elav

Promoter Structure

A 333 proximal promoter region has been identified that confers the neural specificity of elav expression (Yao, 1994). There is no TATA element, and the sequence is not GC rich. The ELAV promoter includes 241 base pairs of the first exon. Deletion of the region within exon 1 results in a precipitous drop in expression (Yao, 1994).

The Snail family member Worniu is continuously required in neuroblasts to prevent Elav-induced premature differentiation

Snail family transcription factors are best known for regulating epithelial-mesenchymal transition (EMT). The Drosophila Snail family member Worniu is specifically transcribed in neural progenitors (neuroblasts) throughout their lifespan, and worniu mutants show defects in neuroblast delamination (a form of EMT). However, the role of Worniu in neuroblasts beyond their formation is unknown. RNA-seq was performed on worniu mutant larval neuroblasts, and reduced cell-cycle transcripts and increased neural differentiation transcripts were observed. Consistent with these genomic data, worniu mutant neuroblasts showed a striking delay in prophase/metaphase transition by live imaging and increased levels of the conserved neuronal differentiation splicing factor Elav. Reducing Elav levels significantly suppressed the worniu mutant phenotype. It is concluded that Worniu is continuously required in neuroblasts to maintain self-renewal by promoting cell-cycle progression and inhibiting premature differentiation (Lai, 2012).

Stem cells must remain proliferative without becoming tumorigenic, and must remain competent to differentiate without actually differentiating. How stem cells maintain stemness - cell survival, cell-cycle progression, and the capacity to differentiate - is a widely relevant question with clinical significance. Drosophila neural progenitors (neuroblasts) have become a good model system to study how neural stem cells self-renew and maintain stem cell identity. Larval neuroblasts undergo repeated rounds of asymmetric cell division, each time generating a smaller differentiating daughter cell and a larger self-renewing neuroblast. During neuroblast division, many proteins are asymmetrically partitioned into the neuroblast or daughter cell, where they often contribute to neuroblast self-renewal or daughter cell differentiation, but much less is known about the transcriptional program that maintains neuroblast self-renewal (Lai, 2012).

Worniu (Wor) is a zinc finger transcription factor in the 'Slug/ Snail' family, and is transcribed in neuroblasts from the time of their birth. Over 50 Snail family members have been characterized in metazoans; they can directly bind DNA, RNA, or protein and regulate a wide range of cellular functions. Snail family members are best known for inducing epithelial-mesenchymal transition (EMT) during mesoderm development and neural-crest cell formation. In Drosophila, four Snail family genes are known: wor, escargot, snail, and scratch. The genes wor, escargot, and snail are expressed in neuroectoderm during embryogenesis to trigger EMT in neuroepithelial cells and transform them into newly-delaminated neuroblast. Wor, Escargot, and Snail also act redundantly to promote expression of the apical polarity gene inscuteable (insc) and the cell-cycle regulator string in newly formed embryonic neuroblasts (Lai, 2012 and references therein).

The only Snail family member known to be expressed continuously in neuroblasts is Wor, but its function beyond neuroblast formation has not been investigated. This study shows that Wor maintains neuroblast self-renewal via dual functions: it promotes cell-cycle progression (specifically the prophase-to-metaphase transition) and it inhibits premature differentiation (by suppressing Elav protein levels). These functions occur in neuroblasts well after their formation, highlighting the potential role of Snail family members in stem cell self-renewal (Lai, 2012).

To analyze the wor mutant phenotype, a deficiency was used that removes wor and several flanking genes, Df(2L)Exel8034, and a specific mutation within the wor gene, wor¹. wor¹ was found to have two missense mutations, one of which alters the amino acid Pro443 to Ser in the conserved zinc finger domain and probably changes the conformation for DNA/RNA/protein binding. Because wor¹/wor¹ had a slightly weaker phenotype compared to wor¹/Df(2L)Exel8034 due to lesser amount of Wor protein in the latter genotype, it is concluded that wor¹ is a strong hypomorph. wor¹/Df(2L)Exel8034 was used for all experiments described in this study (called 'wor mutants') (Lai, 2012).

Wor protein is nuclear and is predicted to be a transcription factor, so the transcriptional profile of wild-type (WT) and wor mutant neuroblasts was compared to identify biological processes that were regulated by Wor. The TU- (thiouracil-) tagging method was used to identify mRNAs that are actively transcribed in WT or wor mutant neuroblasts. TU-tagging is a spatial/ temporal intersectional method to purify nascent RNA from designated tissues during a specific developmental stage. Uracil phosphoribosyltransferase (UPRT) was expressed in larval neuroblasts using wor-gal4, which produced a high level of UPRT in WT and wor mutant larval neuroblasts with some persistence into their newborn progeny. Early third instar larvae were fed 4TU for 5 hr beginning at 72 hr after larval hatching (ALH) and then thio-labeled RNA was purified and RNA-sequencing was performed; a custom computational pipeline was designed to analyze the results. Two replicates were performed from wor mutants and two from WT. An average of 5.49 million reads from WT and 5.35 million reads from wor mutants was mapped. A comparison of the averaged WT versus averaged wor data showed that wor mutants had 13.8% of genes upregulated at least 2-fold and 9.1% of genes downregulated at least 2-fold (Lai, 2012).

Genes upregulated in wor mutants were enriched for gene ontology (GO) terms linked to neuronal differentiation such as G protein coupled receptor signaling, sensory perception, serotonin receptor signaling, and synaptic transmission. In addition, a group of 'neuronal differentiation genes' was recently defined in a transcriptomic analysis of larval brains enriched for neuroblasts or neurons. 253 of the
1,100 'neuronal differentiation' genes were differentially regulated in wor mutants (>2-fold or <2-fold), with a strong bias toward being upregulated. GO analysis of the upregulated genes shows significant overrepresentation of the terms signaling, synaptic transmission, synapse organization, and neuropeptide signaling pathway categories. It is concluded that wor mutant neuroblasts aberrantly upregulate neuronal differentiation genes (Lai, 2012).

The downregulated genes were likewise analyzed. It was asked whether previously defined 'neuroblast genes' or 'cell cycle genes' are downregulated in wor mutant neuroblasts - the converse of the observed upregulation of neuronal differentiation genes. It was found that 104 of the
970 'neuroblast' genes from Carney (2012) were differentially expressed in wor mutants >2-fold or <2-fold, with a strong bias toward being downregulated. The downregulated genes had a highly significant over-representation of the GO terms cell cycle, microtubule cytoskeleton organization, cytokinesis, cell division, and chromosome segregation. Similarly, a downregulation of Drosophila genes annotated as 'cell cycle' was found: of the 586 cell cycle annotated genes (GO:0007049), 67 were differentially regulated in wor mutants versus WT, and most (74.6%) were downregulated. It is concluded that wor mutant neuroblasts fail to properly express 'neuroblast' genes including those regulating the cell cycle (Lai, 2012).

Based on transcriptomic analysis, it was predicted that wor mutant neuroblasts would show defects in neuroblast attributes (cell-cycle progression, cell polarity, and survival) and precocious neural differentiation. All of these phenotypes could lead to the smaller brain size and reduced neuroblast numbers observed in wor mutants. To determine whether wor mutant neuroblasts have a normal cell cycle, EdU incorporation was performed and the number of EdU+ neuroblasts was counted immediately after the pulse. In this and subsequent experiments, larval neuroblasts were identified as large (>8 mm) Dpn+ cells within the central brain; optic lobe neuroblasts were not characterized. Most WT neuroblasts were EdU+, consistent with their reported cell cycle time of
2 hr. In contrast, very few wor mutant neuroblasts were EdU+, indicating a cell-cycle delay between G2-M-G1. To determine if the wor mutants were delayed in mitosis, the mitotic index of WT and wor mutant brains was measured by staining for the M-phase marker phosphohistone H3 (PH3). By late third instar (96-120 hr ALH) there was a striking increase in the PH3+ neuroblasts in wor mutant compared to WT. It is concluded that third instar wor mutant neuroblasts have a delay in completing mitosis (Lai, 2012).

