BACKGROUND
the elav  gene of d. melanogaster was the the first identified member of a family of neuronal rna binding proteins that is conserved in metazoans  <cit> .
the proteins in this family contain three rna recognition motifs , with a hinge region separating the second and third rrms and an optional non-conserved n-terminal region.
the hinge includes signals essential for nuclear export and subcellular localization  <cit> .
rrm are common protein domains found in all life kingdoms.
in humans, there are  <dig> genes encoding rrm containing proteins, which represent 2% of the human gene products.
proteins containing one or several of these domains are capable of interacting in a sequence specific manner with single stranded rna molecules and of directing the assembly of multiprotein complexes  <cit> .
in spite of the remarkable sequence conservation of the rrm domains, rrm-containing proteins perform numerous functions, intervening at all the possible steps of rna metabolism.
the rrm domain is composed of about  <dig> amino acids and contains a conserved octapeptide termed rnp- <dig>  and a conserved hexapeptide termed rnp- <dig>  structural studies indicate that four antiparallel beta-sheets form the rna interaction surface, with rnp- <dig> and rnp- <dig> on the two inner sheets .
in rna-rrm complexes, nucleotides establish contacts with residues in the rnps, with regions in the rrm beyond the rnp domains also involved in rna recognition.
the plasticity of rrms in their sequence-specific recognition of topologically diverse rna is likely to be correlated with their presence in a variety of proteins involved in the diverse steps of post-transcriptional regulation.
there are three elav-related genes in d. melanogaster.
the elav gene encodes a nuclear product present in all neurons throughout development and is required for the differentiation of postmitotic neurons and their maintenance  <cit> .
the rbp <dig>  product is present in neuronal nuclei starting at the third larval instar and also in the cytoplasm of cystocytes during oogenesis.
although neuronal expression is predominant, rbp <dig> mutations reveal a role in cystocyte proliferation and differentiation, but no neuronal defects have been reported  <cit> .
the expression of fne  resembles elav's, but with a slightly delayed onset.
fne is cytoplasmic, but the elav and fne genes interact, suggesting protein shuttling  <cit> .
the products of elav family members are essentially present in the nervous system, in all of the neurons in the case of elav itself, but more generally in subsets of neurons and/or neuroblasts and glial cells.
expression has also been detected in other tissues, in particular in testes and ovaries, or found to be ubiquitous .
diverse molecular functions in the control of rna half life, nuclear export, rna 3' end formation, alternative rna processing, polyadenylation and translation have been proposed for these proteins  <cit> .
multiple functions, both cytoplasmic and nuclear have been demonstrated for hur, an ubiquitously expressed member of the human family  <cit> .
the evolutionary relationship between members of the family are complex.
for instance, the four human proteins share 74–91% identity, while the three drosophila proteins share only 59–68% identity.
the goal of the work reported here was to investigate these relationships.
we found that the elav family has an eventful evolutionary history, somewhat masked by the high level of amino acid conservation of the gene products, but revealed by analysis of the gene structure of the different family members .
we attribute the rapid functional evolution of the family members, as opposed to the high level of sequence conservation, to the plasticity of the rrm domains, where small changes in critical positions have the potential to modify interactions with rna.
RESULTS
the paralogs fne and rbp <dig> share a conserved organization of their coding regions but elav, the third family member, is distinct
all three drosophila paralogs elav, rbp <dig> and fne are essentially expressed in neurons.
elav null mutants are embryonic lethal, while the rbp <dig> null mutation is viable, but surprisingly confers a female sterility phenotype.
fne null mutants, although not fully characterized, are also viable .
in order to understand the evolutionary mechanisms responsible for the generation of these paralogs, we examined their gene structure.
although their organizations are apparently quite distinct, we found remarkable conservation in the correspondance between exons and specific protein regions in rbp <dig> and fne .
there are two differences  the presence of new mini-exons respectively specific for each of the two genes and  the use of a single exon in fne but two in rbp <dig> to encode the third rrm.
strikingly, this organization is totally different in the elav gene, whose complete orf, except for the a of the atg initiation codon, is encoded by a single exon.
conserved exon junctions are present in most elav orthologs
we took advantage of the recent sequencing of complete genomes  <cit>  to survey the gene family in  <dig> species by  identifying all the family members and  comparing the organization of the orf in exons.
in humans, d. melanogaster and c. elegans, we extracted from the databases protein sequences deduced from cdna analyses, and aligned genomic dna with cdna to determine the exon-intron structure.
