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Molecular and Cellular Biology, March 2000, p. 1836-1845, Vol. 20, No. 5
Department of Biology and Center for Complex
Systems, Brandeis University, Waltham, Massachusetts 02454
Received 21 September 1999/Returned for modification 11 November
1999/Accepted 7 December 1999
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.
The Drosophila melanogaster
erect wing gene (ewg) provides a function that is vital
in the nervous system and essential for the development of specific
indirect flight muscles (13, 15, 19). A single 116-kDa
protein is sufficient for both neuron- and muscle-associated functions
(15, 46). Recent data indicates that, although the
functional 116-kDa protein is highly enriched in neurons, the gene is
transcribed comparably in both neuron-rich and neuron-poor tissues and
that neuron-enriched expression of the functional protein is achieved
by posttranscriptional regulation of ewg, which includes
both alternative and inefficient splicing (27). Alternative
splicing in two regions leads to enrichment in heads of the transcript
with an open reading frame (ORF) that encodes the 116-kDa protein
containing an unusual DNA binding domain. Further, misregulation is
biologically consequential, as global expression of the 116-kDa protein
can be lethal (27).
The Drosophila embryonic lethal abnormal visual system
(elav) gene encodes a neuron-specific RNA binding protein
and is expressed in all neurons (44). Since
Drosophila ELAV has been shown to be important for the
formation of the neuron-specific protein isoform of Neuroglian
(26), we investigated if ELAV also has a role in
ewg regulation. ELAV-like proteins are evolutionarily conserved, as several genes encoding proteins with three RNA
recognition motifs with high homology to ELAV in the RNA recognition
motifs have been identified in both vertebrates and invertebrates
(reviewed in reference 2). Data on several proteins
of the ELAV family, from mammals and Drosophila, suggest
that these proteins bind the 3' untranslated region (UTR) and regulate
mRNA stability (11, 12, 17, 29, 41, 42) and translatability
(3, 21). Recently, an autoregulatory function, whereby ELAV
regulates its own expression, requiring 3' UTR sequences has also been
demonstrated (47).
In this paper, we report that, indeed, in photoreceptor neurons, the
generation of the 116-kDa EWG protein is dependent on ELAV and this
dependence is contingent on the presence of ewg introns.
Next, using reverse transcription-PCR (RT-PCR), we show that the
ewg splicing profile is altered in ELAV-deficient
photoreceptors such that transcripts representing splice choices that
lead to the 116-kDa ORF are reduced. We also show that ectopic
expression of ELAV in nonneural tissue is sufficient both to increase
RNAs with neuron-like splicing choices and for the expression of the 116-kDa protein. These data further substantiate an in vivo role of
ELAV in promoting neuron-specific splice isoforms. Further, we show
that alternative splicing of armadillo (arm),
another ubiquitously expressed gene with a neuron-specific isoform
(31), is also regulated by ELAV. Finally, we report our
initial findings that not all genes with neuron-specific alternatively
spliced transcripts are regulated by ELAV.
Fly stocks and genetic crosses.
Flies were raised at 25°C.
Standard genetic marker genes and Balancer chromosomes were as
described in reference 30. Canton-S was used as the
wild-type strain. The alleles and transgenes used in this study are as
follows (abbreviated names are given, followed by full descriptions):
ewgl1, an ewg lethal allele which
does not allow expression of the 116-kDa EWG protein (27);
ewgR19-1 and ewgR19-2, two independent inserts of
ewg genomic transgene
P{ry+=ewg+} that provide full
rescue of ewgl1 (18);
elav-EWG, a transgene,
P{w+=elav-EWG} (in which ewg cDNA
[SC3 ORF] is fused to the elav promoter [53]), which provides full rescue of
ewgl1 and is referred to in reference
13 as EWGNS; elav-ewg, a
transgene, P{w+=elav-ewg}28 (in which
ewg genomic transcribed sequences are fused to the
neuron-specific elav promoter [53]), which
provides full rescue of ewgl1 (25);
elavedr, a specific insert of the transgene
P{w+=elav+} which has a
wild-type elav genomic rescue fragment (ELAV expression of
P{w+=elav+}edr insert is
specifically reduced in photoreceptor neurons, but expression in brain
neurons is less affected due to the transgene insertion site
[25, 26]) and whose expression phenotype is revealed
in combination with elave5, an elav
null allele; edr, the genotype
elave5; elavedr;
UAS-ELAV2e2 and
UAS-ELAV3e1, two inserts of transgene
P{w+=UAS-ELAV} on chromosomes 2 and 3 that
express elav cDNA under UAS transcriptional
control (26); P{w+=elav)DmORF2, a
transgene expressing ELAV under the control of the elav
promoter (54); and c309, a P{GAL4}c309
enhancer trap line with the transgene insertion on the second
chromosome (34).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Neuron-Enriched Splicing Pattern of
Drosophila erect wing Is Dependent on the Presence of
ELAV Protein
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2-3
ry+) males, a source of transposase (43).
