAS-ELAV^2e2 (Ac) and c309/UAS-ELAV^2e2; UAS-ELAV^3e1 (Ad) were stained with anti-EWG serum. Note that ectopic ELAV expression with two doses of UAS-ELAV further increases EWG levels in nonneural cells. (Ac) Expression of elav transcript in wild-type (+) and ectopically expressing (e) wing discs of the genotype c309/UAS-ELAV^2e2; UAS-ELAV^3e1 revealed by RT-PCR. Transcripts of ribosomal protein 49 (rp49) were used as a control. (B) Induction of ectopic EWG expression by ectopic ELAV depends on the presence of ewg introns. Wing imaginal discs of the genotypes ewg^l1 y w sn/Y; c309; UAS-ELAV^3e1 elav-EWG (Ba and b) and ewg^l1 y w sn/Y; c309; UAS-ELAV^3e1 ewgR19-1 (Bc and d) were double labeled for ELAV (Ba and c) and EWG (Bb and d). Note that ELAV can drive EWG expression from ewg intron-containing transgenes. Bar, 50 μm.

FIG. 4

Exclusion of exon 6 of arm is dependent on ELAV levels. (A) Schematic representation of arm splicing. The penultimate exon 6 is excluded in neurons (31). (B) arm splicing was monitored in edr and wild-type eye discs and in wild-type and c309/UAS-ELAV^2e2; UAS-ELAV^3e1 wing discs. c309 is an enhancer trap GAL4 line (expression pattern shown in Fig. 3). Note that exclusion of exon 6 (arrow) correlates with the presence of ELAV.