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The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets ^,

Mathieu Gabut,^1, Jérôme Dejardin,^2, Jamal Tazi,^1* and Johann Soret^1*

CNRS, UMR 5535, Institut de Génétique Moléculaire de Montpellier, Montpellier F-34293, France,¹ CNRS, UPR 1142, Institut de Génétique Humaine, CNRS, Montpellier F-34293, France²

Received 5 October 2006/ Returned for modification 9 November 2006/ Accepted 18 January 2007

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout the growth of a whole organism. Modulating the expression levels of five SR proteins in the developing eye of Drosophila melanogaster revealed that these splicing factors induce various phenotypic alterations in eye organogenesis and also affect viability. Although the SR proteins dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impaired normal axonal projections of photoreceptors and neurogenesis in visual ganglia. Microarray analyses revealed that many transcripts involved in brain organogenesis have altered splicing profiles upon both loss and gain of B52 function. Conversely, a large proportion of transcripts regulated by dASF/SF2 are involved in eye development. These differential and specific effects of SR proteins indicate that they function to confer accuracy to developmental gene expression programs by facilitating the cell lineage decisions that underline the generation of tissue identities.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alternative pre-mRNA splicing represents a powerful means of allowing higher eukaryotes to increase their proteome diversity and to regulate gene expression in a development- and tissue-specific way (31). Among the different factors involved in regulation of alternative splicing, members of the SR family are among the most extensively studied (4). SR proteins are essential splicing factors involved in early steps of spliceosome assembly as well as regulators of alternative splicing. Members of the SR family were also recently shown to participate in mRNA export (20, 26), degradation (27, 53), protein translation (39), and maintenance of genome stability (28).

SR proteins share a very similar domain organization consisting of one or two RNA recognition motifs at the N terminus and a region rich in arginine-serine dipeptides (RS domain) at the C terminus (18). However, they recognize distinct exonic splicing enhancer elements and are not functionally redundant in vitro or in vivo (5, 30). Evidence that SR proteins exert essential roles in vivo was first reported for B52/SRp55 in Drosophila melanogaster. Indeed, B52-null mutants do not develop past the second-instar larval stage even though the splicing of several endogenous transcripts tested was not significantly altered (37). Later, gene targeting of SF2/ASF in the DT40 chicken B-cell line and of SRp20 in mice was reported to cause lethality (21, 50). More recently, conditional deletion of SC35 in the thymus and in the heart was shown to cause a defect in T-cell maturation and to induce dilated cardiomyopathy in mice (13, 49). Interestingly, a similar deletion of SF2/ASF in the heart resulted in a significantly different phenotype (51), demonstrating that these two related splicing factors play fundamentally distinct roles in cardiac tissue.

While members of the SR protein family have been well characterized at the biochemical level, relatively little is known concerning their physiologically relevant target pre-mRNAs. Identifying the repertoire of genes specifically regulated by the different splicing factors is crucial not only to decipher their role in biological pathways but also to understand how they could be involved in human diseases which are increasingly associated with alterations of splicing processes (15).

As a way to address this question, we have taken advantage of Drosophila as a model system to investigate the consequences of tissue-specific expression of different members of the SR family. When expressed at similar elevated levels in the developing eye, SR proteins induce distinct developmental defects. Coimmunoprecipitation experiments performed with extracts from dASF/SF2 or B52 transgenic flies were used to isolate target transcripts, and these were detected by microarrays analyses. Each SR protein was found to interact with distinct sets of targets, including numerous genes whose functions are related to the phenotypes observed. In the case of B52 transgenic flies, which exhibit the more severe phenotype, we show that the high lethality is correlated with developmental defects in the optical lobe and with splicing alterations of B52-interacting mRNAs that are known to be involved in brain development.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fly strains. Flies were raised in standard corn meal yeast extract medium. To generate the transgenic flies, we inserted the green fluorescent protein (GFP) cDNA fused to the cDNA sequences of dASF/SF2, dSC35, d9G8, and B52 into the pUAS-T expression vector. Canton S w¹¹¹⁸ embryos were injected with the pUAS-T:GFP-SR constructs, and homozygous lines containing a unique insertion site were established after several rounds of crossing. The following transgenic lines were used: w¹¹¹⁸; P{UAS-GFP-B52#5} (transgene on chromosome 3), w¹¹¹⁸; P{UAS-GFP-B52#6} (transgene on chromosome 2), w¹¹¹⁸; P{UAS-GFP-dASF#1} (transgene on chromosome 2), w¹¹¹⁸; P{UAS-GFP-dSC35#5} (transgene on chromosome 2), and w¹¹¹⁸; P{UAS-GFP-d9G8#2} (transgene on chromosome 3). As control line, we used the w¹¹¹⁸; P{UAS-GFP.nls}8 line (transposable element on chromosome 3, Bloomington stock no. 4776), expressing the GFP reporter gene fused to a nuclear localization signal (NLS) (kindly provided by F. Maschat). Males from the UAS-GFP-SR transgenic lines were crossed with virgin GMR (Glass Multiple Receptor)-GAL4 females (transgene on chromosome 2), and the resulting heterozygous generations were raised at 25°C. The emergence rate was evaluated over 500 pupae and the sex ratio evaluated for the corresponding progenies. The B52 binding site (BBS) transgenic line (P{w+, UAS-BBS (5.12)}/TM2, Ubx) used to validate the B52 targets has been described previously (42). To validate the in vivo inhibition of B52 activity, these flies were crossed with the transgenic line P{GMR-Gal4.[UAS-B52]}2/CyO, which constitutively expresses B52 under the control of the GMR promoter.