Due to transposase activity of 2-3, the P
element containing the elav rescue construct is excised in
cells yielding somatic clones of ELAV-deficient tissue. Clones were
detected by double staining eye imaginal discs of third-instar larvae
for both ELAV and EWG. Clones were viewed in an MRC-600 confocal microscope.
Immunohistochemistry. Imaginal discs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 40 min and washed several times in PBS containing 0.1% bovine serum albumin and 0.3% Triton X-100. Antibody incubations were done overnight at 4°C. Anti-EWG rabbit serum (15) was used at a 1:300 or 1:400 dilution, and anti-ELAV monoclonal antibody 9F (16) was used at a 1:20 dilution. Anti-APPL rabbit serum (33) was used at a 1:200 dilution. Secondary antibodies were from Jackson Immunoresearch Laboratories Inc. (West Grove, Pa.) and were used at a dilution of 1:50 or 1:100. Photography was done using a Zeiss Axiophot fluorescence microscope and Tmax-400 film.
RT-PCR assays.
Total RNA was isolated with Trizol reagent
(Life Technologies, Gaithersburg, Md.) in accordance with the
manufacturer's instructions. For precipitation of the RNA, 10 to 20 µg of glycogen (Boehringer Mannheim, Indianapolis, Ind.) was used.
Sixty eye or 60 wing imaginal discs from wandering third-instar larvae
were dissected, collected in Trizol reagent, and used for
oligo(dT)-primed RT after treatment with RNase-free DNase I (Life
Technologies). RT was done with the Superscript II cDNA synthesis kit
(Life Technologies) according to the manufacturer's instructions,
except that the RNA was kept at 50°C for 5 min before the RT reaction
was started. After RT the cDNA was treated with RNase H. Control
experiments were carried out with no reverse transcriptase. PCR was
performed using the ewg and rp49 primers
described in reference 27. An additional ewg primer was FV, 5'-GCTTGTCCTCATTTTATATTGAG-3'.
From 60 discs 1/20 of the disc tissue was used for PCR, yielding
very similar amounts of PCR product from different genotypes for the
same amount of disc tissue. Semiquantitative PCR was done for 30 cycles, and PCR products were visualized on agarose gels. The sizes of
the expected products for each primer pair used are catalogued in Table
1. In addition to the size, the identity
of PCR products was further verified by restriction digests or direct
sequencing with ABI automated sequencing equipment. Quantitative
amplification was done in the presence of 5 µCi of
[
-32P]dCTP, and PCR products were separated on
nondenaturing 5% polyacrylamide gels. Quantitation was done on a
Molecular Dynamics PhosphorImager using Imagequant at cycles 22 and 24, which was in the linear range for all primer pairs. The Appl
primers were
5'-CGCCTCGCCGCGATGGGAGC-3' and
5'-GATCCCTTGCACTTAGCCGCAT-3'.