To determine more precisely the nature of the M-phase delay in wor mutants, live imaging of neuroblast mitosis was performed within the intact brain. Third instar larval neuroblasts expressing both His2A:RFP to monitor chromosomes and Zeus:GFP to image spindle microtubules were imaged. Wild-type neuroblasts showed the expected mitosis length of
20 min. In contrast, wor mutant neuroblasts showed a dramatically extended prophase and/or prometaphase. Failure in centrosomal separation and bent mitotic spindles were observed. In two cases neuroblasts were observed that 'escaped' prophase arrest, and these had a relatively normal length of anaphase. It is concluded that wor mutant neuroblasts show an arrest or delay in the prophase/metaphase transition, a stage of the cell cycle where microtubules are dramatically reorganized (see Discussion) (Lai, 2012).

Cell-cycle delays have been observed in neuroblasts lacking aPKC or Dap160 apical cortical polarity proteins, and wor-escargot-snail triple null mutants lack apical localization of Insc in embryonic neuroblasts. Apical and basal polarity proteins were stained, and a failure of all proteins to be properly localized during prophase was observed; yet localization was normal by metaphase, most likely by a microtubule-dependent mechanism. It is concluded that Wor is required to establish neuroblast polarity at prophase (Lai, 2012).

wor mutants have fewer neuroblasts compared to the WT brains, which could be caused by neuroblast apoptosis or differentiation. To determine if this reduction was due to neuroblast apoptosis, a genetic sensor for caspase-mediated apoptosis was used, in which caspase activity induces nuclear localization of GFP by cleaving a membrane tether. wor mutant second instar brains were found to have multiple large GFP+ cells at the location of central brain neuroblasts, indicating an elevated level of caspase-mediated cell death. A more general cell death marker, TUNEL staining was used, and the Dpn antibody was used to unambiguously identify neuroblasts. No TUNEL+ Dpn+ neuroblasts were found in the WT brains; in contrast TUNEL+ Dpn+ neuroblasts were observed in wor mutants. RNAi depletion of the Dronc caspase gave a significant but partial rescue of the neuroblast numbers (wor¹/Deficiency; wor-gal4 UAS-dronc RNAi); partial rescue is probably because wor-gal4 is only expressed in a subset of neuroblasts or because of incomplete knockdown by RNAi. It is concluded that the loss of neuroblasts seen in wor mutants is largely due to apoptosis.

Based on the transcriptomic analysis, it was predicted that wor mutant neuroblasts would show precocious neural differentiation. To determine if wor mutant neuroblasts initiate premature differentiation, well-characterized evolutionarily conserved neural differentiation marker Embryonic lethal abnormal visual system (Elav; Hu family in mammals) was stained; the Elav protein is normally only detected in mature postmitotic neurons where it promotes neuron-specific alternate splicing. Wild-type larval neuroblasts transcribe elav but have low or no Elav protein, whereas many wor mutant neuroblasts showed detectable Elav protein. It is concluded that wor mutant neuroblasts have an abnormally high level of the Elav neuronal differentiation marker, consistent with premature differentiation (Lai, 2012).

Elav is a RNA-binding protein known to promote neuronal-specific splicing of at least three direct target genes: neuroglian (nrg), erect wing (ewg), and armadillo (arm). RNA-seq reads spanning the junctions of alternatively-spliced exons of all three genes were counted, and it was found that the neural-specific, Elav-dependent splice isoforms for all three transcripts were increased in wor mutants compared to WT. Thus, the increased level of Elav in wor mutant neuroblasts appears sufficient to bias splicing toward the neuronal-specific isoforms for all three of its known target genes (Lai, 2012).

To determine the effect of increased Elav levels on the self-renewal of wor mutant neuroblasts, tests were performed to see whether wor mutant phenotypes could be rescued by reducing Elav levels. wor-gal4 was used to drive to drive UAS-elav-RNAi in larval neuroblasts, and a complete rescue of the wor mutant cell cycle phenotype and a substantial rescue of the wor mutant cell polarity phenotype were observed. Thus, the increased level of Elav protein in wor mutant neuroblasts results in most of the cell cycle and cell polarity defects. Reducing Elav levels was not able to restore normal neuroblast numbers, suggesting that it is an Elav-independent pathway. To provide an independent test for the role of Elav in neuroblast cell cycle and cell polarity, Elav levels were increased in otherwise Wt neuroblasts, and cell cycle and cell polarity phenotypes similar to wor mutants were observed, without altering neuroblast number. It is concluded that Wor keeps Elav protein levels low in neuroblasts, which is necessary for establishing neuroblast cell polarity and cell-cycle progression - both key stem cell features (Lai, 2012).

Having established that Wor is necessary to maintain neuroblast properties (proliferation, polarity, survival), it was of interest to see if ectopic Wor was sufficient to induce neuroblast attributes in GMCs or prevent neuronal differentiation. prospero gal4 was used to overexpress Wor in larval neuroblasts and their progeny (abbreviated as Wor^OXN hereafter). Unexpectedly, the Wor^OXN larval brains were smaller than WT brains, their larval neuroblasts were smaller in diameter, and the neuroblasts exhibited a severe cell cycle delay. No change was observed in the number of Dpn+ central brain neuroblasts. To determine the cause of the Wor^OXN phenotype, tests were performed for ectopic Prospero (Pros) protein in neuroblasts, because Pros is known to inhibit cell-cycle progression in larval neuroblasts (Lai, 2012).

Whereas both WT neuroblasts and wor mutant neuroblasts lack nuclear Pros, Wor^OXN neuroblasts had clearly detectable nuclear Pros. Furthermore, when Pros levels were reduced in Wor^OXN larvae (Wor^OXN; pros¹⁷/+) partial but significant rescue of the cell cycle and cell size phenotypes was found, and a slight increase in neuroblast numbers. This latter result suggests that Wor overexpression has the ability to transform GMCs/neurons into neuroblasts, but that this is usually masked by Pros-mediated cell-cycle arrest. It is concluded that overexpression of Wor does not lead to a transformation of GMC/neurons into neuroblasts, and that WT neuroblasts must precisely regulate Wor levels; too little Wor leads to Elav-induced premature differentiation, whereas too much Wor leads to Pros-induced cell-cycle arrest (Lai, 2012).

Because wor mRNA and protein are specifically detected in neuroblasts, not in neurons or glia, the brain phenotypes described in this study are most likely to be due to cell autonomous function of Wor within neuroblasts. This study has shown that Wor prevents premature differentiation of neuroblasts, a conclusion based in part on the upregulation of neuronal differentiation transcripts in wor mutant neuroblast lineages. The observed increase in neuronal differentiation transcripts is likely to be an underestimation, because wor mutant neuroblast lineages have three times fewer UPRT+ neurons than WT neuroblast lineages (due to the neuroblast cell-cycle delay in wor mutant neuroblasts). The reduced number of neurons in the wor mutant clones makes it all the more striking that neuronal differentiation transcripts were found to be upregulated in wor mutant neuroblast lineages (Lai, 2012).