Antibodies and immunofluorescence staining of larval tissues. Eye imaginal discs and brains from third-instar larvae were fixed for 20 min in PBT (1x phosphate-buffered saline, 0.1% Tween 20) with 4% formaldehyde. The tissues were extensively washed in PBT and incubated in PBT-1% bovine serum albumin (BSA) for 1 h at 4°C. Following incubation with the primary antibody diluted in PBT-1% BSA overnight at 4°C, the tissues were washed three times with PBT and incubated with the secondary antibody diluted in PBT-1% BSA for 2 h at 4°C. After three washes in PBT, the tissues were conserved in mounting agent (Vectashield mounting medium H1000; Vector) at 4°C. The polyclonal anti-cleaved-caspase-3 antibodies (no. 9661; Cell Signaling Technology) (kindly provided by P. Lassus), monoclonal anti-Cut 2B10 antibodies, and monoclonal anti-Fas2 1D4 antibodies from the Developmental Studies Hybridoma Bank (provided by F. Girard) were used at a 1/250 dilution. Cy3-conjugated anti-mouse (no. 315-166-047) and anti-rabbit (no. 111-166-047) secondary antibodies were diluted according to the manufacturer's instructions (Jackson Immunoresearch).

Microscopy and images analysis. Eye photographs were obtained using a Nikon Coolpix 990 charge-coupled device camera mounted on an MZFLIII binocular (Leica). All eye photographs represent the average eye pigmentation and phenotypes from each fly population, for both sexes, at 25°C.

Larval eye imaginal disc and brain images were taken with an LSM510 META confocal microscope (Carl Zeiss) using either a 25x plan Neofluar multi-immersion objective (numerical aperture, 0.8) or a 40x apochromat water immersion objective (numerical aperture, 1.2). Samples were sequentially illuminated at 488 and 543 nm, and subsequent GFP and Cy3 fluorescence was detected using 505-530 BP and 560LP filters, respectively.

Larval extract preparation and immunoprecipitation. Anterior quarters of 600 third-instar larvae were dissected in cold 1x phosphate-buffered saline and pooled in 1 ml of cold NETN buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5 mM MgCl2, 0.25% NP-40, 0.5% Triton X-100) with 400 µg tRNA from Escherichia coli (Roche), 20 µl RNasin (Promega), and 100 µl protease inhibitor cocktail (complete, EDTA-free; Roche). Tissue extracts were homogenized with a plastic pestle in a 1.5-ml microcentrifuge tube and sonicated twice for 5 seconds. The homogenates were centrifuged at 3,500 rpm at 4°C for 5 min. Protein G-Sepharose beads (Amersham Biosciences) were presaturated by incubating with NETN buffer with 1% BSA for 1 h at 4°C on a rotator and sequentially washed with coating buffer (50 mM Tris [pH 7.5], 150 mM NaCl). One hundred microliters of protein G-Sepharose beads was then coated with 15 µl of monoclonal anti-GFP antibody (Roche) in 500 µl of coating buffer with 1% BSA for 1 h at room temperature on a rotator and washed successively three times with coating buffer and NETN buffer. Coated beads were incubated for up to 2 h at 4°C with the supernatants supplemented with 5 µl of RNasin, 6 µl of 100 mM dithiothreitol, 18 µl of 0.5 M EDTA, and 60 µl of protease inhibitor cocktail (Roche). Following centrifugation for 5 min at 3,500 rpm, the supernatant was removed. The beads were washed six times in cold NETN buffer and resuspended in 100 µl of RNase-free water. The beads were then treated with proteinase K, and immunoprecipitated RNAs were isolated following phenol-chloroform extraction and precipitation. For Western blot analyses, total extracts from anterior quarters of 50 third-instar larvaes were prepared as described above.

Western blots. Both total extracts and protein samples collected during the immunoprecipitation protocol were run on an 11% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto nitrocellulose by electroblotting for 90 min in 10 mM 3-(cyclohexylamino)propanesulfonic acid (pH 11.0) transfer buffer containing 10% ethanol. Blots were probed with monoclonal antibody 104, polyclonal anti-dASF/SF2 (Eurogentec), or monoclonal anti-GFP antibodies (Roche Diagnostics) and revealed with the LumiLight Western blot substrate (Roche) chemiluminescence kit.

Total RNA isolation, poly(A)⁺ RNA purification, and reverse transcription-PCR (RT-PCR). Anterior quarters of 600 third-instar larvae were homogenized with a plastic pestle in a 1.5-ml microcentrifuge tube, and total RNA extraction was performed with 2 ml of TriReagent (Sigma). First-strand cDNAs were synthesized from 5 µg of total RNA with a first-strand cDNA synthesis kit (Amersham). For PCR analyses, 1/15 of the reaction mixture was amplified with Taq polymerase (Invitrogen), and the cycle number was kept to a minimum to maintain linearity. Primer sequences are available upon request. PCR products were separated on 1.5 to 3% agarose gels containing ethidium bromide and visualized under UV light. Densitometric analyses of the amplified products were performed with the Gnome software.

For transcriptome microarray analyses, poly(A)⁺ RNAs were purified from 600 µg of total RNA, using the Oligotex mRNA midi kit (QIAGEN) according to the manufacturer's instructions.