The elav primers were 5'-GTTCGCTTTGTTTGTCCAGCC-3' and 5'-TGTTGCCGCCACTGCTGCGGC-3'. The arm
primers were 5'-GCAGGATTACAAGAAGCGGCT-3' and
5'-CTCCAGACCCTGCATCGAATC-3'. The nrg primer was
5'-CGGAAAGTACGATGTCCACG-3', with return primers being
5'-TAAATCAAAGTCCTTTGCGTCC-3' and
5'-TGATGCGCCGCAGCGGAATTGT-3'. The creb primers
were 5'-AAGATCTTCACCGAGATCAGCG-3' and
5'-GCGCTTACGGGTCTGATCCT-3', spanning alternatively spliced
exon 6 as described in reference 55. The
pres primers were as described in reference
35. The fra primers were
5'-TGGTGGATGGTCACAATCTGAA-3' and
5'-CTTCACTATCTGCGCCACCAGA-3'. The PTP4E primers were
5'-CATCCTGCAGCACATTCACAAG-3' and
5'-AGATTCCCGTCTCAGCTATGCC-3'. The PTP10D primer was
5'-ATATCTGCATCCACCAGTGCCT-3', with the return primers
being 5'-AGATGGTTGGGTTCTGTTGGGT-3' and
5'-GCTGGTCATCATGGAGTATCGC-3'. The PTP99A primers were
5'-GAATTCGAGGATGTGACAACGG-3' and
5'-ACAGGGCAAACGAATTGTTGAA-3'.
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Plasmid construction.
The EcoRI-PstI
fragment of genomic ewg was cloned into pCaSpeR 4 (14). The elav promoter (53) was
digested with NotI and EcoRI and inserted via a
NotI-EcoRI adapter oligonucleotide into pBluescript (Stratagene) and subsequently cloned into pCaSpeR 4 containing the genomic ewg fragment as an EcoRI
fragment, and Drosophila embryos were transformed by
standard techniques as described in reference 54,
but 2-3 was used as the transposase source (43).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Figures
1A
and 2A summarize the
aspects of ewg exon and intron structure and splicing
patterns relevant to this study and described previously
(27). The primary transcript of ewg, which has 10 exons, A to J (exon A is not shown in Fig. 1), is alternatively spliced in two regions (Fig. 2A). We have previously shown that 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, which is depicted in Fig. 1 (SC3 ORF). Additionally, splicing
of introns 3a, 3c, and 6 is inefficient, as these introns are
retained in polyadenylated ewg RNA (27). Despite
similar abundance of other splice isoforms, only one protein of 116 kDa could be detected (27).
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In this analysis, we consider splicing of introns 2 to 6, which encompass the entire coding region, with a special focus on the alternative splicing that leads to the 116-kDa ORF. From previous studies, we know that due to alternative splicing, several conceptual isoforms are encoded by the ewg locus (27). All putative EWG isoforms share exons B and C and thus should be recognized by the anti-EWG antiserum. Inclusion of exon E or I, as well as retention of introns 3a, 3c, and 6, changes the 116-kDa ORF. Since our analysis failed to detect isoforms other than the 116-kDa protein, we concluded that other isoforms are either minor, unstable, or not made (27). These observations together with the ability of the 116-kDa protein to restore all three characterized phenotypes associated with ewg mutations, viability, erect wings, and formation of the flight muscles (15, 27), suggest that the other isoforms are unlikely to be functionally significant. However, since generalized increased expression of the 116-kDa protein is lethal, the splicing regulation of ewg is important.
In these studies, as a source for neuron-enriched RNA we used third-instar larval eye-antenna imaginal discs. In the eye discs, the differentiating field of photoreceptors shows robust ELAV expression (Fig. 1Ba); however, it does contain many cell types in addition to the photoreceptor neurons (51). As a source of nonneural tissue we used wing imaginal discs, which at this stage of development have not yet differentiated neurons and therefore are devoid of any ELAV protein (see below).
Presence of ewg introns confers ELAV regulation of EWG expression. To analyze whether ELAV has a role in neuron-enriched expression of the 116-kDa EWG protein, we used a synthetic elav mutant genotype, edr, which has dramatically reduced ELAV expression in developing photoreceptors, though expression in central nervous system neurons is only slightly affected (26). Members of our group had previously noted (26) that photoreceptors with elavedr as their exclusive source of ELAV have a low level of EWG compared to wild-type photoreceptors, while expression of many other proteins remains unaffected (Fig. 1B). To further test that ELAV indeed regulates EWG expression, we generated ELAV-null somatic clones in the eye discs as described in Materials and Methods. Figure 1C shows a somatic clone in a disc simultaneously immunoreacted for ELAV and EWG. The merged image reveals that EWG and ELAV immunoreactivity is coincidentally absent from the same cells. Thus, EWG expression in photoreceptors is dependent on ELAV expression and the effect of ELAV is cell autonomous.