A second reason it is concluded that Wor prevents premature differentiation of neuroblasts is the finding that wor mutants have increased levels of the differentiation marker Elav within neuroblasts. How does Wor normally keep Elav protein levels low in neuroblasts? Wor may repress elav at the transcriptional or post-transcriptional levels. Although no change was seen in elav transcript abundance between WT and wor mutant neuroblast lineages by RNA-seq, wor mutants have three times fewer UPRT+ neurons than wor mutants (Lai, 2012).

The extra neurons in WT should result in more elav transcripts; the fact that equal levels were seen suggests that wor mutant neuroblasts may have increased levels of elav transcription. On the other hand, Wor may repress Elav at a posttranscriptional level. Wild-type embryonic and larval neuroblasts transcribe the elav gene but little of the mRNA is translated; it is likely that elav is also posttranscriptionally regulated in larval neuroblasts, and this step could be subject to direct or indirect regulation by Wor. Thus, Wor may regulate elav at the transcriptional and/or posttranscriptional level to keep Elav protein low in neuroblasts (Lai, 2012).

How does Elav promote premature differentiation of neuroblasts? Elav may act by inducing neuronal-specific splicing of its direct targets neuroglian, erect wing, and armadillo (which was observed to be upregulated in wor mutants), or additional targets that have yet to be identified. In addition, other RNA splicing factors, many of which are up- or downregulated at least 2-fold in wor mutants, may coregulate Elav targets and/or splicing of additional pre-mRNAs. Genomic analysis of alternative splicing junction usage in wor mutants showed a profound change of global splicing events: 15.0% of all potentially alternatively-spliced exons (14,476 junctions from 3,430 genes) showed >2-fold change in wor mutants compared to WT. Because the function of different splice isoforms are so poorly understood, it can only be speculated that some or all of the upregulated splice isoforms promote neural differentiation and inhibit cell cycle in wor mutants. Neuronal differentiation seen in wor mutant neuroblasts is not complete, because wor mutant neuroblasts maintain expression of neuroblast markers such as Dpn, Ase, and Miranda. Thus, wor mutant neuroblasts have a mixed fate, in which both neuroblast and neuronal genes are expressed (Lai, 2012).

Wor is required to promote cell polarity at prophase. The defect in apical protein localization seen in wor mutants is similar to that seen in the absence of an external polarizing cue in embryonic neuroblasts, or in sgt1 mutant larval neuroblasts. It is also coincident with the prophase cell-cycle delay observed by live imaging, but the relationship between loss of polarity proteins and prophase delay is unknown. Wor is also required to prevent neuroblast apoptosis. In mammals, Snail family members are known to protect cells from apoptosis triggered by loss of survival signals. It remains unknown whether Wor acts in a similar manner; all that can be said is that Wor acts via an Elav-independent pathway to maintain neuroblast survival (Lai, 2012).

Wor is required for cell-cycle progression from prophase to metaphase. It is interesting that loss of wor causes cell-cycle delays at the precise time when the microtubule cytoskeleton is dramatically reorganized into a bipolar spindle. In addition, the RNA-seq data shows that wor mutants are depleted for 'microtubule cytoskeleton organization' annotated transcripts. Mammalian Snail family proteins confer migratory properties to epithelial cells during EMT or metastasis, which also involves a dramatic reorganization of the cytoskeleton. Thus, Wor may have a conserved function in regulating the microtubule cytoskeleton. Because reducing Elav levels can rescue cell-cycle progression, Wor appears to regulate the microtubule cytoskeleton indirectly, via keeping Elav protein levels low. High levels of Elav in neuroblasts may induce microtubule organization characteristic of mature neurons, such as using a single centrosome to nucleate unidirectional microtubule outgrowth into the axon. Thus, neuroblasts with high levels of Elav may be unable to efficiently duplicate their centrosomes or form a bipolar mitotic spindle, leading to the observed prophase arrest phenotype (Lai, 2012).

Targets of Activity

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins might mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponds with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV is expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, the generation of the neural isoform of Neuroglian is observed. It is concluded that Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing. Although performed in vivo, these experiments do not exclude an indirect effect of ELAV, as for example, on the stability of a neuroglian-specific splicing factor (Koushika, 1996).

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements (AREs) in their 3' untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes, which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame (ORF); (2) with a constitutively spliced intron inserted into the ORF, and (3) with the intron nASI whose splicing is regulated in neurons by ELAV, inserted into the ORF. The two sets differ from one another only in their 3'UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. The results show that: (1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3'UTR, but not of transgenes with Act5C 3'UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm, ELAV is localized to both the cytoplasm and the nucleus (Toba, 2002).

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways

Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. This study shows that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach it was demonstrated that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis it was demonstrated that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth. These results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network (Haussmann, 2008).

Several pathways have been identified that stimulate synaptic growth at NMJs of Drosophila larvae (Wnt/Wingless, TGF-β/BMP and jun kinase). Overexpression of AP-1 and mutants in regulatory genes involved in Wnt/Wingless and TGF-β/BMP pathways (spinster, highwire, shaggy and the proteasome) can increase bouton numbers, suggesting that synaptic growth is regulated through the balance of stimulatory and restrictive signals. This study has identified such a restrictive role for the transcription factor EWG and, through the analysis of EWG-regulated genes, for the N pathway in the regulation of synaptic growth. Using genetic mosaics, it was further demonstrated that EWG's role in synaptic growth regulation is cell-autonomous, suggesting that the transcriptional regulator EWG mediates this restrictive effect through the alteration of transcription pre-synaptically (Haussmann, 2008).

Analysis of genes differentially expressed in ewg^l1mutants revealed a rather unexpected set of genes involved in synaptic growth regulation, besides an expected number of metabolic genes due to homology of EWG to human NRF-1. Most genes that could account for the phenotype of ewg^l1mutants, and that are thus expressed in the nervous system, are involved in transcriptional and post-transcriptional regulation of gene expression. Although changes of transcript levels in ewg^l1mutants were mostly moderate, their significance was validated through mRNA profiling with rescued ewg^l1mutants under the same conditions of RNA preparation and microarray hybridization. In addition, differences in gene expression in ewg^l1mutants were validated using quantitative RT-PCR and biochemical assays with regard to predicted changes in glycogen levels based on differential regulation of genes involved in gluconeogenesis. Furthermore, genetic interaction experiments in double mutants with increased bouton numbers support that these co-regulated genes are functionally connected in regulating synaptic growth (Haussmann, 2008).

The group of neuronal genes among those differentially regulated in ewg^l1mutants that have been demonstrated to have roles in synaptic growth or could account for it, is remarkably small. In particular, from the large number of cell adhesion molecules and cytoskeletal proteins present in the Drosophila genome only a handful is differentially regulated. Similar results have also been obtained in response to JNK and AP-1 signaling. These results are in contrast to changes in gene expression induced by acute or chronically enhanced neuronal activity in Drosophila seizure mutants, which also result in synaptic overgrowth. Here, the vast majority of differentially regulated genes are for cell adhesion molecules and cytoskeletal proteins or their regulators, and genes involved in synaptic transmission and neuronal excitability; transcriptional or post-transcriptional regulators comprise only a minor portion. These differences could be explained by separate pathways regulating growth independent of neuronal activity (Haussmann, 2008).