in other cases we used the predicted protein sequences, either published or computed for our purpose, as detailed in the methods.
we examined species from the chordata , arthropoda  and nematoda , for a total of  <dig> genes .
first, we found that the size of elav families varies  among the  <dig> species that we studied, with no clear relationship between family size and brain/animal complexity .
for instance, dipterans possess three elav genes, while the hymenopteran apis mellifora, with ten times as many neurons as drosophila, possesses only one gene.
levels of identity between the proteins encoded by the  <dig> genes are high, with the lowest score  obtained in the comparison of d. melanogaster elav with the unique c. elegans protein.
between humans and drosophila, there is 54–64% amino acid identity in the elav-related proteins, 38% identity for the arginase proteins  and 33% identity for the engrailed proteins .
the levels of elav-related protein identity are thus remarkable.
the crystal structure of the first two rrm of human hud associated with cfos rna, identifies  <dig> amino acids whose side chain is making direct rna contacts  <cit> .
these residues are conserved in all  <dig> elav-like proteins that we examined, except for the arginine in rnp <dig> of the second rrm, which appears to be specific to the human proteins and to one of the b. mori elav-like, bm- <dig>  in the other species there is a conserved substitution by a lysine.
second, we found remarkable conservation of exon structure.
from vertebrates to invertebrates, we identified eight exon junctions in the rrms/hinge region .
we named them j <dig> to j <dig>  from the most upstream to the most downstream.
all are present in several phyla, except for j <dig> and j <dig> which are specific for fne and rbp <dig> from drosophila and are implicated in the generation of mini-exons in the sequence coding  these proteins .
overall, the j <dig> junctions  are unambiguously homologous since  the level of protein sequence conservation is such that the amino acid positions where the junctions intervene are clearly aligned and conserved  and  nucleotide sequence analysis shows that at a given exon junction, the splice is at the same position in the codons: specifically between the first and the second bases of the spliced codons  or exactly between codons .
there are two exceptions to this strict conservation.
first, j <dig> is interrupted in rbp <dig> of d. melanogaster by the intronic insertion of an alternative mini-exon, without alteration of the j <dig> 5' or 3' splice sites.
second, in fne, j <dig> is split by the intronic insertion of a mini-exon, the j <dig> donor splice site is additionally shifted downstream while the j <dig> acceptor splice site is maintained .
interestingly, the junctions j <dig> and j <dig> occupy the same position relative to rnp- <dig> in rrm <dig> and rrm <dig>
the junctions j <dig> and j <dig> map in a moderately conserved coding region, essential for nuclear export and proper subcellular localization , including only a conserved hexamer .
both j <dig> and j <dig> split the spliced codons between the second and the third bases.
in this region, three types of events affecting the splicing seem to have occured independently: 1) the introduction of a mini-exon , that can be alternatively spliced ,  the shift of the 5' splice site   the shift of the 3' splice .
noticeably, the regions close respectively to j1/j <dig>  j4/j <dig> and j6/j <dig> as well as the entire hinge region between rrm <dig> and rrm <dig> appear more variable than the rest of the protein.
intronless elav-like genes are present in diptera and lepidoptera
interestingly, for six of the analyzed genes , the entire conserved region of the protein is encoded by a single exon.
based upon both their gene structure and the level of protein sequence identity, the dipteran intronless genes constitute a homogeneous elav-type group.
in contrast, although intronless like elav, the b. mori genes encode proteins more similar to fne/rbp <dig> than to elav.
this observation suggests that distinct evolutionary forces shaped the b. mori genes and the dipteran elav-like intronless-genes, respectively.
to evaluate this hypothesis, we performed a phylogenetic analysis of the  <dig> elav orthologs/paralogs, using the upgma algorithm, with bootstrap analysis .
this analysis shows with high confidence  that in dipterans, the proteins encoded by the intronless genes  cluster together, while the two b. mori genes products cluster with the fne/rbp <dig> sequences similar results were obtained when performing sequence alignments using the neighbor joining method .
because the d. melanogaster elav gene is nested in the third intron of the arginase gene  <cit> , we probed the gene environment of the intronless elav orthologs that we report here.
we found that the nested elav/arg gene organization is unique to drosophila, specifically d. melanogaster and  <dig> additional drosophila species whose genomes have recently been sequenced  <cit>  .