The ELAV-dependent regulation of EWG expression in photoreceptors could result from either transcriptional or posttranscriptional mechanisms, for example, regulation of alternative splicing or mRNA stability. To distinguish between these mechanisms, ewg transgenes that contain either the genomic transcribed region or the minigenes that express the SC3 cDNA under the control of a heterologous promoter were used. SC3 cDNA has a 1,962-nucleotide-long (1,962-nt-long) 5' UTR and a 578-nt-long 3' UTR (15). 3'-RACE experiments confirmed that the 3' end of the SC3 cDNA coincides with the 3' end of the transcript (data not shown). Minitransgene elav-EWG expresses the SC3 cDNA with only 27 bp of the 5' UTR fused to the neuron-specific elav promoter (Fig. 1A) and terminates 10 nt after a polyadenylation signal (AAUAAA) (13). ewgR19 is a genomic transgene starting 1,852 nt 5' to exon B, including exons B to J and introns 2 to 6 (Fig. 1A) (18). elav-ewg is a genomic transgene encompassing exons B to J and introns 2 to 6 (EcoRI-PstI fragment from the ewg gene fused to the elav promoter [Fig. 1A]) (53). Also, in constructing elav-EWG and elav-ewg, no additional 3' UTR sequences were added to avoid masking the regulation of ewg expression. All transgenes provide full rescue of the lethal phenotype of ewgl1, a null allele (13, 27). EWG immunoreactivity for these three transgenes was assessed in ewgl1 and edr male eye discs (Fig. 1D). In edr, EWG signal results from summation of both the endogenous ewg+ gene and the transgene, while in the ewgl1 background only the transgene expression is observed, as ewgl1 is a protein-null allele (27). In edr, the endogenous EWG signal is reduced compared to wild-type expression (Fig. 1Bc and d), but the signal from elav-EWG is robust and similar to that in the ewgl1 genetic background (Fig. 1Da and b). Therefore, ELAV does not regulate elav-EWG expression. In contrast, the expression of both ewgR19 and elav-ewg depends on ELAV, as indicated by the reduced EWG signal from these transgenes in edr compared to that in the ewgl1 genetic background (Fig. 1Dc and f). These results show that ELAV-dependent EWG expression in photoreceptors is observed when transgenes contain introns 2 to 6, even when the transcription is driven by a heterologous promoter, as in the case of elav-ewg. The requirement for introns makes splicing an attractive step for ELAV control, especially in the light of the known alternative splicing of ewg transcripts (27). Also, since elav-ewg and elav-EWG have the same 3' UTR, but only elav-ewg responds to reduced ELAV levels, 3' UTR-mediated regulation by affecting stability is unlikely.Analysis of splice forms indicative of 116-kDa ORF of ewg. Generation of the neuron-enriched 116-kDa ORF, which requires inclusion of exon D and exclusion of exons E and I, is achieved by excision of introns 3a, 3c, and 6 (27). Previous analysis had revealed that neuron-rich head RNA has higher concentrations of splice forms indicative of alternatively spliced introns 3a, 3c, and 6, while splicing of introns 2, 3b, 4, and 5 was similar in head and body RNAs. To determine if ELAV affects ewg splicing patterns, the concentration of different spliced isoforms of ewg in wild-type and ELAV-deficient third-instar edr eye discs was assessed by quantitative RT-PCR using nine primer pairs. The number and size of the expected bands for each primer pair are catalogued in Table 1, and the experimental results are shown in Fig. 2B.