Particularly striking is the large number of genes involved in RNA processing among genes differentially expressed in ewg^l1mutants. Although local regulation of gene expression is required in growth cones of navigating axons, a prominent role for pre-synaptic regulation of gene expression at the RNA level is only just emerging, but is a hallmark of post-synaptic plasticity. Several RNA binding proteins have been implicated in memory storage . osk and CPSF (cleavage and polyadenylation specificity factor) are among the genes differentially regulated in ewg^l1mutants. Other genes involved in RNA processing differentially regulated in ewg^l1mutants comprise the whole spectrum of regulation at the post-transcriptional level, from nuclear organization (otefin), alternative pre-mRNA processing (Pinin, CPSF, Rox8) and export/import (Segregetion distorter, Nxf2, CG11092, Karyopherin, Transportin) to transport, localization and translation (oskar, swallow, ribosomal protein genes S5 and Rpl24), and likely also include the regulation of mRNA stability (Rox8) (Haussmann, 2008).

An intriguing connection between ewg and signaling pathways involved in regulating synaptic growth is indicated by differentially regulated components of the Wg and N pathways (gro and Hairless) in ewg^l1mutants. Consistent with a role of the co-repressor gro in Wg and N mediated transcriptional regulation of synaptic growth, Wg and N signaling pathways do not operate independently of ewg in genetic interaction experiments. The transcriptional regulatory networks of EWG, Wg and N seem to be highly interwoven. Overexpression of pan, the transcriptional mediator of canonical Wg signaling, which is repressed by gro, does not lead to a further expansion of synaptic growth in ewg mutants, suggesting a requirement for ewg-regulated genes. This effect could be mediated by deregulated N signaling, which is also repressed by gro, but antagonistic to Wg in synaptic growth. Thus, removal of gro, as in ewg, will relieve the repressive effect of N and antagonize the stimulatory effect of pan. In the complementary situation, removal of N increased bouton numbers further in the absence of EWG, which is consistent with an increase in Wg signaling as a result of down-regulated gro in ewg mutants. Antagonism between N and Wg pathways has also been found in wing discs, where N inhibits armadillo (arm), the transcriptional co-activator of canonical Wg signaling. Intriguingly, gro has also been found to be a target of receptor tyrosine kinase signaling and, thus, can combine additional pathways with N and Wg signaling. In addition to transcriptional hierarchies, chromatin remodeling has also been implicated in synaptic plasticity. Strikingly, CG6297, a Drosophila homologue of the histone deacetylase RPD3, is differentially expressed in ewg^l1mutants and physically interacts with gro (Haussmann, 2008).

How ewg exerts its effect on TGF-β signaling is less clear. A prominent regulatory step in this pathway is the regulated degradation of the SMAD co-factor Medea by Highwire. Several genes involved in regulating protein stability are differentially down-regulated in ewg mutants (CG6759, CG3431, CG4973, CG7288, CG3455, CG9327 and CG9556). Lower expression levels of these genes might interfere with stabilization of Medea and explain why the effect of activated TGF-β signaling is not additive in the absence of EWG (tkvA GOF ewg LOF). Bouton numbers in wit null mutants are marginally increased in the absence of EWG, suggesting further that genes regulated by SMADs are involved in mediating synaptic overgrowth in ewg^l1mutants. Potentially, ewg could also regulate TGF-β signaling through the endosomal pathway involving spinster and/or spichthyin (Haussmann, 2008).

Functionally related genes have been shown to be co-regulated, suggesting additional ELAV targets in EWG-regulated gene networks. Indeed, ELAV negatively regulates alternative splicing of the penultimate exon in armadillo (arm). Exclusion of this exon, which truncates the carboxyl terminus of arm, reduces Wg signal transduction, which is in agreement with ewg's antagonistic role relative to Wg signaling. Another known ELAV target gene is neuroglian (nrg), where a role in synapse formation has recently been demonstrated in the giant fiber system. Taken together, the establishment of a gene network regulated by EWG will now serve as valuable tool to identify further ELAV regulated modules that shape the synapse (Haussmann, 2008).

The transcription factor EWG is a major target of the RNA binding protein ELAV, which regulates EWG protein expression via a splicing mechanism. EWG is required pre-synaptically and cell-autonomously at third instar neuromuscular junctions to restrict synaptic growth, demonstrating that restrictive activities at gene expression levels are also required for synaptic growth regulation. EWG mediates regulation of synaptic growth primarily by increasing transcript levels of genes involved in transcriptional and post-transcriptional regulation of gene expression. Genes at the end of the gene expression hierarchy (effector genes) represent only a minor portion of EWG-regulated genes. Since analysis of mutants in genes differentially regulated in ewg^l1mutants revealed that these genes are involved in both stimulatory and restrictive pathways of synaptic growth, and since ewg genetically interacts with a number of signaling pathways (Wingless, Notch, TGF-β and AP-1), the results suggest that synaptic growth in Drosophila is regulated by the interplay of multiple signaling pathways rather than through independent pathways (Haussmann, 2008).

Posttranscriptional Regulation of ELAV

ELAV is posttranscriptionally regulated by multiple splicing and below-threshold levels of ELAV protein severly effect neural differentiation (Yao, 1993).

Analysis of elav germline transformants shows that one copy of elav minigenes lacking a complete 3' untranslated region (3' UTR) rescues null mutations at elav, but that two copies are lethal. When they could be recovered as adults, flies carrying two copies of the altered elav transgene have reduced fertility. In less extreme cases, flies carrying two transgenes do not show significantly decreased viability, but still show decreased fertility. Overall, viability is reduced in males more than in females, and fertility is decreased in females more than in males. Deleterious effects were seen both in normal and elav null backgrounds. Additional in vivo experiments demonstrate that elav expression is regulated through the 3' UTR of the gene and indicate that this level of regulation is dependent on Elav itself. Elav expression is found to be independent of elav gene dosage. Similar to the endogenous elav locus, transgenes carrying the 3' UTR show dosage independence of elav expression. In contrast, minigenes carrying a truncated elav 3' UTR produce Elav in a dosage-dependent fashion. Thus, the 3' UTR is responsible for normalizing the amount of Elav produced, regardless of the gene copy number. Because Elav is an RNA-binding protein, the simplest model to account for these findings is that Elav binds to the 3' UTR of its own RNA to autoregulate its own expression. Although increased levels of Elav in the proteins of head extracts from flies carrying two copies of transgenes could not be detected, it is possible that elevated Elav expression could happen either earlier in development and/or in a subset of neurons. Alternatively, it is possible that the production of an abnormal ELAV mRNA, and not of the final gene product, triggers the deleterious phenotypes associated with two transgene copies. Going past a threshold of transgene mRNA reached when two copies of the transgene are present could lead to the observed deleterious effects, presumably by titrating a factor that binds the transgene RNA and forms an unprocessable complex. Clearly, additional experiments are required to test these possibilities (Samson, 1998).

Elav interaction with mRNA

Elav family member proteins are characterized by three RNA recognition motifs (RRMs, also RBD), the first two of which are in tandem and the third of which is separated by an interdomain hinge region. The RRM consists of 80-90 amino acid (aa) residues with two highly conserved short motifs, an RNP1 octamer and an RNP2 hexamer, and is found in numerous proteins involved in post-transcriptional processes. The crystal structures of RRMs from U1A, hnRNP A1, U2B'', and Sxk reveal that the tertiary structure of the RRM domain consists of four ß pleated sheets packed against two alpha-helices. Within the Elav family of proteins, the three RRM domains are highly conserved, whereas the N-terminal domain and the hinge can vary. An evolutionary analysis of the RRM domains has revealed a close association between RRM1 and RRM2, suggesting that they arose by a duplication event, but the third RRM domain was on a separate branch of the phylogenetic tree. Outside the Elav family, the closest related RRM domains are found in the protein encoded by the Drosophila melanogaster Sex-lethal gene, which contains two tandem RRMs that are related to the first two Elav RRMs (Lisbin, 2000 and references therein).

elav-encoded function, although vital at the organismal level, is not vital for cell survival. The elav-null embryonic nervous system has a disorganized appearance in that many processes are irregular as evidenced in defective commissures. Otherwise the embryos and the neuronal soma appear normal. Elav regulates neural-specific alternative splicing of at least three broadly expressed genes, nrg, ewg, and arm. In each case, the level of a neural-specific isoform-encoding transcript is influenced by Elav levels. Additionally, by analogy with other family members, Elav could also affect mRNA stability by interacting with the 3' untranslated region or regulating translatability (Lisbin, 2000 and references therein).