Labeled cDNA and aRNA synthesis. One-tenth of immunoprecipitated RNA samples was amplified for 8 h, and corresponding Cy3- and Cy5-labeled antisense RNAs (aRNAs) were synthesized using the Amino Allyl Message Amp II aRNA amplification kit (Ambion) according to the manufacturer's instructions.

First-strand cDNA was synthesized with PowerScript reverse transcriptase (Clontech) from 1.5 µg of poly(A)⁺ RNA mixed with 1 µl mRNA spike control and 1.6 µl of anchored oligo(dT)20 (2.5 µg/µl; Invitrogen) in the presence of 40 U of RNase OUT (Invitrogen); 10 mM dithiothreitol; dATP, dCTP, and dGTP (0.5 mM each); 0.2 mM dTTP; and 0.3 mM aminoallyl-dUTP (Ambion) in Powerscript incubation buffer. The mixture was incubated at 42°C for 3 h, and the RNA template was degraded by alkaline hydrolysis (0.32 M NaOH at 65°C for 15 min). After neutralization with HEPES (free acid, 0.67 M), the aminoallyl-modified cDNAs were purified on QIAquick PCR purification columns (QIAGEN) according to the manufacturer's instructions, except that QIAquick wash buffer was replaced with 80% ethanol and cDNA was eluted in water. A second purification was done by ethanol precipitation.

The aminoallyl-modified cDNAs were chemically coupled to Cy3 and Cy5 N-hydroxysuccinimidylesters in 50 mM Na₂CO₃ solution (pH 9.0) for 1 h at room temperature in the dark. The coupling reaction was terminated by the addition of hydroxylamine (1.33 M). The fluorescent cDNAs were subsequently purified from unreacted CyDye using QIAquick PCR purification columns (QIAGEN).

Microarray hybridization. The microarrays designed with the Drosophila Operon 1.1 collection (Operon Biotechnologies), consisting of 14,593 oligonucleotides (70-mer) representing the 13,664 genes and 17,899 transcripts annotated in the Gadfly 3.1 database (Berkeley Drosophila Genome Project [http://www.bdgp.org]), are fully described in the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/projects/geo/) under the reference entry GPL4275.

Prior to hybridization, excess oligonucleotides were removed from the arrays by shaking the arrays twice for 1 min in 0.2% SDS. Arrays were then washed two times in distilled water. Labeled cDNA was added to microarray hybridization buffer version 2 (Amersham Biosciences) with a final concentration of 50% formamide, denatured at 95°C for 3 min, and applied to the microarrays in individual chambers of an automated slide processor (Amersham Biosciences). Hybridization was carried out at 37°C for 16 h. Hybridized slides were washed at 37°C successively with 1x SSC (0.15 M NaCl plus 0.015 M sodium citrate)-0.2% SDS for 10 min, twice with 0.1x SSC-0.2% SDS for 10 min, with 0.1x SSC, for 1 min and with isopropanol before air drying.

Data acquisition. Microarrays were scanned at 10-µm resolution in both Cy3 and Cy5 channels with a GenePix 4200AL scanner with a variable photo multiplier tube voltage to obtain maximal signal intensities with <0.1% probe saturation. ArrayVision software was used for feature extraction. Spots with high local background or contaminating fluorescence were flagged manually. The local background, calculated for each spot as the median of the fluorescence intensities of four squares surrounding the spot, was subtracted from the foreground fluorescence intensity.

Statistical analysis of GO annotations. Gene ontology (GO) statistics were compared using the program GOstat2 (http://gostat.wehi.edu.au/) (3) in the pools of dASF/SF2 and B52 potential target genes and in the list of 13,664 genes present on the Drosophila microarray. Only overrepresented GO annotations with a maximal P value of 0.5 corrected by the method of Benjamini and Hochberg (3) were considered in the output lists, for subsets of GO hierarchies including development, RNA, apoptosis, and proteolysis.

Microarray accession number. Sample data have been deposited in the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/projects/geo/) under reference entry GSE5872.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
GMR-driven expression of distinct SR proteins differentially affects Drosophila eye development and viability. In order to identify potential mRNA targets associated with different SR proteins, we used Drosophila, a model organism particularly suitable to establish molecular links between specific alterations of gene expression and developmental phenotypes. We took advantage of the Gal4 upstream activation sequence (UAS) binary system (8) to construct transgenic flies expressing the SR family members d9G8, dSC35, dASF/SF2, and B52 fused at the N terminus to GFP, which allows accurate quantification of SR protein expression levels, visualization of their expression patterns, and immunoprecipitation of SR protein-associated mRNAs.

To drive expression in differentiating photoreceptor cells (R cells), transgenic flies carrying a single homozygous UAS-GFP-SR element were mated to flies from the GMR-Gal4 line (16). The latter express the yeast transcription factor in the posterior region of the eye imaginal disc under the control of GMR activating sequences derived from the glass gene promoter. As a control, we used transgenic flies carrying a GFP-NLS construct (expressing the GFP fused to an NLS). While no obvious phenotypes were noticed for the homozygous transgenic lines in the absence of Gal4 induction, expression of each of the GFP-SR proteins driven by GMR-Gal4 resulted in progeny flies that displayed defects in eye development of different degrees of severity (Fig. 1A). Mild phenotypes included pigmentation alterations and defects in ommatidia structures (GMR/GFP-d9G8#2 and GMR/GFP-dSC35#5), while stronger defects were characterized by reductions in eye size (GMR/GFP-dASF#1) and significant necrosis (GMR/GFP-dASF#1 and GMR/GFP-B52#5). Variations in the sex ratios and viability of the flies were also observed, again at various degrees, between the different Gal4-expressing progenies. As already reported (23, 24), viability was strongly impaired in B52 transgenic flies (Fig. 1B).