Using primers that flank intron 2 and introns 4 and 5, we confirmed that these introns were efficiently spliced in the wild-type and edr eye discs and that the amplified bands indicated equal concentrations of cDNA from the edr and wild-type eye disc samples (Fig. 2B, primers 4F-5R). Further confirmation that the two RNA samples had similar concentrations came from the control rp49 primers, which, as expected, yielded comparable bands (data not shown) (28). To analyze splicing within the 3b region, three sets of primers were used, as follows: 3aF-3aR (flanking intron 3a), 3aF-3cR (flanking the entire region), and 3cF-3cR (flanking intron 3c). In edr, the PCR products indicative of 3a-spliced and unspliced transcripts remain comparable to wild-type eye discs (Fig. 2B, primers 3aF-3aR). The splicing in the 3c region is complicated by the presence of exon E, which is excluded from the 116-kDa ORF. Primers 3cF-3cR and 3aF-3cR yield four and six products respectively, as described in Table 1 and depicted in Fig. 2B, which shows the band of the product expected from the 116-kDa ORF and another band that includes exon E. A modest effect of ELAV depletion is discerned on intron 3c, as in edr eye disc RNA, transcripts with only exon D included (Fig. 2B, primers 3cF-3cR and 3aF-3cR) show a reduction of about threefold, while those that contain both D and E show no change when compared to the wild-type eye disc RNA, as evidenced in 3cF-3cR and 3aF-3cR reactions. These results show that ELAV depletion results in a reduction of transcripts that splice intron 3c, but splicing of introns 3a, 3c-1, and 3b is relatively unaffected. Based on the spliced product/spliced-plus-unspliced product ratio for introns 3a and 3c (about 0.55 for each) and assuming that the two events are independent, we estimate that the overall splicing efficiency which leads to inclusion of exon D is only about 30%. In the intron 6 region, a dramatic (greater than 10-fold) reduction of intron 6-spliced transcripts (Fig. 2B, primers GF-GR) is seen in edr. To analyze retention of intron 6 we used primer pairs 6F-RV and FV-6R; in each case, one of the primers is within intron 6a. Marginal differences were observed; they were, however, in the range of variability, suggesting that intron 6 is spliced only from a small amount of all transcripts. Therefore, a likely interpretation of these results is that ELAV's presence enhances splicing of intron 6. In summary, ELAV depletion is accompanied by reduction of intron 6 splicing and a more modest reduction of intron 3c splicing.Ectopic ELAV induces expression of EWG protein in nonneural
tissue.
Next we tested if EWG protein expression can be induced
when ELAV is ectopically expressed in nonneural wing imaginal discs. In
third-instar wing discs ewg RNA is broadly expressed
(14), while EWG protein is not detected at this stage (Fig.
3Ab). ELAV was ectopically expressed in a
subset of wing disc cells that do not normally express ELAV using the
GAL4/UAS system (8) as described previously
(26). Wing discs immunoreacted for EWG show the presence of
EWG protein when ELAV is expressed ectopically (Fig. 3Ac). To test if
EWG levels correlate with the concentration of ELAV, wing discs with
one and two doses of UAS-ELAV transgenes were compared. An
increase in the concentration of ELAV clearly shows a dose-dependent
enhancement of the EWG signal (Fig. 3Ac and d). Thus, ELAV is
sufficient to induce EWG expression, in a dose dependent manner, in
nonneural wing disc tissue.
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Ectopic ELAV induces expression of 116-kDa ORF-like splice profile in nonneuronal tissue. To address whether the generation of EWG protein is accompanied by changes in the splicing profiles, polyadenylated RNA from wild-type and ectopic ELAV-expressing (c309/UAS-ELAV2e2; UAS-ELAV3e1) wing discs was analyzed using quantitative RT-PCR. As expected, elav transcript levels in the wild-type wing discs were below the level of detection, but a robust signal was seen in ectopically expressing wing discs (Fig. 3Ae). Wild-type and ELAV-expressing wing disc profiles do not differ in the splicing of ewg introns 2, 3b, 4, and 5 (data not shown for intron 2) (Fig. 2C, primers 3aF-3cR [lowest band] and 4F-5R). However, differences were observed in products of the alternatively spliced ewg introns 3a, 3c, and 6 (see below).