Although the full extent of elav-modulated genes is currently not known, it is reasonable to suggest that the misregulation of a cohort of elav-regulated genes could collectively disable the embryonic nervous system. This discussion focuses is on the Elav-RNA interaction, which is the point of convergence for all elav-related functions. The analysis of AGD mutations (mutations expected to selectively impair the RNA-binding ability of the RRM without destroying the overall domain structure) reveals that aromatic amino acid substitutions in the RNP1 domain of each RRM result in proteins unable to carry out their in vivo function, since they show no rescue on their own. A recent report on the crystal structure of Sxl complexed with tra target RNA further validates the importance of aromatics in protein-RNA interactions (Handa, 1999). This SXL study has demonstrated that Phe in both RRM1 and RRM2 and Val in RRM2, precisely the amino acids mutated in the present study, are involved in stacking interactions with RNA. The complete conservation of these amino acids between Elav and Sxl, and the effectiveness with which the substitution mutations in these residues disable the protein, suggest that these mutations abrogate or at least greatly diminish the RNA-protein interaction. Since the AGD mutation in the third RRM also does not provide rescue on its own, the RNA-binding ability of the third RRM is also essential. However, 3AGD did provide modest rescue in conjunction with the hypomorphic allele ts1, suggesting that the 3AGD can associate with either the target RNA or in some other way facilitate the Elav^ts1-RNA interaction. These data indicate that each RRM contributes to the Elav function through its RNA-binding property. The AGD mutations do not appear to perturb subcellular localization of mutant Elav. Thus RNA binding by Elav, as disrupted by these mutations, is not essential for nuclear localization (Lisbin, 2000).

Domain replacement is a form of mutagenesis that samples many mutations at once while increasing the likelihood of maintaining the basic structure and stability of the protein. In assays used in this study, the RRM3 domain replacement proteins were fully functional. The complete functional rescue by ER3 and EH3, chimeric proteins that replace the third RNA-binding domain with RRM domains from other proteins, leads to the conclusion that the RRM3 domains are functionally homologous. It also demonstrates a considerable degree of pliancy within the amino acids, as in the case of RBP9 RRM3 (RBP9 is the D. melanogaster protein most homologous to Elav), where there are 21 amino acid changes in 80 residues. Moreover, 2 conservative amino acid changes in the RNP1 sequence, Tyr to Phe and Ser to Thr, that are in positions demonstrated to contact RNA bases in the Sxl/tra RNA cocrystal, apparently do not significantly alter Elav target recognition (Lisbin, 2000).

In contrast to RRM3 replacements, the RRM1 and RRM2 replacements provide at best a marginal rescue. The ER1 protein provides a modest rescue of e5 allele (~5%) and robust rescue of the temperature sensitive ts1 allele (~65%), while ES1 provides only a supplemental function in the ts1 allele rescue (~8%), and no function in the e5 rescue. The rescue demonstrated in the ts1 background by these proteins suggests that they can participate in some protein-RNA interactions, albeit at a lower than normal efficiency. It is also interesting to note that in contrast to ES1, ES1/2 provides rescue of both e5 and ts1 alleles, underscoring the possible importance of amino acids in the C-terminal half of RRM1 for functional specificity. Similar to ES1, ES2 also exhibits the ability to provide supplemental function in the ts1 background, but no ability to rescue e5 allele (Lisbin, 2000).

The low function of the RRM1 replacement is surprising given the high homology among these proteins. Elav and Sxl RRM1 and RRM2 are strikingly similar. While Sxl specifically interacts with the UGUUUUUUU sequence, Sxl can also presumably recognize a poly(U) stretch without the intervening G, since it is lacking in some in vivo Sxl targets. Elav-like proteins have been reported to bind to a variety of poly(U)-rich sequences in vitro, further suggesting similar modes of target recognition. The data from Handa, 1999, allow an assessment of homology between RRM1 and RRM2 of Elav and Sxl among just the amino acid residues that participate directly in RNA binding. The six residues from the RNP1 and RNP2 sequences of both RRMs that are involved in base stacking interactions in the Sxl-RNA crystal are conserved in Elav. The only other residue involved in base stacking, Arg195, lies in loop5 of RRM1 and is not conserved in Elav (Thr). Of the amino acid side groups that contact either the backbone or base of the Sxl target RNA, 8 of 12 are identical. This homology of RNA-interacting residues argues for a similar mechanism of RNA recognition. The RBP9 RRM1's similarity to Elav is even more striking, since RNP1 and RNP2 domains are identical to Elav's and overall it differs in only 15 of 80 residues (discounting the 13 amino acids in loop3) (Lisbin, 2000).

Based on the Sxl model it is reasonable to entertain the idea that RRM1 and RRM2 of Elav together make up a single binding site. Single domain replacement proteins ES1 (8%), ES2 (17%), and ER1 (67%) are able to provide some supplemental function. However, when both RRM domains are replaced as in ES12 and ER12, the chimeric proteins are completely nonfunctional. This could perhaps suggest that in a single domain replacement chimeric protein, the remaining Elav domain is able to serve as the main anchoring domain. An untested possibility is that the concentration of ES12 and ER12 proteins in the nucleus is insufficient to support function. It is also possible that in these chimeras, inter-RRM1-RRM2 interactions are compromised, but conservation of the two Sxl RRM1 residues that interact with RRM2 (Tyr131 and Lys197) in both Elav and RBP9 makes this unlikely (Lisbin, 2000).

Given the homology between corresponding domains of Elav, RBP9, and Sxl, results of these domain replacement studies are indeed puzzling. On the one hand, RRM3 replacements are fully functional, which is consistent with the notion that RRM3 acts as a module. On the other hand, the very limited function of RRM1 and RRM2 replacements suggests that perhaps the function of these two domains is distinct from that of RRM3 and involves additional intra- and/or inter-protein-RNA or protein-protein interactions. Moreover, these results imply that although the RNA-binding property of each RRM is essential for Elav function, residues other than the RNP1 and RNP2 must also be important for the specificity of Elav function (Lisbin, 2000).

Drosophila neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Using a minigene reporter, it has been shown that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, criteria are proposed for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI (Lisbin, 2001).