View larger version (46K): [in this window] [in a new window]   FIG. 1. Expression of distinct SR proteins in the Drosophila developing eye induces phenotypes differing in their severity. (A) Stereomicroscopic views of adult compound eyes from representative male and female individuals of the indicated transgenic lines. Very few adult females and no males were obtained when males from the UAS-GFP-B52#6 were mated to virgin GMR-GAL4 females. (B) Emergence rate and sex ratio analyses of the progenies obtained following mating of males from the indicated transgenic lines with virgin GMR-GAL4 females. (C) Western blot analysis of the GFP-SR fusion proteins present in extracts purified from anterior quarters of third-instar transgenic larvae. Equal amounts of total proteins (30 µg per lane) were loaded onto the gel and probed with an anti-GFP monoclonal antibody following transfer onto nitrocellulose. The positions of molecular mass markers are indicated on the right. (D) The same nitrocellulose filter was reprobed with an anti-ß-tubulin antibody to normalize for loading and transfer levels. (E) Western blot analysis with monoclonal antibody 104, revealing both the endogenous and exogenous B52 proteins in the GFP-NLS and GFP-B52 larval extracts (as described for panel C). (F) Western blot analysis with a polyclonal anti-dASF antibody, revealing both the endogenous and exogenous dASF/SF2 proteins in the GFP-NLS and GFP-dASF larval extracts.

To assess whether the severity of the phenotypes depended upon the ratio between the exogenous and corresponding endogenous SR proteins, Western blot analyses were performed with dASF/SF2-specific antibodies and monoclonal antibody 104 on protein extracts obtained from anterior parts of third-instar larvae. As shown in Fig. 1E and F, the ratios of exogenous to endogenous SR protein expression were very similar in the GMR/GFP-dASF#1 and GMR/GFP-B52#6 larvae. Assuming that the GMR-expressing tissues represent up to 10% of the third-instar larva anterior quarters, these experiments indicated that the expression level of the GFP-SR proteins in these tissues is 5 to 10 times higher than that in their endogenous counterparts.

Taken together, these observations demonstrate that, when expressed at similar levels, distinct SR splicing factors induce different developmental defects.

B52, but not dASF/SF2, GMR-driven expression severely impairs brain development in Drosophila. To obtain a more detailed insight into the developmental defects giving rise to the observed phenotypes, direct and immunofluorescence analyses were performed on eye imaginal discs from third-instar larvae of the control GMR/GFP-NLS, GMR/GFP-dASF#1, and GMR/GFP-B52#6 transgenics. In all cases, GFP fluorescence was restricted to the differentiating R cells localized in the posterior part of the eye imaginal disc (Fig. 2) after the passage of the morphogenetic furrow (MF) (16). In GMR/GFP-dASF#1 larvae, the organization of the differentiating R cells was slightly modified compared to that in control GMR/GFP-NLS counterparts (Fig. 2A and B). As shown in Fig. 2C to F, this organization was dramatically altered in GMR/GFP-B52#6 larvae.

View larger version (47K): [in this window] [in a new window]   FIG. 2. GFP-dASF and GFP-B52 induce disorganization of R and cone cells in eye imaginal discs of transgenic third-instar larvae. (A) Direct fluorescence analysis of the GFP-NLS fusion protein expressed in the larval eye reveals the clusters of developing R cells that emerge in a rectangular array. (B and C) The R-cell organization is slightly altered in the GMR/GFP-dASF#1 larval eye (B) and strongly impaired in GMR/GFP-B52#6 transgenics (C). Magnification, x20 or x100 (insets). (D to F) Direct fluorescence analysis and DAPI (4',6'-diamidino-2-phenylindole) staining of the whole developing eye confirm the differential alterations of R-cell organization in GMR/GFP-dASF#1 (E) and GMR/GFP-B52#6 (F) larval eyes compared to that observed in the control (D). The position of the MF is indicated by arrowheads. Magnification, x20 or x100 (insets). (G to I) Anti-Cut immunostaining of cone cells indicates that the number of these differentiated cells is significantly reduced in the developing eyes of GMR/GFP-dASF#1 (H) and GMR/GFP-B52#6 (I) third-instar transgenic larvae compared to that of the control (G). Magnification, x40. (J to L) Anti-caspase 3 immunostaining of the control larval eye (J) reveals apoptotic cells located on the posterior side of the MF (arrowheads). In GMR/GFP-dASF#1 transgenics (K), numerous apoptotic cells are also disseminated throughout the posterior part of the eye imaginal disc, whereas in GMR/GFP-B52#6 larvae, apoptotic cells concentrate at the most posterior part of the eye (L). Magnification, x20.

Since these phenotypes could result from increased apoptosis of the corresponding precursor cells, immunofluorescence staining was performed with an anti-active-caspase-3 antibody, which detects activated Drosophila effector caspases drICE and DCP-1 (52). As expected, apoptotic cells were detected on the posterior side of the MF in the control larvae (Fig. 2J), whereas significant apoptosis was observed throughout the posterior part of the eye imaginal disc in GMR/GFP-dASF#1 larvae (Fig. 2K). In GMR/GFP-B52#6 progeny, a high number of apoptotic cells were concentrated in the more posterior part of the eye disc (Fig. 2L), where R-cell organization and cone cell differentiation were primarily altered.