The RT-PCR profiles observed in ectopic ELAV-expressing wing discs are consistent with the expectation that ELAV's presence promotes 116-kDa ORF-like transcripts. In the intron 3c region, a higher concentration (about 10-fold) of transcripts, indicative of a 116-kDa-like ORF, is seen in wing discs ectopically expressing ELAV (Fig. 2C, compare bands in images corresponding to primers 3cF-3cR and 3aF-3cR). Also, a two- to threefold increase in the spliced 3a product, accompanied by a comparable decrease in the unspliced product, is seen in wing discs ectopically expressing ELAV (Fig. 3C, compare bands in images corresponding to primer 3aF-3aR). Overall splicing efficiency which leads to inclusion of exon D is only about 5% but increases to about 25% upon ectopic ELAV expression. There is a barely discernible intron 6-spliced product in wild-type wing discs, but ectopic ELAV expression results in an increase of orders of magnitude in the intron 6-spliced transcript while transcripts that retain exon I are unchanged (Fig. 2C, primers 6F-6R). Similar to edr and wild-type eye disc RNA, levels of retained intron 6 were unchanged under ectopic ELAV expression (Fig. 2C, primers FV-6R and RV-6F). In summary, ectopically expressed ELAV in wing discs mimics the splice profile of wild-type eye discs. Wild-type wing discs imitate the ELAV-deficient edr eye discs, except that inclusion of exon D is reduced and splicing of intron 6 is barely detectable (Fig. 2B and C). Thus, ectopic ELAV in nonneural cells leads to the neuronal splicing pattern. Further, the dose-dependence of ectopic EWG expression in the wing disc on ELAV levels implies that ELAV is a rate-limiting factor in this cell type (Fig. 3Ac and d).ELAV regulates neuronal splicing of arm and
nrg.
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) (Fig. 4A)
(31). 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 edr eye
discs, as well as from wild-type eye discs and wing discs ectopically
expressing ELAV was analyzed by RT-PCR. The amount of n-arm
was reduced in ELAV-deficient eye discs, and in the ectopically
expressing wing discs expression of n-arm was clearly
induced (Fig. 4B). No change was observed in the band representing
u-arm splicing (Fig. 4B). rp49 levels were equal
and elav levels were comparable to those shown in Fig. 3Ae.
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 nrg
transcripts (25, 56).
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ELAV does not regulate all neuron-enriched splicing events. Thus far, we have identified three transcripts, ewg, nrg, and arm, where ELAV affects alternative splicing to produce neuron-enriched or -specific protein isoforms (26). This made us wonder if all genes which are alternatively spliced and which are expressed in neuronal and nonneuronal tissues are regulated by ELAV. Therefore, we analyzed alternative splicing of several ubiquitously expressed genes which are alternatively spliced. While searching for this class of genes, genes which could be involved in the nervous system defects of elav-mutant embryos were favored (22, 45). The DCC homologue frazzled (24), receptor phosphatases PTP4E, PTP10D, and PTP99A (39, 52), CREB (55), and presenilin (35) were tested for ELAV-dependent alternative splicing in edr and wild-type eye discs. None of these genes seem to be ELAV dependent, since their splicing patterns were unaffected, i.e., not reduced in elavedr eye discs (data not shown). Thus, not all genes which are alternatively spliced and expressed in neuronal and nonneuronal tissues are regulated by ELAV.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of EWG is regulated posttranscriptionally. Previous studies demonstrate that, in spite of the broad transcription of the ewg gene, the 116-kDa EWG isoform is almost exclusively generated in neurons and that the other predicted isoforms, if synthesized, are present at very low levels (27). One exception to this is the expression of EWG protein in the developing myoblasts (13) that has been shown to be important in the development of the dorsal longitudinal indirect flight muscles. Whether the myoblast EWG is the same 116-kDa protein is not known; however, a minitransgene expressing SC3 cDNA under the control of heat shock promoter 70 rescues the muscle phenotype (15). In this study, we show that abundance of certain splice isoforms indicative of the 116-kDa EWG ORF are reduced when ELAV is reduced. Additionally, we show that ectopically expressed ELAV can induce EWG expression. How is this positive regulation effected? The broad expression of ewg primary transcript makes transcriptional regulation unlikely to be the primary cause. Nevertheless, we formally excluded that possibility by demonstrating that intron-containing transgenes are ELAV responsive even when driven by a heterologous promoter, while intronless transgenes driven by the same heterologous promoter are ELAV independent.