A model is invisioned in which, in the absence of ELAV, the default 3' splice site is solely used, despite lacking a consensus branch-point sequence and a proximal polypyrimidine tract. It is hypothesized that the conserved intron elements, along with the default exon and polyadenylation site(s) and perhaps other as yet unrecognized sequences, conspire to positively maintain splicing exclusively to an otherwise weak default 3' splice site. In the presence of ELAV, however, this positive maintenance is disrupted or partially disrupted, either by directly competing with poly(U)-binding proteins, or by countering the effects of the conserved sequences, leading to 3' splice site recognition of the more consensus-like but distal neural-specific 3' splice site. This model has predictive value. For example, the exclusive use of the default 3' splice site could be compromised by deletion of one or both conserved intron elements. Further studies will be needed to address these questions (Lisbin, 2001).

found in neurons, a member of the Drosophila elav gene family, interacts with elav

elav, a gene necessary for neuronal differentiation and maintenance in Drosophila, encodes the prototype of a family of conserved proteins involved in post-transcriptional regulation. found in neurons (fne), a gene encoding a new ELAV paralogue, has been identified. FNE binds RNA in vitro. fne transcripts are present throughout development and contain long untranslated regions. Transcripts and proteins are restricted to neurons of the CNS and PNS during embryogenesis. These features are reminiscent of elav. However, fne expression is delayed compared to elav's, and FNE protein appears cytoplasmic, while ELAV is nuclear. GAL4-directed overexpression of fne in neurons leads to a reduction of stable transcripts produced from both the fne and elav endogenous loci, suggesting that fne autoregulates and also regulates elav (Samson, 2003).

fne encodes a predicted protein with three RNA recognition motifs, RRMs. In vitro binding assays demonstrate the RNA binding capability of FNE. Comparison with ELAV in the same assays indicates that the proteins have distinct nucleic acid binding properties, although they both show a high affinity for polyuridilyc acid. Conceptual translation of the Drosophila genomic sequence relying upon the prediction of best splicing sites, generates a shorter protein (Accession AAF48215) missing 18 residues between RNP-2 and RNP-1 of RRM1 of the FNE encoded by cDNA-28h, suggesting that there might be a second form of FNE protein. New fne cDNA/EST sequences will be necessary to verify this possibility. No alternative forms of ELAV have been reported, but do exist for RBP9 and for some of the vertebrate paralogs (Samson, 2003).

Strikingly, similarity is higher among the four vertebrate ELAV proteins than it is among the three fly proteins. In addition, FNE/RBP9 resemble vertebrate ELAV as well as, if not better than, Drosophila ELAV. These observations underline the complex evolutionary relationships among the different ortho-/paralogues. One scenario is that fne and rbp9 derive from a duplication of an ancestral gene that had already diverged from elav, possibly before the separation of vertebrates and invertebrates. The human genes might thus derive from two rounds of duplication of the fne/rbp9 ancestor. The functional relationships among the different members of the family within and between species remain unclear, but the specific differences seen in their subcellular localization and in their nucleic acid binding properties in vitro are suggestive of different or complex functions (Samson, 2003).

Similar to elav, fne transcripts contain long untranslated regions. The identified fne cDNA contains a 1068 nucleotide-long ORF, spanning 2.8 kb of genomic sequence. Transcripts whose sizes range from 4 to at least 8 kb, reveal the presence of unusually long untranslated region(s) in fne RNA. Genome analysis predicts fne untranslated regions of up to 500 nucleotides 5' and up to 6 kb 3' to the gene. The significance of unusually long UTRs in the case of fne is not known. The elav gene contains a 6 kb long 3' UTR whose important role in normal elav function has been demonstrated. In general, 3' UTRs act in cis to regulate mRNA translation, stability, and localization through association with regulatory proteins or with antisense RNA. 3' UTRs also play a role in trans in myoblast growth and differentiation. These possibilities remain to be explored in the case of fne and elav (Samson, 2003).

fne is expressed in most, if not all neurons of the central and peripheral nervous system during embryogenesis. Consistent with the enrichment of fne transcripts in heads and the similarity between elav and fne transcript patterns, it is believed that fne expression remains specific to the nervous system in later stages. FNE protein appears in embryonic neurons shortly after ELAV, and it is also produced in elav^null embryos. As opposed to ELAV, FNE is essentially cytoplasmic. The apparent subcellular localization of proteins only partly reflects their presence and function in cell compartments. For instance, HuR appears nuclear but shuttles between nuclei and the cytoplasm, where it is thought to be involved in mRNA stabilization. Nevertheless, the different subcellular localizations of FNE and ELAV suggest different molecular functions (Samson, 2003).

Overexpression of fne in neurons causes a significant developmental arrest during larval stages. ELAV ectopic expression in the wing discs is known to lead to neo-function. Although the possibility that fne overexpression causes neo-function cannot be excluded, the elav promotor was chosen to restrict overexpression within normal fne cellular and temporal specificities and mimic a hypermorphic mutation. Indeed, the properties of the GAL4 expressing lines predict that the onset of fne overexpression is delayed compared to its normal onset, and that depending upon lines, the specificity of fne overexpression differs. This probably accounts at least in part for the differences in the severity of the phenotypes that are observed in different genetic combinations (Samson, 2003).

Overexpression of fne in neurons causes a decrease in the stable levels of both endogenous elav and fne transcripts in embryos. Although it is not possible to exclude that this reflects the occurrence of embryonic neuronal death, this possibility is not favored because anti-ELAV immunocytochemistry shows apparently normal CNS and PNS in embryos. Rather, this data suggests that fne overexpression decreases the level of expression of the endogenous fne locus by autoregulation, and that of the elav locus by feedback regulation (since fne is expressed later than elav). It is not clear whether alteration of the stable transcript patterns has a significant repercussion on protein expression during embryonic stages, since both ELAV and FNE appear to be stable proteins. It may be that the effect is delayed, consistent with the larval lethality that was observed (Samson, 2003).

It is possible that fne autoregulates and regulates elav via direct binding to the RNAs produced by these genes. Consistent with this possibility, both ELAV and FNE were found to bind in vitro to a sequence present in the elav 3' UTR. A role for RBP9 in transcript downregulation has been previously reported. Additional experiments will be necessary to determine the mechanism by which FNE affects elav expression in neurons (Samson, 2003).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

Elav possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3'-untranslated region (3'-UTR) is necessary for autoregulation. RNA-binding assays and in vitro selection was used to identify the ELAV best binding site in the elav 3'-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K_d 40 nM). This finding supports the model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. The best binding site was narrowed to a 20 nt long sequence A(U₅)A(U₃)G(U₂)A(U₆) in an alternative 3' exon. A model was proposed and tested in which the regulated use of this alternative 3' exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne (Borgeson, 2005).

ELAV-binding sites have been reported in regulated introns of nrg and erect wing (ewg). In nrg RNA, they are defined as long tracts including at least two 8-10 nt-long U-rich stretches. In ewg RNA, ELAV-binding sites consist of AU-rich elements including three to four tandemly repeated A(U_4-6) sequences. The current approach allowed mapping the ELAV best binding site in its RNA to a shorter U-rich sequence that is only 20 nt long (SSE). It was found that the affinity of ELAV for a 65 nt long RNA including SSE is in the nanomolar range, similar to that of ELAV for a 164 nt long RNA derived from ewg proposed to bind several ELAV molecules. It is also similar to ELAV affinity for an RNA including the 114 nt long EXS6 from nrg RNA that cross-links to ELAV (Borgeson, 2005).

Competition experiments highlight the higher affinity of ELAV for the SSE site than for a mutant version of the site where a C replaces the single G. Thus, the interaction between ELAV and the RNA depends in part upon recognition of specific bases, not just of the phosphate sugar-backbone (Borgeson, 2005).

Sequence analysis of 500 nt framing SSE with the Stem Loop program as well as attempts to identify secondary structures with the programs Mfold and PlotFold in the 65 nt long SSE1FDelta RNA failed to identify any secondary structure with base pairing of SSE. ELAV thus binds ssRNA, reminiscent of the binding of the Sxl and HuD proteins, whose two RRMs share extensive structural similarity with ELAV RRM1 and RRM2 and which also bind ssRNA with no base pairing (Borgeson, 2005).