in the  <dig> other non-drosophila species examined here, there is no close linkage between the arginase gene and the elav gene family members.
in particular, the mosquitos, similar to d. melanogaster, each have three elav-like genes, including one intronless version, but unlike d. melanogaster they have an intronless arg, which obviously rules out the possibility of a nested gene.
in b. mori, although the two intronless bm- <dig> and bm- <dig> genes map at loci distinct from the arg locus, an intron putatively homologous to the third intron of the d. melanogaster arginase gene is present .
discussion
the d. melanogaster gene elav is specific to the dipteran phylum and results from retrotransposition
the elav gene from drosophila was the first identified member of this family, is considered as its prototype  <cit> , and most of the subsequently discovered orthologs are named after it.
however, the present analysis highlights unique characteristics of this gene that suggest it is of recent evolutionary origin, after the separation of dipterans and lepidopterans.
aside from elav, only the dipteran genes ae- <dig>  ag- <dig> and cp- <dig> encode proteins that are more similar to elav than to fne and rbp <dig>  in addition to the intronless elav-likes, dipteran genomes carry two genes encoding proteins of the type fne/rbp <dig>  also found in the seven other genomes analyzed.
thus elav, ae- <dig>  ag- <dig> and cp- <dig> represent a newly evolved gene form specific to dipterans.
in addition, the elav gene structure is suggestive of retrotransposition, a process considered significant in the evolution of genomes, including drosophila  <cit> .
the genes ae- <dig>  ag- <dig> and cp- <dig> from mosquitoes share with elav not only a higer level of similarity between their products, but also the property of having their orf in a single exon.
the absence of introns  is atypical: we identified conserved exon junctions that are a landmark present in most of the elav-related genes.
furthermore, the elav gene of drosophila is nested in the arginase gene.
in humans, retrotransposition is an important contributor to the generation of nested genes  <cit> .
we thus propose that elav originated from a recent retrotransposition event.
it is possible that the same retrotransposition is at the origin of both the lepidopteran intronless fne/rbp9-like genes and the dipteran elav-like genes.
a duplication of the retrotransposed gene in the ancestor to b. mori and different fates for the ancestral gene copies in the two groups would bring about the present situation.
alternatively, we do not exclude that independent retrotranspositions happened in lepidopteran and dipteran ancestral lineages.
interestingly, the nested arg/elav arrangement found in d. melanogaster is not conserved in the mosquitoes, where the host gene  became intronless.
this parallels the nested arrangement of the intronless sina gene in an intron of the rh <dig> gene, as found in mosquitoes and nine species of the drosophila genus.
the remaining three species of the genus have an intronless rh <dig>  with a loss of the ancestral rh <dig> copy where sina was originally embedded  <cit> .
these situations show the lability of nested gene arrangements.
elav: the genesis of a new function
it was unexpected to find that the copy number of elav family members varied from species to species.
given the maintenance of this gene family in all metazoans, we assume that there is a function for at least one, if not all, of the genes in each species.
mutants have been reported in only three species.
the knockout of neuronal hud in mice causes motor and sensory defects  <cit> .
it is not excluded that the mild phenotype of this mutant is the consequence of gene redundancy.
in c. elegans, cholinergic synaptic transmission is altered in mutants of the single elav ortholog exc- <dig>  which is expressed in a subset of neurons and other non-neuronal cells  <cit> .
in both cases, viability and apparent morphology are normal.
in drosophila melanogaster, the vital gene elav is required in all neurons  <cit> , whereas rbp <dig> is essential for female fertility  <cit>  but does not affect viability.
we recently generated null mutations of the fne gene , whose preliminary analysis indicates that they are viable in adults and lead to no apparent morphological defects.
aside from elav itself, characterized mutations of the elav gene family are viable, suggesting a non-vital function of the ancestral gene.
considering that elav appears to be a new member of the family, its vital function is quite striking.
this situation is reminescent of that of sex-lethal , a gene fundamental to sex determination in drosophila, but which does not act as a sex determining factor in non-drosophilids.
the drosophilid genomes indeed contain two sxl paralogs , while non-drosophilids have one.
it has been proposed that there was a duplication of the ancestral gene in drosophilids and acquisition of a new function by one of the copies  <cit> .
we believe that a retrotransposition of the elav/fne/rbp <dig> ancestor gene at the time of the separation of dipterans/lepidopterans led to a gene duplication and the evolution of a new function for elav.