Mechanism of ELAV-mediated regulation. Our major findings are that transcripts indicative of the 116-kDa ORF are reduced when ELAV is reduced in the photoreceptors of the eye imaginal discs and are induced when ELAV is ectopically expressed in the imaginal wing discs. We have previously shown that a similar transcript preference is also seen in the neuron-rich adult heads over neuron-poor bodies (27). Together, these two findings suggest that the observations made in the photoreceptors may be extended to adult neurons. Since ELAV is not expressed in the myoblasts, the mechanism of ewg regulation in myoblasts is not addressed here.
Our data was collected by RT-PCR analysis with oligo(dT)-primed cDNA and therefore only relates to that fraction of ewg pre-mRNA that was amplified from the first-strand cDNA synthesis. An additional limitation of these data is that third-instar eye discs, the source for neuron-rich RNA, are composed not only of neurons with high levels of ELAV expression but also of other nonneuronal cell types that have no ELAV. We used eye discs because of the unique genetic advantage offered in having an ELAV-depleted cell type. The wing discs present a more homogeneous population of cells devoid of ELAV. ELAV could influence splicing efficiency of specific splice sites directly, or affect the stability of specifically spliced transcripts through 3' UTR interaction, similar to its mammalian family members HuR (17, 42), HuD (11, 12), and Hel-N1 (29). Below we evaluate key data presented in this paper in the context of these two alternatives. Although direct action by ELAV is the simplest alternative, our data do not rule out a scenario in which ELAV orchestrates these events through ELAV-regulated trans-acting protein(s). First, observations with the ewg transgene expression suggest that the 3' UTR is neither sufficient nor necessary for ELAV-mediated ewg regulation. Genomic transgenes are ELAV regulated, while minitransgenes without introns which express the 116-kDa isoform and have the same 578-nt-long 3' UTR as the SC3 cDNA are not ELAV responsive. Furthermore, ewg transgene EWGHS (P{w+=hsp
EWG),
in which the SC3 DNA is driven by the hsp70 promoter, also generates a
116-kDa protein in nonneural tissues and is immune to ELAV in the eye
disc (25). The requirement for the intron-containing genomic region for ELAV dependence in photoreceptor neurons and in nonneural wing imaginal discs suggests that the 3' UTR alone is not
sufficient. Moreover, if ELAV-mediated regulation stabilizes the
116-kDa ORF encoding transcripts only in neurons, then the 116-kDa ORF
transcripts transcribed from a cDNA with the same 3' UTR under a
heterologous promoter should be unstable in ELAV-deficient eye discs,
which does not appear to be the case.
Second, although transcripts that include exon J or exons J and I both
share the same 3' ends, only transcripts that include J alone show a
correlation with ELAV. For example, the majority of spliced RNA species
in the 6F-6R reaction in wing discs (Fig. 2C) include both exons J and
I. The spliced RNA species which includes exon J alone is almost not
detected. If transcript stabilization were the mechanism, one would
expect to see both transcripts increased when ELAV is ectopically expressed.
Third, data presented in this paper show that, in ELAV's presence,
intron 6-spliced transcripts increase. However, the transcripts that
retain intron 6 do not show a dramatic change, nor is there a major
effect on transcripts that retain exon I. This was observed in
comparisons of both edr and wild-type eye discs and
wild-type wing discs and wing discs ectopically expressing ELAV (Fig.
2B and C). These observations are consistent with the scenario in which
ELAV enhances splicing but overall splicing of intron 6 is still very
inefficient. Similar arguments also hold for splicing of intron 3c.
These data allow a rough estimate of the efficiency of splicing in the
wild-type eye disc RNA amplified from a polyadenylated RNA population,
resulting in the 116-kDa splice form, to be about 1 to 2%. This
calculation assumes that splicing of introns 3a, 3b, and 6 occurs
through independent events and that all products in a given reaction
are amplified with equal efficiency (intron 3a-spliced product, about
50%; intron 3c-spliced product, about 50%; and intron 6-spliced
product, less than 20%).