Taken together, these observations strongly suggest that the GMR-driven dASF/SF2 expression interferes with the differentiation program and/or triggers apoptosis of cone cells, whereas expression of GFP-B52 affects both R and cone cells. While this difference could account for eye phenotypes of distinct severity, it cannot explain the 100% lethality systematically observed at the third-instar larval stage in the GMR/GFP-B52#6 progeny.

Several studies previously revealed that development of the adult insect optic lobe, and more precisely the lamina ganglion, is strictly dependent on innervation from the compound eye (41). To determine whether the alterations of R-cell differentiation observed in GMR/GFP-B52#6 larvae impair the development of the optic lobe, we first analyzed the expression of GFP-NLS and GFP-SR proteins in this tissue. As shown in Fig. 3A and B, GFP-NLS expression was readily observed in axons of the R1 to R6 cells, which terminate their outgrowth in the lamina (32). GFP-NLS was also detected, although at a lower level, in R7 and R8 axons targeted to the distal medulla. These observations indicate that, in spite of its fusion to an NLS domain, the GFP protein is not restricted to the R-cell nucleus. Similar observations were obtained for the GFP-dASF protein (Fig. 3E and F) revealing on the one hand that GFP-SR protein localization is not restricted to the nuclei of R cells, a result that is similar to one recently published (17), and on the other hand that GFP-dASF/SF2 expression does not significantly affect the localization of the R-cell axons in the lamina. In sharp contrast, GFP-B52 induced a strong alteration in the projection of R-cell axons.

View larger version (52K): [in this window] [in a new window]   FIG. 3. R-cell axons project aberrantly in optic lobes from GMR/GFP-B52#6 larvae and impair correct development of visual ganglia. Direct fluorescence analyses and DAPI staining indicate that GFP-SR fusion proteins are expressed in R-cell axons that extend from the optic stalk (os) and project into the lamina (la), and medulla (me) visual ganglia in both the control (A and B) and GMR/GFP-dASF#1 (E and F), but not in GMR/GFP-B52#6 (I and J), transgenic larvae. Direct fluorescence analysis and anti-fasciclin II immunostaining reveals R-cell axons as well as lamina monopolar cells that form arborizations in the distal medulla in both the control (C) and GMR/GFP-dASF#1 (H) transgenic larval optic lobes. In GMR/GFP-B52#6 larvae (K), lamina monopolar cells are not detected. The mushroom bodies also revealed by the anti-fasciclin II antibody are indicated (mb). Direct fluorescence analysis and anti-caspase 3 immunostaining indicate that optic lobes from GMR/GFP-B52#6 larvae contain major apoptotic foci (L) compared to those of control (D) and GMR/GFP-dASF#1 (G) transgenics. Magnifications, x40 (A, B, E, F, I, and J) and x25 (C, D, G, H, K, and L).

dASF/SF2 and B52 are associated with distinct sets of mRNA targets. To identify dASF- and B52-specific mRNA targets accounting for the distinct phenotypes induced in the transgenic flies, we analyzed transcripts coimmunoprecipitated with the GFP-SR proteins, assuming that their specificity is not significantly different from that of their endogenous counterparts. These experiments were performed using a monoclonal anti-GFP antibody in extracts from anterior quarters of third-instar larvae (Fig. 4A), and copurified mRNA species were used as probes to hybridize long oligonucleotide (QIAGEN Operon) microarrays specific for the 13,664 genes of the GadFly 3.2 Drosophila genome database (http://flybase.bio.indiana.edu). In this assay, cRNAs prepared from GFP-NLS-coimmunoprecipitated mRNAs were used as a nonspecific interaction control.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Coimmunoprecipitation experiments linked to microarray analyses identify potential dASF/SF2 and B52 target genes. (A) Western blot analysis of the GFP-SR fusion proteins present in the imput (I), supernatant (S), and pellet (P) fractions of the immunoprecipitation step. Protein extracts purified from anterior quarters of the indicated transgenic third-instar larvae were processed as described in Materials and Methods. Samples of the different fractions were probed with an anti-GFP monoclonal antibody. The three bands systematically observed in the pellet fractions correspond to mouse immunoglobulins revealed by the secondary antibody used for enhanced chemiluminescence detection. The positions of the molecular mass markers are indicated on the right, and the positions of the different GFP-tagged proteins are indicated on the left. (B) Sizes of the sets of dASF/SF2 and B52 candidate target genes belonging to three major functional classes (upper part) and subjected to alternative splicing (lower part). (C) Sizes of the sets of dASF/SF2 and B52 candidate target genes regulated by alternative splicing and belonging to three major functional classes (upper part) and histogram representation of the percentages of dASF/SF2 and B52 alternatively spliced candidate target genes included in these functional classes (lower part). (D) Histogram representation of the percentages of dASF/SF2 and B52 candidate target genes involved in eye and brain development (left) and frequencies of alternatively spliced (AS) target genes within the same subclasses (right). To categorize the functions of identified genes, QIAGEN Operon annotations have been cross-checked with those provided by the Flybase and PubMed databases, including mutant phenotypes, expression patterns, and gene functions.