conserved rna binding proteins: a reservoir for accelerated functional evolution
we have pointed out that the elav-like proteins, including elav itself, have maintained a high level of sequence conservation between species, higher than that of engrailed, a conserved transcription factor with a homeodomain, or that of arginase, a ubiquitous metabolic enzyme that arose before the divergence of procaryotes and eucaryotes.
this is intriguing in light of the extensively documented diversity of the properties of individual members of the family.
first, although there is expression in the nervous system of at least one of the elav family members in every investigated metazoan , expression is also detected in other tissues and is even sometimes ubiquitous  <cit> .
second, the functions of these proteins are multiple, whether at the cellular level, where they include cell differentiation/survival  <cit>  and cell proliferation/control of the cell cycle  <cit>  or at the biological level, with impacts on motor/sensory activity, memory, fertility or viability  <cit> .
finally, the apparent subcellular localization of these proteins is diverse , in agreement with diverse molecular functions  <cit> .
the data thus reveal a diversification of the functions and of the specificity of expression of elav family members and implies a diversification of the interactions with other macromolecules, most evidently the rnas whose metabolism is regulated by the rrm containing proteins.
the dna duplications and retrotranspositions that occured in the elav gene families constitute a starting point for the diversification of gene function.
changes in cell or tissue specificity of expression are often linked to modifications of non-translated regulatory regions.
however, changes affecting the sub-cellular localization, known to be dependent upon the hinge region between rrm <dig> and rrm <dig>  or changes in the interactions with proteins or rna must depend upon the protein product of the elav-like genes.
sequence alignments of the elav-like proteins shows that they are overall very conserved.
but we were puzzeld by the fact some of the conserved exon junctions  are adjacent to sequences that are among the most variable of the proteins.
they include short insertions of amino acids,  exon addition and amino acid variations.
the intron sequence indeed provides a potential source of sequence variability: it is conceivable that intron extremities become integrated into coding sequences by shifting of the exon boundaries.
alternatively, the intron can serve as the site of insertion of a new exon.
an additional surprising point was the fact that these variable micro regions are almost directly upstream of important conserved motifs, specifically rnp- <dig>  and the octapeptide in the region essential for nuclear export and subcellular localization.
the modification of residues outside of the rnp has the potential to alter the interactions between the rrm and an rna  <cit> .
additionally, alterations of the region responsible for nuclear export/cellular localization modify this function .
we thus propose that the maintenance of the exon junctions is vital to the evolution of the elav family, in particular the generation of new functions.
as a consequence, one would predict that rrm <dig>  rrm <dig> and the hinge region have prominent roles in functional specificity.
it may be significant in this respect that rrm <dig> replacements in elav by rrm <dig> from rbp <dig> or hud are fully functional, while rrm <dig> or rrm <dig> replacements by corresponding rrms from rbp <dig> or sxl are largely non-functional  <cit> .
more generally, it seems that rrm-containing proteins could serve as favorable targets for the rapid evolution of gene functions.
because of the structural versatility of the rrm domain, it can be adapted for sequence specific recognition of many different nucleic acid structures and different protein partners  <cit> .
the sxl protein, a crucial regulator of sex determination in drosophila contains  <dig> rrm, and appears to be the result of such a rapid adaptation of function.
in the search for genetic changes that distinguish our brains from that of our ancestors, the focus has been on the identification of non-synonymous changes in coding regions and the modification of regulatory sequences  <cit> .
our work suggests that the very conserved rrm-containing proteins may have contributed to human brain evolution, especially when considering the fundamental importance of the regulation of rna metabolism in neurons, where alternative splicing  <cit>  and localized rna translation and degradation  <cit>  take place with impacts on cortex development, neuronal regeneration and plasticity.
CONCLUSIONS
the elav gene family encodes proteins with three rna recognition motifs  acting as neuronal post-transcriptional regulators in all metazoans.
since they show remarkable sequence conservation, the documented diversity of their molecular roles is unexpected.
we report the occurence of elav-like gene duplications and deletions in metazoans, and show that the vital elav gene of drosophila is newly emerged, specific to dipterans and of retrotransposed origin, challenging its status of prototype for the family.
these findings, together with the plasticity of the interactions between rrm and rna, suggests that the elav-like proteins may have played an important role in the evolution of the gene functions crucial in brain evolution.

