BIOLOGICAL OVERVIEW

The quantity of a gene product may be controlled in a number of ways: regulation of the gene's rate of transcription initiation, the rate of protein synthesis and degradation, and the stability of the RNA are all factors that determine the level of any given protein in a cell. For years these ideas have reverberated like mantras in molecular biology circles, but evidence for the mechanisms of posttranscriptional regulation were sorely lacking. Recently it has been found that whole classes of cytoplasmic mRNAs can be made inactive, that proteins affect mRNA stability and proteins regulate the structure of mRNA. ELAV is one of a class of proteins that binds to mRNA and presumably has a regulatory function.

ELAV is required for correct differentiation and maintenance of the nervous system. The gene encodes an RNA binding protein expressed in all neurons after birth, qualifying elav as a pan-neural gene. The subcellular distribution of ELAV was investigated using ELAV-specific antibodies and scanning confocal laser microscopy. ELAV is predominantly localized within the nucleus where it concentrates within discrete domains described as dots and webs. To characterize these discrete domains an analysis of Drosophila coiled bodies was initiated. Coiled bodies are non-capsular nuclear bodies that appear to be composed of coiled fibrils. Most coiled bodies disassemble prior to or during mitosis. After cell division, the reassembly of coiled bodies occurs during G1 phase and is preceded by the reformation of nucleoli. Coilin is a 80-kD nuclear protein, identified with autoimmune serum, that is found as an integral component of coiled bodies. The polyclonal antibody R288 raised against human coilin was used to identify coiled bodies in cells of the Drosophila larval central nervous system. Double-labeling immunohistochemistry shows that, similar to vertebrate and plant systems, small nuclear ribonucleoproteins are enriched within these structures. The nuclear distribution of ELAV is reminiscent of the distribution of a number of splicing factors, including snRNPs, snRNAs, U1 70K and U2AF, as well as the snRNA-specific 2,2,7 trimethyl guanosine cap within mammalian nuclei (see Sans fille for more information on Drosophila splicing factors). Further analysis of ELAV reveals that subnuclear domains enriched with this molecule localize within and close to coiled bodies and close to subnuclear domains enriched with splicing factors. Deletion of the ELAV alanine/glutamine-rich amino-terminal auxiliary domain has no discernible effect on localization; proteins produced from elav lethal alleles distribute normally. This morphological study provides the first hint of a role for ELAV in the generation of alternatively spliced neural-specific mRNAs (Yannoni, 1997).

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

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

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

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

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

GENE STRUCTURE

Bases in 5' UTR - 491

Exons - three

PROTEIN STRUCTURE

Amino Acids - 519

Structural Domains

The protein contains three repeats of a pair of sequences defined as RNA-binding consensus sequences, an octopeptide (RNP1), and an appropriately spaced hexapeptide, RNP2 (Yao, 1991).

The neuron specific Drosophila Elav protein belongs to the Elav family of RNA binding proteins, characterized by three highly conserved RNA recognition motifs; an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. The role of the Elav hinge in localization and function has been examined in vivo using transgenes encoding Elav hinge deletions. Subcellular localization of the hinge mutant proteins reveals that residues between amino acids 333-374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human Elav family member, HuR. However, this defined sequence is insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays have revealed that mutant proteins that fail to localize to the nucleus are unable to provide Elav vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag (Yannoni, 1999).

date revised: 20 Nov 97

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