An ELAV-binding site with a U-rich sequence and a low dissociation constant with ELAV was thus identified in the elav 3'-UTR. The characteristics of this site resemble those of the previously identified ELAV targets, supporting the validity of the binding site that was identified in the elav 3'-UTR, and thus the model that elav autoregulation depends upon direct binding of ELAV to the 3'-UTR of elav RNA (Borgeson, 2005).

ELAV protein and its homologues contain three conserved RNA-binding domains. Mutations in any of the three domains abolish ELAV function. However domain swap experiments (with RRMs from the Drosophila paralogue RBP9, the Drosophila relative Sxl or the human orthologue HuD) showed that chimeric ELAV proteins with substituted RRM3, but not RRM1 and/or RRM2, are functional (Borgeson, 2005).

The binding properties of individual RRMs were evaluated to determine if distinct RRMs are responsible for the specific target recognition of nrg and elav-binding sites. It was found that both RRM1 and RRM3 are capable of significantly binding RNA on their own, with RRM1 showing a higher affinity than RRM3. No RNA-binding activity was detected for RRM2 with the targets that were tested (nrg, elav sites, and the OL RNA), even using different batches of proteins. The possibility cannot be excluded that the ELAV-RRM2 fusion protein, in spite of the fact that it is abundantly and stably produced, may be non-functional, possibly owing to altered folding. Another alternative is that ELAV-RRM2 can bind RNA only when another partner RRM is available, as in the case of HuD or HuC, whose second RRMs increases the affinity of RRM1 for an AU-rich sequence and stabilizes the complex (Borgeson, 2005).

Conserved residues between HuD and Sxl interact with different bases in their different targets [U(A/U)UUUAUUUU for HuD and UGUUUUUUUUU for Sxl], and it thus seems possible that the same ELAV RRMs are involved in nrg and elav target recognition. However, the binding assay involving one protein and two binding sites shows that RRM1 and RRM3 display different relative affinities for the nrg and elav RNA-binding sites. As a correlate, if the properties of individual RRMs are maintained in the complete protein, ELAV binding with specific RNAs may rely upon particular interactions with different RRMs. In vivo, it is conceivable that the complexes might interact with different sets of factors and thus enter distinct pathways of post-transcriptional processing, depending upon which RRM is involved in RNA contact (Borgeson, 2005).

The neuronal protein FNE is encoded by an elav paralogue, and the expression of the two genes is interconnected. In particular fne over-expression causes a decrease of elav (and fne) stable trancript levels, suggesting feedback regulation. Both FNE and ELAV can bind the elav SSE-binding site independently, and it has been shown that the two proteins can interact directly, since they have been identified in a large-scale two-hybrid screen of Drosophila proteins as high confidence partners. These two properties could be involved in the feedback regulation. FNE could regulate elav by binding to elav RNA directly or FNE could titrate ELAV, both antagonizing the occupation of RNA-binding sites by ELAV. This would require that ELAV, which is predominantly nuclear, and FNE, which is predominantly cytoplasmic, would be present in the same cellular compartment, at least transiently. HuR, an apparently nuclear human orthologue of ELAV shuttles between the nucleus and the cytoplasm (Borgeson, 2005).

When mixing FNE and ELAV with RNA, binding complexes with RNA including both ELAV and FNE are formed. One explanation for this observation is that a low affinity-binding site is also present in the RNA target and bound at high protein concentrations, allowing bridging of protein complexes via the RNA. But, since the proteins interact directly, it seems more probable that direct protein-protein interaction, before or after RNA binding, is responsible for the formation of these complexes (Borgeson, 2005).

The processes required for the generation of mature mRNAs are intimately linked and depend upon cis-regulatory regions scattered over the entire length of an RNA. Analysis of elav cDNAs provides some insights into the remarkable complexity of the transcripts, presumably reflecting post-transcriptional regulation (Borgeson, 2005).

The locus produces multiple abundant and/or stable transcripts whose size far exceeds that of the only coding elav RNA so far identified. Such a situation was first reported in the case of the gene suppressor of white apricot, which encodes an SR-like protein, and produces mature short transcripts that are detected only briefly during development, while larger polyadenylated unspliced RNAs are the only detected transcripts during most of development (Borgeson, 2005).

In addition, four polyadenylation sites were identified in elav, as was reported for some of the vertebrate Hu loci, where they have been proposed to play specific roles in post-transcriptional regulation. Indeed, this type of organization in the 3' end of transcripts is not rare, since 28.6% of 8700 analysed human 3'-UTRs contain two to four polyadenylation sites (Borgeson, 2005).

Although a model where ELAV binding in the proximity of polyadenylation sites and slowing the recruitment of cleavage factors or of poly(A) polymerase can explain the data reported in the case of nrg and ewg, it does not fit in the case of the elav gene. The sequences of ELAV-binding sites are not dramatically different, but they differ in their nature. ELAV binding to nrg and ewg RNAs occurs in alternative 3' introns and is responsible for the generation of alternative forms of proteins, while binding to elav RNA occurs in an alternative non-coding 3' exon and does not cause protein diversity. The SXL protein, whose two RRMs share extensive structural similarity with ELAV also possesses several distinct functions, in this case at the level of splicing and of translation regulation (Borgeson, 2005).

ELAV binds to a site retained in a non-coding elav RNA (RE58603), that differs by the inclusion of an alternate 3'-terminal exon from the functional form of elav mRNA (LD33076). RT-PCR data are in agreement with the proposed model hypothesizing that the coding transcript is produced as long as the level of ELAV protein remains below a given threshold and thus unbound to SSE. The non-coding transcript is alternatively produced by alternative choice of the terminal exon/polyadenylation site once ELAV protein level passes this threshold and binds SSE. This model provides a mechanism for the regulation of ELAV protein level, which has been shown genetically to be critical and dependent upon the elav 3'-UTR (Borgeson, 2005).

The dual RNA-binding mechanism, suggested by the specific preferences for different RNAs by individual RRMs, might be linked to dual functional properties, for instance via association with different additional partner proteins, which might include FNE. Future work will aim to examine this possibility in order to identify the mechanism(s), such as regulation of exon definition and/or cleavage/polyadenylation responsible for elav regulatory functions (Borgeson, 2005).

Neural-specific elongation of 3' UTRs during Drosophila development

The 3' termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. This study reports that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3' UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. These extended 3' UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3' lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function (Hilgers, 2011).

This study identified ~30 genes that exhibit developmental regulation of their 3' UTRs. As a class, the expressed transcripts contain some of the longest 3' UTRs in the Drosophila genome and are comparable to the largest 3' UTRs known in mammals. All of the genes undergo this posttranscriptional transition shortly after the onset of zygotic transcription, with the first detection of the long isoforms at 2-4 h AF. Perhaps the loss or gain of specialized RNA-processing factors during the MZT leads to the extension of the 3' UTRs. Alternatively, depletion of one or more components of the general mRNA poly(A) machinery at the MZT or in neural tissues could lead to weakened poly(A) and mRNA cleavage efficiency, therefore promoting the synthesis of longer transcripts. Such a mechanism, diminished levels of the essential poly(A) factor Cstf-64, promotes the formation of longer isoforms of IgM in B lymphocytes (Hilgers, 2011).