 the elav gene of d. melanogaster was the the first identified member of a family of neuronal rna binding proteins that is conserved in metazoans. the proteins in this family contain three rna recognition motifs, with a hinge region separating the second and third rrms and an optional non-conserved n-terminal region. the hinge includes signals essential for nuclear export and subcellular localization. rrm are common protein domains found in all life kingdoms. in humans, there are genes encoding rrm containing proteins, which represent 2% of the human gene products. proteins containing one or several of these domains are capable of interacting in a sequence specific manner with single stranded rna molecules and of directing the assembly of multiprotein complexes. in spite of the remarkable sequence conservation of the rrm domains, rrm-containing proteins perform numerous functions, intervening at all the possible steps of rna metabolism. the rrm domain is composed of about amino acids and contains a conserved octapeptide termed rnp- and a conserved hexapeptide termed rnp- structural studies indicate that four antiparallel beta-sheets form the rna interaction surface, with rnp- and rnp- on the two inner sheets. in rna-rrm complexes, nucleotides establish contacts with residues in the rnps, with regions in the rrm beyond the rnp domains also involved in rna recognition. the plasticity of rrms in their sequence-specific recognition of topologically diverse rna is likely to be correlated with their presence in a variety of proteins involved in the diverse steps of post-transcriptional regulation. there are three elav-related genes in d. melanogaster. the elav gene encodes a nuclear product present in all neurons throughout development and is required for the differentiation of postmitotic neurons and their maintenance. the rbp product is present in neuronal nuclei starting at the third larval instar and also in the cytoplasm of cystocytes during oogenesis. although neuronal expression is predominant, rbp mutations reveal a role in cystocyte proliferation and differentiation, but no neuronal defects have been reported. the expression of fne resembles elav's, but with a slightly delayed onset. fne is cytoplasmic, but the elav and fne genes interact, suggesting protein shuttling. the products of elav family members are essentially present in the nervous system, in all of the neurons in the case of elav itself, but more generally in subsets of neurons and/or neuroblasts and glial cells. expression has also been detected in other tissues, in particular in testes and ovaries, or found to be ubiquitous. diverse molecular functions in the control of rna half life, nuclear export, rna 3' end formation, alternative rna processing, polyadenylation and translation have been proposed for these proteins. multiple functions, both cytoplasmic and nuclear have been demonstrated for hur, an ubiquitously expressed member of the human family. the evolutionary relationship between members of the family are complex. for instance, the four human proteins share 74–91% identity, while the three drosophila proteins share only 59–68% identity. the goal of the work reported here was to investigate these relationships. we found that the elav family has an eventful evolutionary history, somewhat masked by the high level of amino acid conservation of the gene products, but revealed by analysis of the gene structure of the different family members. we attribute the rapid functional evolution of the family members, as opposed to the high level of sequence conservation, to the plasticity of the rrm domains, where small changes in critical positions have the potential to modify interactions with rna.  the paralogs fne and rbp share a conserved organization of their coding regions but elav, the third family member, is distinct all three drosophila paralogs elav, rbp and fne are essentially expressed in neurons. elav null mutants are embryonic lethal, while the rbp null mutation is viable, but surprisingly confers a female sterility phenotype. fne null mutants, although not fully characterized, are also viable. in order to understand the evolutionary mechanisms responsible for the generation of these paralogs, we examined their gene structure. although their organizations are apparently quite distinct, we found remarkable conservation in the correspondance between exons and specific protein regions in rbp and fne. there are two differences the presence of new mini-exons respectively specific for each of the two genes and the use of a single exon in fne but two in rbp to encode the third rrm. strikingly, this organization is totally different in the elav gene, whose complete orf, except for the a of the atg initiation codon, is encoded by a single exon. conserved exon junctions are present in most elav orthologs we took advantage of the recent sequencing of complete genomes to survey the gene family in species by identifying all the family members and comparing the organization of the orf in exons. in humans, d. melanogaster and c. elegans, we extracted from the databases protein sequences deduced from cdna analyses, and aligned genomic dna with cdna to determine the exon-intron structure. in other cases we used the predicted protein sequences, either published or computed for our purpose, as detailed in the methods. we examined species from the chordata, arthropoda and nematoda, for a total of genes. first, we found that the size of elav families varies among the species that we studied, with no clear relationship between family size and brain/animal complexity. for instance, dipterans possess three elav genes, while the hymenopteran apis mellifora, with ten times as many neurons as drosophila, possesses