The results obtained from two independent immunoprecipitation experiments (see Table S2 in the supplemental material) indicate that mRNA species significantly enriched (enrichment factor of >1.5) in coimmunoprecipitates from GMR/GFP-dASF#1 and GMR/GFP-B52#6 larval extracts correspond to an equivalent but small number of genes (76 and 77, respectively). Among them, only 10 were consistently common to both GFP-dASF and GFP-B52 immunoprecipitates, confirming the lack of general redundancy between the dASF/SF2 and B52 activities. The majority of the genes identified by transcripts associated with GFP-dASF and GFP-B52 were found to belong to the same three functional classes (Fig. 4B, upper part) involved in RNA metabolism (dASF, 23.9%; B52, 30.9%), development (dASF, 32.2%; B52, 22.2%), and apoptosis or proteolysis (dASF, 7.8%; B52, 13.6%). Moreover, compared to the 13,664 genes represented in the array, both the dASF and B52 potential target genes revealed a significant enrichment in gene ontology terms associated with developmental processes, and more precisely in neural system development, RNA or mRNA metabolism, splicing, apoptosis, and proteolysis (see Table 3 in the supplemental material).

Around 60% of the genes corresponding to enriched target transcripts were not previously reported to be alternatively spliced (Fig. 4B, lower part), an observation possibly reflecting the involvement of SR proteins in constitutive splicing as well as in different steps of mRNA life such as export, stability, and translation (40). When only alternatively spliced genes were considered, 80 to 90% of them were found to be distributed in the functional classes concerning RNA metabolism, development, and apoptosis (Fig. 4C, upper part). Interestingly, the alternatively spliced genes associated with dASF and B52 are particularly well represented in the "development" class, where they correspond to 70 to 80% of the genes identified in this category (Fig. 4C, lower part). Taken together, these observations suggest that SR proteins may participate in developmental processes mainly through their activity in alternative splicing regulation.

Since the developmental defects induced by GFP-dASF and GFP-B52 in transgenic larvae differ essentially at the level of the optic lobe neurogenesis, we then searched for genes specifically involved in eye and nervous system development among the GFP-dASF and GFP-B52 targets (Table 1). As shown in Fig. 4D, approximately 70% of these genes are related to brain development in GFP-B52 targets, whereas the predominant fraction of these GFP-dASF targets are involved in eye development. Again, 70 to 80% of these targets correspond to alternatively spliced genes, raising the possibility that alterations of their splicing pattern are responsible for the observed phenotypes.

View larger version (29K): [in this window] [in a new window]   FIG. 5. RT-PCR validation of selected targets. (A) Validation of dASF/SF2 and B52 common target genes. (B) Validation of dASF/SF2 target genes. Alterations of splicing events annotated in Flybase (http://flybase.bio.indiana.edu) for the indicated genes were assessed in GMR/GFP-NLS, GMR/GFP-dASF#1, and GMR/GFP-B52#6 transgenic larvae by semiquantitative RT-PCR experiments. The schematic genomic organization of the gene region subjected to alternative splicing is represented on the right, and the different mRNA isoforms are designated according to the Drosophila melanogaster Exon Database (DEDB). Constitutive and alternative exons are represented by white and shaded boxes, respectively. The correspondence between mRNA isoforms and amplified products is shown on the right, and oligonucleotide pairs used to amplify the different isoforms are indicated by arrows. Quantitative ratios of the different mRNA isoforms were determined by densitometric analyses.

Similarly, the splicing profile of the potential B52 target Pyd (Polychaetoid), which is involved in eye development, as well as those of Nup154, Tm1 (Tropomyosin 1), and mnb (minibrain), which play a role in nervous system development, was essentially affected in GMR/GFP-B52#6 larvae (Fig. 6). To confirm that these genes indeed correspond to genuine B52 targets, their splicing patterns were also analyzed in transgenic larvae expressing an RNA aptamer which corresponds to tandemly repeated high-affinity BBS, which was previously reported to efficiently titrate this SR protein and inhibit B52-dependent splicing in vitro (42). The expression of this specific B52 inhibitor, under the control of the GMR driver, also induces eye developmental defects in transgenic flies (see Fig. S1A in the supplemental material) and is able to rescue the eye phenotype induced by B52 overexpression (see Fig. S1B and C in the supplemental material), thereby confirming its efficacy to counteract the activity of this SR protein in vivo. As shown in Fig. 6, the splicing profiles of the four genes were significantly altered in UAS-BBS transgenics, in a manner either similar to that observed in GMR/GFP-B52#6 larvae (Nup 154 and TM1) or clearly different (Pyd and mnb). Thus, both increased B52 expression and its in vivo depletion, targeted in the same tissue, resulted in modifications of alternative splicing patterns for multiple genes during Drosophila visual system development.

View larger version (30K): [in this window] [in a new window]   FIG. 6. RT-PCR validation of selected B52 targets. Alterations of splicing events annotated for the indicated genes were assessed in GMR/GFP-NLS, GMR/GFP-dASF#1, GMR/GFP-B52#6, and UAS-BBS transgenic larvae as described for Fig. 5. The schematic genomic organization of the gene region subjected to alternative splicing is represented on the right. The correspondence between mRNA isoforms and amplified products is shown on the right, and oligonucleotide pairs used to amplify the different isoforms are indicated by arrows. Quantitative ratios of the different mRNA isoforms were determined by densitometric analyses.