Previous studies suggest that Drosophila 3' UTRs are longest during early development. The genes identified in this study do not conform to this general trend but are consistent with recent whole-genome studies in vertebrates that suggest a statistical enrichment for longer 3' UTRs at later stages in development. In mammals, the expression of long 3' UTR isoforms has been correlated with the loss of cell proliferation and the onset of differentiation. The genes described in this study do not fit this model and may instead be responding to a specific developmental cue during neurogenesis. The key correlation for the large 3' extensions identified in this study is neural expression, irrespective of the state of proliferation. However, the occurrence of 3' elongation events at additional genes in other tissues cannot be excluded because the datasets used for this analysis made use of whole-embryo RNA samples at various developmental stages (Hilgers, 2011).

A significant fraction of the genes with extended 3' UTRs encode proteins implicated in RNA binding or processing, including ago1, adar, pumilio, brat, mei-P26, shep, imp, fne, and elav. Some of these genes, like ago1, are broadly expressed in a variety of tissues. Nonetheless, the extended isoforms of ago1 mRNAs are specifically enriched in neural tissues, a known hotbed of posttranscriptional regulation, including regulation by miRNAs and differential splicing. For example, Dscam is thought to produce tens of thousands of spliced isoforms in the Drosophila CNS. Furthermore, in Drosophila, directed transport of mRNAs, like bicoid, requires functional elements within the 3' UTR. Whether RNA binding factors such as Pum participate in a network of cross-regulation by repression, activation, or transport awaits further study (Hilgers, 2011).

It is currently unclear whether the long forms of mRNAs as seen in mammalian cells produce less protein than the short forms in Drosophila. However, enrichment of Pum recognition motifs in the extended 3' UTRs of elav, brat, and pumilio suggests regulation by repression because Pum and Brat are known to form localized translation repression complexes essential for anterior-posterior body patterning in early embryogenesis. Such regulation may have particular relevance in the Drosophila nervous system because Pum is required for dendrite morphogenesis. It is proposed that neural-specific isoforms of the genes identified in this study comprise elements of an interactive RNA-processing network that mediates some of the distinctive posttranscriptional processes seen in the nervous system (Hilgers, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

elav is expressed in all neurons after they are born and remain expressed through embryogenesis to adulthood. ELAV is not present in neuroblasts, ganglion mother cells or glia. It is localized to the nucleus (Yao, 1993).

Combinatorial expression of Prospero, Seven-up, and Elav identifies progenitor cell types during sense-organ differentiation in the pupal antenna

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. Three broad categories of sense-organs are known to be specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero, Elav, and Seven-up shows that there are three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates (Sen, 2003).

Development of single sensory units have been traced by using enhancer-trap insertions into the neurilized genes (neu^A101 and neu-Gal4). Olfactory progenitor cells delaminate from the epithelium as single isolated cells with apically located nuclei and are arranged in distinct domains in the early antennal disc. By 8 h APF, these progenitors begin association with one to three additional cells forming well-defined clusters. These cell clusters do not arise by division of the olfactory progenitor since the first evidence of cell division as seen by phosophohistone-3 (PH3) immunoreactivity is after 12 h APF. The cells within the cluster are referred to as secondary progenitors, since their division gives rise to all the cells of an individual sense-organ. Most of the clusters divide between 16 and 22 h APF (Sen, 2003).

This analysis was restricted to clusters of secondary progenitors composed of three cells, although two and four cell clusters can also be identified by expression of GFP driven by neu-Gal4 (henceforth referred to as Neu-GFP). At 14 h APF, clusters are oriented in a single plane and have not yet begun cell division. Expression of Pros and Elav was examined by using specific antibodies, while Svp was monitored by following ß-galactosidase activity in the enhancer-trap line svp^P1725. None of these markers express in primary olfactory progenitor cells but appear shortly after formation of groups of secondary progenitors. Double-labeling of 14-h APF discs with anti-Pros and anti-Elav reveals that two of the three cells within a cluster express both of these markers. Pros expression appears prior to that of Elav within the same cell. One of these cells also expresses the Svp reporter (henceforth called Svp-lacZ). The combinatorial expression of genes allowed identification of three progenitor types. PIIa does not express any of the markers and is recognized only by expression of Neu-GFP; PIIb expresses Neu-GFP, Pros, Elav, and SvplacZ, while PIIc expresses Neu-GFP, Pros, and Elav. Clusters composed of only two cells lack the PIIc progenitor and those with four cells contain two PIIc progenitors. Hence, differential expression of genes could provide cells within a single cluster the potential to exhibit independent fates (Sen, 2003).

The distribution of Pros, Elav, and SvplacZ was examined during division of the secondary progenitors. Staining with phenylene-diamine allowed identification of interphase nuclei, while entry and exit from mitosis was monitored by changes in Neu-GFP distribution. During mitosis, only one cell per cluster exhibits asymmetric cortical Pros crescents. The neighboring cell shows either compact or a uniform cytosolic localization. The failure to observe two cortical Pros crescents per cluster even in colcemid-arrested discs suggested either that PIIb and PIIc divide at different times or that Pros is asymmetrically segregated in only one of these cells (Sen, 2003).

By 36 h APF, postmitotic cells of the sensory units occupy positions comparable to those in the adult; the shaft and socket cells are identifiable by their external cuticular projections. Pros is present in sheath and socket cells, while Elav is exclusively neuronal. Clonal experiments have shown that the sheath cell arises from PIIb lineage possibly inheriting Pros asymmetrically from the progenitor. The socket, however, is derived from PIIa, which does not express Pros, indicating de novo synthesis (Sen, 2003).

The majority of peripheral antennal glia arise during development of the coeloconic sensilla. PIIb has been identified as the glial progenitor in a number of gliogenic sense-organs. In the olfactory sense-organs, PIIb can be unequivocally recognized by expression of Pros, Elav, and Svp-lacZ (Sen, 2003).

Clusters in the region of the antenna populated by coleoconic sense-organs were selected for detailed analysis. PIIb divides to produce a large cell that remains within the epithelial layer and a smaller basal cell. The basal cell transiently expresses Pros and low levels of the Svp reporter and also stains with antibodies against the glial cell marker Reverse Polarity. The nascent glial cell loses Pros and Svp expression and rapidly migrates away to become associated with the fasiculating sensory neurons (Sen, 2003).

The gliogenic lineage described above occurs only within the coeloconica sensilla (i.e., ~70 out of 450 sensilla). PIIIb, like PIIb in all other clusters, expresses Pros, Svp, and Elav. Mitosis of all secondary progenitors is completed by 22 h APF, and marker distribution in progeny was examined at 25 h APF. At this time, sensory cells orient along the apicobasal axis resembling positions in the mature sensillum (Sen, 2003).

Effects of Mutation or Deletion

A developmental analysis of genetic mosaics shows that elav gene function is autonomously essential in the eye and for normal development of the optic lobes, but not necessary in most imaginal discs with the exception of the eye disc (Campos, 1985). In viable mutants there are behaviorial abnormalities, and aberrant axon development (Jimenez, 1987).

There are three known viable alleles of the elav gene, namely elavts1, elavFliJ1, and elavFliJ2, which manifest temperature-sensitive phenotypes. The modification of the elavFliJ1 allele corresponds to the change of glycine426 (GGA) into a glutamic acid (GAA). elavts1 and elavFliJ2 were both found to have tryptophan419 (TGG) changed into two different stop codons, TAG and TGA, respectively. Protein analysis from elavts1 and elavFliJ2 reveals not only the predicted 45-kD truncated ELAV protein due to translational truncation, but also a predominant full-size 50-kD ELAV protein, both at permissive and nonpermissive temperatures. Apparently there is a functional suppression of the nonsense mutation (Samson, 1995) .

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date revised: 25 August 2022

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