only one gene. levels of identity between the proteins encoded by the genes are high, with the lowest score obtained in the comparison of d. melanogaster elav with the unique c. elegans protein. between humans and drosophila, there is 54–64% amino acid identity in the elav-related proteins, 38% identity for the arginase proteins and 33% identity for the engrailed proteins. the levels of elav-related protein identity are thus remarkable. the crystal structure of the first two rrm of human hud associated with cfos rna, identifies amino acids whose side chain is making direct rna contacts. these residues are conserved in all elav-like proteins that we examined, except for the arginine in rnp of the second rrm, which appears to be specific to the human proteins and to one of the b. mori elav-like, bm- in the other species there is a conserved substitution by a lysine. second, we found remarkable conservation of exon structure. from vertebrates to invertebrates, we identified eight exon junctions in the rrms/hinge region. we named them j to j from the most upstream to the most downstream. all are present in several phyla, except for j and j which are specific for fne and rbp from drosophila and are implicated in the generation of mini-exons in the sequence coding these proteins. overall, the j junctions are unambiguously homologous since the level of protein sequence conservation is such that the amino acid positions where the junctions intervene are clearly aligned and conserved and nucleotide sequence analysis shows that at a given exon junction, the splice is at the same position in the codons: specifically between the first and the second bases of the spliced codons or exactly between codons. there are two exceptions to this strict conservation. first, j is interrupted in rbp of d. melanogaster by the intronic insertion of an alternative mini-exon, without alteration of the j 5' or 3' splice sites. second, in fne, j is split by the intronic insertion of a mini-exon, the j donor splice site is additionally shifted downstream while the j acceptor splice site is maintained. interestingly, the junctions j and j occupy the same position relative to rnp- in rrm and rrm the junctions j and j map in a moderately conserved coding region, essential for nuclear export and proper subcellular localization, including only a conserved hexamer. both j and j split the spliced codons between the second and the third bases. in this region, three types of events affecting the splicing seem to have occured independently: 1) the introduction of a mini-exon, that can be alternatively spliced, the shift of the 5' splice site the shift of the 3' splice. noticeably, the regions close respectively to j1/j j4/j and j6/j as well as the entire hinge region between rrm and rrm appear more variable than the rest of the protein. intronless elav-like genes are present in diptera and lepidoptera interestingly, for six of the analyzed genes, the entire conserved region of the protein is encoded by a single exon. based upon both their gene structure and the level of protein sequence identity, the dipteran intronless genes constitute a homogeneous elav-type group. in contrast, although intronless like elav, the b. mori genes encode proteins more similar to fne/rbp than to elav. this observation suggests that distinct evolutionary forces shaped the b. mori genes and the dipteran elav-like intronless-genes, respectively. to evaluate this hypothesis, we performed a phylogenetic analysis of the elav orthologs/paralogs, using the upgma algorithm, with bootstrap analysis. this analysis shows with high confidence that in dipterans, the proteins encoded by the intronless genes cluster together, while the two b. mori genes products cluster with the fne/rbp sequences similar results were obtained when performing sequence alignments using the neighbor joining method. because the d. melanogaster elav gene is nested in the third intron of the arginase gene, we probed the gene environment of the intronless elav orthologs that we report here. we found that the nested elav/arg gene organization is unique to drosophila, specifically d. melanogaster and additional drosophila species whose genomes have recently been sequenced. in the other non-drosophila species examined here, there is no close linkage between the arginase gene and the elav gene family members. in particular, the mosquitos, similar to d. melanogaster, each have three elav-like genes, including one intronless version, but unlike d. melanogaster they have an intronless arg, which obviously rules out the possibility of a nested gene. in b. mori, although the two intronless bm- and bm- genes map at loci distinct from the arg locus, an intron putatively homologous to the third intron of the d. melanogaster arginase gene is present. discussion the d. melanogaster gene elav is specific to the dipteran phylum and results from retrotransposition the elav gene from drosophila was the first identified member of this family, is considered as its prototype, and most of the subsequently discovered orthologs are named after it. however, the present analysis highlights unique characteristics of this gene that suggest it is of recent evolutionary origin, after the separation of dipterans and lepidopterans. aside from elav, only the dipteran genes ae- ag- and cp- encode proteins that are more similar to elav than to fne and rbp in addition to the intronless elav-likes, dipteran genomes carry two genes encoding proteins of the type fne/rbp also found in the seven other genomes analyzed. thus elav, ae- ag- and cp- represent a newly evolved gene form specific to dipterans. in addition, the elav gene structure is suggestive of retrotransposition, a process considered significant