Taken together, these observations indicate that genes identified by coimmunoprecipitation experiments coupled to microarray analyses likely correspond to genuine dASF and B52 targets and confirm that these SR proteins regulate alternative splicing of specific subsets of pre-mRNAs that can be linked to altered phenotypes.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Beside previous studies reporting that changes in the expression levels of SR splicing factors can affect the development of transgenic flies (1, 23, 24, 34, 37), this work constitutes the first comparative analysis of the developmental defects induced by increased expression of different SR proteins. Moreover, this in-depth characterization of dASF/SF2- and B52-induced phenotypes combined with the systematic identification of the potential targets of these splicing factors in the affected tissues provides important clues to decipher their role in biological pathways.

Our immunofluorescence experiments strongly suggest that the GMR-driven dASF/SF2 expression interferes with the differentiation program and/or triggers apoptosis of cone cells, whereas expression of GFP-B52 affects both R and cone cells. Interestingly, B52 overexpression in SL2 cells was recently shown to strongly repress the expression of dE2F1, an activator of cell proliferation, while the loss of expression of this SR protein is associated with splicing default of the dE2F2 gene, an antagonist of dE2F1 function (34). Consistently, the eye phenotypes of dE2F1 mutants resulting from cell cycle defects are suppressed by B52 mutations that probably affect the expression of dE2F2 mRNA. These results are in good agreement with our observation that the dE2F2 mRNA level is clearly increased in GMR/GFP-B52#6 larvae and with immunofluorescence experiments performed with antibodies raised against phosphorylated histone H3, which indicated that the number of dividing cells in the second mitotic wave prior to MF passage is significantly reduced in GMR/GFP-B52#6 eye imaginal disc compared to the corresponding control tissue (data not shown).

Besides the disorganization of ommatidia in the developing eye, a striking difference between the phenotypes induced by dASF/SF2 and B52 was the very high lethality provoked by the latter SR protein in third-instar larvae. Direct fluorescence analyses as well as immunostaining experiments have revealed that in B52 transgenic larvae, the R-cell axons project aberrantly within the optic lobe, an alteration likely responsible for the absence of normal lamina neurogenesis. Indeed, numerous studies have reported that retinal axon projections induce the organization of laminar and medullar neurons (41) and coordinate the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (12). Strikingly, visual system mutants showing a reduced or absent retina also have a completely missing lamina and a greatly reduced medulla, indicating that retinal innervation is required for further development of these ganglia (33). Interestingly, the phenotype induced by B52 is very similar to that of eyeless mutants, which lack a lamina and display a significant size reduction of the other visual ganglia (10). In some mutants, eye and optic lobe defects are associated with an important lethality, suggesting that abnormal neurogenesis in the optic lobe impairs vital functions.

In the past few years, global assays aimed at identifying cellular RNA targets of splicing regulators have involved coimmunoprecipitation strategies followed by microarray analysis (25, 47), genomic systematic evolution of ligands by exponential enrichment (SELEX) (22), and, more recently, development of alternative splicing microarrays (5, 36, 48). In the present study, immunoprecipitation of RNA species bound to GFP-dASF and GFP-B52 proteins expressed in the Drosophila eye identified an equivalent number of candidate target genes (76 and 77, respectively). Among them, only 40% were already known to be alternatively spliced. Since many genes subjected to alternative splicing are not yet annotated (11, 43), a higher proportion of the candidate targets could actually correspond to alternatively spliced genes, as reflected by our RT-PCR experiments which revealed two neurotactin mRNA isoforms that had not been previously described (see Fig. S3 in the supplemental material). Moreover, 15 to 20% of the dASF/SF2 and B52 potential targets correspond to still-uncharacterized genes whose splicing status remains to be established.

Identification of constitutively spliced genes as candidate targets can also reflect the involvement of SR proteins in constitutive splicing. Indeed, it was recently reported that dASF/SF2 and B52 control 105 and 22 constitutive splicing events, respectively, in SL2 cells (5). On the other hand, such candidates could be subjected to other activities of SR proteins, such as regulation of mRNA stability and translation (27, 39). It is noteworthy that one of the B52 targets found in our screen (CG9080) has already been identified by genomic SELEX and shown to contain a BBS in the 3' untranslated region of the corresponding mRNA (22).

We also observed that only 6% of the identified genes correspond to targets of both dASF/SF2 and B52. Since splicing microarray analyses revealed that 5% of the splicing events are similarly affected by knockdown of either SR protein (5), these observations confirm the unique character of each SR protein and suggest that the functional overlap between these splicing factors is similar in the different tissues of a whole organism.

Results reported in this study indicate that our strategy efficiently identified specific target genes whose expression is modulated by dASF/SF2 and B52. To date, two different approaches have been used to identify B52 targets or alternative splicing events controlled by dASF/SF2 and B52. Genomic SELEX has identified 15 B52 candidate target genes, among which four exhibit splicing defects in a B52-null mutant (22). Splicing microarrays have revealed that dASF/SF2 and B52 control 319 and 107 splicing events, respectively, in SL2 cells (5), with 6 out of 6 tested being validated by RT-PCR experiments. In the present study, 36 dASF/SF2 and/or B52 potential target genes were analyzed by the same approach (Fig. 5 and 6; see Fig. S3 and S4 in the supplemental material), and 18 (50%) are significantly affected at the level of their splicing pattern in the corresponding transgenic larvae. Moreover, by using transgenic flies expressing an RNA aptamer capable of titrating out the endogenous B52 protein (42; this study), we have confirmed that B52 candidates validated by RT-PCR experiments (Fig. 6; see Fig. S4 in the supplemental material) represent genuine B52 targets. Of note, our RT-PCR experiments were designed to analyze sequence-defined splicing events reported in databases, and it is possible that candidates considered negative are nevertheless affected in other regions or are regulated by SR proteins at steps different from alternative splicing.