Fourth, if a specific transcript is more abundant due to stabilization,
common regions of transcripts (exons B, C, and F to H) should also show
correlation with altered ELAV levels. ELAV levels do not seem to affect
these common regions either in edr eye discs or in wing
discs ectopically expressing ELAV. However, it is possible that, even
in ELAV's presence, the splicing of intron 6 is so inefficient that
the exon J-containing transcript constitutes too small a fraction of
the commonly spliced transcript.
Taken together, these considerations present a strong case for
ELAV-mediated processes influencing specific splice site choices rather
than transcript stabilization to regulate EWG protein expression.
ELAV does not affect all splice site choices in ewg. Many of the splice events, for example, 3b-spliced product or the inclusion of exons E and I, are unaffected by ELAV levels. This could be because these events occur only in nonneuronal cells, e.g., epithelial cells in eye discs. Alternatively, the default splicing could be mediated by another mechanism which does not compete with ELAV. Similar findings were reported in Caenorhabditis elegans for regulated alternative splicing of unc-52 transcripts by mec-8, an RNA-binding protein with two RNA recognition motifs (RRMs) (32). In mec-8 mutants only a subset of alternative splice events of unc-52 transcripts are abolished in a region of several alternative splice events and levels of unaffected alternatively spliced transcripts remain unaltered.
Tissue-specific alternative splicing has been postulated to be controlled by both tissue-specific factors and differential concentrations of general splicing factors (10). Cell culture studies have shown that ubiquitously expressed general splicing factor SR proteins, which, like ELAV, also contain RRMs, influence splice site choice depending on their concentration (9, 48, 49). Also, factors other than SR proteins have been shown to influence alternative splicing (10, 50). The KH-RNA binding motif containing mammalian KSRP is expressed ubiquitously, although at a higher concentration in neurons, where it enhances c-Src neuron-specific splicing in cell culture (38). Drosophila PSI, also a KH-RNA binding motif-containing protein, is expressed predominantly in the soma, where it represses the splicing of the P element third intron (1). Ectopic expression of PSI in the germ line represses splicing of an intron-containing reporter transgene (1). The role of ELAV in alternative splicing does not preclude it from regulating mRNA function in the cytoplasm, e.g., translatability or stability. Indeed, Drosophila Sex-lethal, an RRM-containing protein with significant homology to ELAV RRMs (7), has been shown to have a role both in splicing (4, 36, 37) and in mRNA translatability (5, 23).One biological role of ELAV is promoting generation of specific alternatively spliced protein isoforms in neurons. ELAV ensures that the correct alternatively spliced protein isoforms of certain genes are generated in neurons. Currently we have identified three target genes, ewg, nrg, and arm. 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 panneurally expressed (6, 20, 31). 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 as it lacks the Wingless interacting domain (40); moreover, it preferentially interacts with DE-cadherins (31). Even with these differences in properties, the current evidence suggest that the u-Arm is sufficient to provide the n-Arm function (31). Perhaps a more detailed phenotypic analysis may reveal a specific role for n-Arm.
ewg, also a vital gene, is broadly transcribed (27), but the protein product, a likely transcriptional regulator, is almost exclusively neural (15). 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 (27). These ELAV-regulated genes provide insight into the regulatory role of ELAV in neurons. Experiments reported here, for the first time, demonstrate that the prevalence of neuron-specific ewg, nrg, and arm transcripts positively correlates with ELAV levels, and these results were achieved through increased use of specific splice sites.| |
ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant NS 37169. Confocal microscopy was made possible by NIH shared instrumentation grant RR 05615. M.S. was supported by a fellowship from the Swiss National Science Foundation.
We thank S. Selleck, L. Manseau, and the Bloomington stock center for fly stocks, J. Loureiro and M. Peifer for antibodies, and L. Torroja for discussions. We also thank K.-M. Sun for constructing elav-ewg and E. Dougherty for imaging assistance.
S.P.K. and M.S. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Biology Department, MS 008, Brandeis University, Waltham, MA 02454. Phone: (718) 736-3175. Fax: (781) 736-3107. E-mail: white{at}binah.cc.brandeis.edu.
Present address: Department of Anatomy and Neurobiology, Washington
University School of Medicine, St. Louis, MO 63110.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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