in the evolution of genomes, including drosophila. the genes ae- ag- and cp- from mosquitoes share with elav not only a higer level of similarity between their products, but also the property of having their orf in a single exon. the absence of introns is atypical: we identified conserved exon junctions that are a landmark present in most of the elav-related genes. furthermore, the elav gene of drosophila is nested in the arginase gene. in humans, retrotransposition is an important contributor to the generation of nested genes. we thus propose that elav originated from a recent retrotransposition event. it is possible that the same retrotransposition is at the origin of both the lepidopteran intronless fne/rbp9-like genes and the dipteran elav-like genes. a duplication of the retrotransposed gene in the ancestor to b. mori and different fates for the ancestral gene copies in the two groups would bring about the present situation. alternatively, we do not exclude that independent retrotranspositions happened in lepidopteran and dipteran ancestral lineages. interestingly, the nested arg/elav arrangement found in d. melanogaster is not conserved in the mosquitoes, where the host gene became intronless. this parallels the nested arrangement of the intronless sina gene in an intron of the rh gene, as found in mosquitoes and nine species of the drosophila genus. the remaining three species of the genus have an intronless rh with a loss of the ancestral rh copy where sina was originally embedded. these situations show the lability of nested gene arrangements. elav: the genesis of a new function it was unexpected to find that the copy number of elav family members varied from species to species. given the maintenance of this gene family in all metazoans, we assume that there is a function for at least one, if not all, of the genes in each species. mutants have been reported in only three species. the knockout of neuronal hud in mice causes motor and sensory defects. it is not excluded that the mild phenotype of this mutant is the consequence of gene redundancy. in c. elegans, cholinergic synaptic transmission is altered in mutants of the single elav ortholog exc- which is expressed in a subset of neurons and other non-neuronal cells. in both cases, viability and apparent morphology are normal. in drosophila melanogaster, the vital gene elav is required in all neurons, whereas rbp is essential for female fertility but does not affect viability. we recently generated null mutations of the fne gene, whose preliminary analysis indicates that they are viable in adults and lead to no apparent morphological defects. aside from elav itself, characterized mutations of the elav gene family are viable, suggesting a non-vital function of the ancestral gene. considering that elav appears to be a new member of the family, its vital function is quite striking. this situation is reminescent of that of sex-lethal, a gene fundamental to sex determination in drosophila, but which does not act as a sex determining factor in non-drosophilids. the drosophilid genomes indeed contain two sxl paralogs, while non-drosophilids have one. it has been proposed that there was a duplication of the ancestral gene in drosophilids and acquisition of a new function by one of the copies. we believe that a retrotransposition of the elav/fne/rbp ancestor gene at the time of the separation of dipterans/lepidopterans led to a gene duplication and the evolution of a new function for elav. conserved rna binding proteins: a reservoir for accelerated functional evolution we have pointed out that the elav-like proteins, including elav itself, have maintained a high level of sequence conservation between species, higher than that of engrailed, a conserved transcription factor with a homeodomain, or that of arginase, a ubiquitous metabolic enzyme that arose before the divergence of procaryotes and eucaryotes. this is intriguing in light of the extensively documented diversity of the properties of individual members of the family. first, although there is expression in the nervous system of at least one of the elav family members in every investigated metazoan, expression is also detected in other tissues and is even sometimes ubiquitous. second, the functions of these proteins are multiple, whether at the cellular level, where they include cell differentiation/survival and cell proliferation/control of the cell cycle or at the biological level, with impacts on motor/sensory activity, memory, fertility or viability. finally, the apparent subcellular localization of these proteins is diverse, in agreement with diverse molecular functions. the data thus reveal a diversification of the functions and of the specificity of expression of elav family members and implies a diversification of the interactions with other macromolecules, most evidently the rnas whose metabolism is regulated by the rrm containing proteins. the dna duplications and retrotranspositions that occured in the elav gene families constitute a starting point for the diversification of gene function. changes in cell or tissue specificity of expression are often linked to modifications of non-translated regulatory regions. however, changes affecting the sub-cellular localization, known to be dependent upon the hinge region between rrm and rrm or changes in the interactions with proteins or rna must depend upon the protein product of the elav-like genes. sequence alignments of the elav-like proteins shows that they are overall very conserved. but we were puzzeld by the fact some of the conserved exon junctions are adjacent to sequences that are among the most variable of the proteins. they include short insertions of amino acids, exon addition and amino acid