Comparison of the dASF/SF2 and B52 potential targets identified by the different approaches used so far indicates that three alternative splicing events regulated by dASF/SF2 in SL2 cells (5) affect the genes lola, flo, and garz, which we also identified as targets for this SR protein. On the other hand, three out of the four B52 targets (Rx, Mio, and RhoGap16F) validated by Kim et al. (22) were not identified in our screen. This is not surprising, since genomic SELEX identified all genomic sequences containing a high-affinity BBS, whereas our coimmunoprecipitation strategy was aimed at characterizing mRNAs bound by B52 and expressed in a particular cell type or tissue. Indeed, Rx is significantly expressed in the Drosophila larval brain, but not in R cells (14), and to our knowledge, expression of Mio and RhoGap16F has not been reported to occur in R cells. Among the four B52 targets found by both the genomic SELEX and splicing microarray analyses (fur1, lola, RhoGAp16F, and Syndecan), only lola (longitudinals lacking) has been identified in the present study. While these observations possibly reflect, as observed for fur2 (38), the lack of expression of fur1 in R cells, the situation is more surprising in the case of Syndecan, since expression of this gene has been detected in R-cell axons (35). This target, however, was not validated in B52 mutant larvae (22), suggesting that the event detected on splicing microarrays is restricted to specific tissues. On the other hand, such observations were not unexpected in the light of the many fundamental differences between the RNAi approach examining splicing changes upon protein depletion and our coimmunoprecipitation strategy aimed at identifying mRNAs bound to overexpressed proteins. Along this line, one can speculate that RNAi might primarily affect mRNAs with low-affinity sites since protein reduction is not complete, while overexpression might push occupancy of lower-affinity sites on a different set of mRNAs. In agreement with this hypothesis, a search for high-affinity binding sequences specifically recognized by the human ASF/SF2 homolog (44) and the B52/SRp55 protein (see reference 42 and references therein) did not reveal a specific enrichment of these motifs around the splice sites of the regulated exons in the validated dASF/SF2 and B52 targets (data not shown). However, the restricted number of splicing events considered might explain the lack of statistical significance and the only marginal enrichment of highly scoring putative binding sites for both SR proteins that we observed in our computational analysis.

Our analyses indicate that genes such as neurotactin, chromosome bows, and longitudinals lacking that are involved in axon guidance and neurogenesis constitute potential dASF/SF2 targets. Splicing alterations of the corresponding pre-mRNAs, however, do not appear to affect the correct projection of R-cell axons in the transgenic flies. On the other hand, one can envisage that splicing alterations of lola, which also corresponds to a B52 candidate target, are much less deleterious in dASF/SF2 than in B52 transgenics.

Another interesting B52 target involved in neurogenesis and whose alternative splicing is clearly altered in GMR/GFP-B52 transgenic larvae is minibrain (mnb). This gene encodes a serine/threonine protein kinase which appears to play an essential role during postembryonic neurogenesis (45). Mutations in the mnb gene induce an abnormal spacing of neuroblasts in the larval brain due to a reduced production of neuronal progeny. The human homolog (MNBH/DYRK1A) of the mnb gene is overexpressed in brains from fetuses with Down's syndrome (19), and the phenotypes of DYRK1A transgenic and knockout mice support the idea that DYRK1A contributes to some of the neuropathological traits described in Down's syndrome (2).

Although further experiments will be needed to precisely identify the genes whose splicing alterations account for the specific phenotypes induced by dASF/SF2 and B52, the present study indicates that the coimmunoprecipitation approach linked to microarray analyses represents a straightforward strategy to discover transcripts and associated pathways regulated by individual SR proteins and to understand how they participate in normal and pathological developmental processes.

	ACKNOWLEDGMENTS

 
We are indebted to the personnel of the Montpellier Rio Imaging (MRI) facility for excellent technical assistance in direct and confocal microscopy experiments. Thanks are also due to D. Severac and the personnel of the Montpellier transcriptome platform for help in microarray analyses, to J. Lis for providing us with the BBS transgenic line, to S. Sakr for skillful assistance in RT-PCR analyses, and to Y. Barash for motif predictions and statistical analyses. We are grateful to G. Cavalli for helpful discussions and to B. Blencowe for critical reading of the manuscript.

M. Gabut is a recipient of a fellowship from the Association pour la Recherche contre le Cancer (ARC). This work was supported by grants from the Agence Nationale de la Recherche (ANR-05-BLAN-0261-01) and European Alternative Splicing Network of Excellence (EURASNET, FP6 Life Sciences, Genomics and Biotechnology for Health).

	FOOTNOTES

 
* Corresponding author. Mailing address: CNRS, UMR 5535, Institut de Génétique Moléculaire de Montpellier, Montpellier F-34293, France. Phone: 33 (0) 4 67 61 36 85. Fax: 33 (0) 4 67 04 02 31. E-mail for Johann Soret: johann.soret{at}igmm.cnrs.fr. E-mail for Jamal Tazi: jamal.tazi{at}igmm.cnrs.fr

Published ahead of print on 5 February 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada M5S 3E1.

Present address: Harvard Medical School, Department of Genetics, and Massachusetts General Hospital, Department of Molecular Biology, Boston, MA 02114.

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