variations. the intron sequence indeed provides a potential source of sequence variability: it is conceivable that intron extremities become integrated into coding sequences by shifting of the exon boundaries. alternatively, the intron can serve as the site of insertion of a new exon. an additional surprising point was the fact that these variable micro regions are almost directly upstream of important conserved motifs, specifically rnp- and the octapeptide in the region essential for nuclear export and subcellular localization. the modification of residues outside of the rnp has the potential to alter the interactions between the rrm and an rna. additionally, alterations of the region responsible for nuclear export/cellular localization modify this function. we thus propose that the maintenance of the exon junctions is vital to the evolution of the elav family, in particular the generation of new functions. as a consequence, one would predict that rrm rrm and the hinge region have prominent roles in functional specificity. it may be significant in this respect that rrm replacements in elav by rrm from rbp or hud are fully functional, while rrm or rrm replacements by corresponding rrms from rbp or sxl are largely non-functional. more generally, it seems that rrm-containing proteins could serve as favorable targets for the rapid evolution of gene functions. because of the structural versatility of the rrm domain, it can be adapted for sequence specific recognition of many different nucleic acid structures and different protein partners. the sxl protein, a crucial regulator of sex determination in drosophila contains rrm, and appears to be the result of such a rapid adaptation of function. in the search for genetic changes that distinguish our brains from that of our ancestors, the focus has been on the identification of non-synonymous changes in coding regions and the modification of regulatory sequences. our work suggests that the very conserved rrm-containing proteins may have contributed to human brain evolution, especially when considering the fundamental importance of the regulation of rna metabolism in neurons, where alternative splicing and localized rna translation and degradation take place with impacts on cortex development, neuronal regeneration and plasticity.  the elav gene family encodes proteins with three rna recognition motifs acting as neuronal post-transcriptional regulators in all metazoans. since they show remarkable sequence conservation, the documented diversity of their molecular roles is unexpected. we report the occurence of elav-like gene duplications and deletions in metazoans, and show that the vital elav gene of drosophila is newly emerged, specific to dipterans and of retrotransposed origin, challenging its status of prototype for the family. these findings, together with the plasticity of the interactions between rrm and rna, suggests that the elav-like proteins may have played an important role in the evolution of the gene functions crucial in brain evolution. 
we propose that the conserved exon junctions constitute potential sites for sequence/function modifications, and that rrm binding proteins, whose function relies upon plastic rna-protein interactions, may have played an important role in brain evolution. remarkably, three of the conserved exon junctions are both close to structural elements, involved respectively in protein-rna interactions and in the regulation of sub-cellular localization, and in the vicinity of diverse sequence variations. in an attempt to understand the history of this family, we searched for orthologs in eleven completely sequenced genomes, including those of humans, d. melanogaster and c. elegans, for which cdnas are available. the remaining genes show remarkable conservation of their exon organization, and their products most resemble fne and rbp proteins encoded by the two elav paralogs of drosophila. its members encode structurally conserved neuronal proteins with three rna recognition motifs but they paradoxically act at diverse levels of post-transcriptional regulation. the drosophila gene embryonic lethal abnormal visual system is the prototype of a gene family present in all metazoans.
 the drosophila gene embryonic lethal abnormal visual system is the prototype of a gene family present in all metazoans. its members encode structurally conserved neuronal proteins with three rna recognition motifs but they paradoxically act at diverse levels of post-transcriptional regulation. in an attempt to understand the history of this family, we searched for orthologs in eleven completely sequenced genomes, including those of humans, d. melanogaster and c. elegans, for which cdnas are available.  we analyzed orthologs/paralogs of elav, and found evidence of gain/loss of gene copy number. for one set of genes, including elav itself, the coding sequences are free of introns and their products most resemble elav. the remaining genes show remarkable conservation of their exon organization, and their products most resemble fne and rbp proteins encoded by the two elav paralogs of drosophila. remarkably, three of the conserved exon junctions are both close to structural elements, involved respectively in protein-rna interactions and in the regulation of sub-cellular localization, and in the vicinity of diverse sequence variations.  the data indicate that the essential elav gene of drosophila is newly emerged, restricted to dipterans and of retrotransposed origin. we propose that the conserved exon junctions constitute potential sites for sequence/function modifications, and that rrm binding proteins, whose function relies upon plastic rna-protein interactions, may have played an important role in brain evolution. 