BACKGROUND
there is an ancient  <cit>  but still active debate in the molecular biologist community about the relative contribution of structural genomic modifications  <cit>  that could account for the phenotypic differences observed between primate species, particularly in the emergence of new brain structure and functions  <cit> .
very debated results were found when determining the ka/ks ratio, a tentative indicator of positive darwinian selection, in the coding region of genes expressed in the mammalian brain  <cit> .
however, genome-wide comparative studies of mammalian promoters suggested an accelerated evolution of primate promoters during the last  <dig> million years  <cit> .
recently, divergence between human and chimpanzee sequences have been re-evaluated to almost 5%, resulting mainly from indel events  <cit>  and copy number variants  that strongly contributed in shaping primate genomes  <cit> , offering therefore a wide variety of sites at which primate lineage-specific genetic novelty could happen.
indeed, recent segmental duplications are particularly enriched in genes that display expression differences between humans and chimpanzees  <cit> .
in addition, an accelerated rate of contraction or expansion in gene families, including brain-expressed genes, operated in primates when compared with other mammals  <cit> .
on a larger scale, cnvs contributed significantly to diverse expression phenotypes in primates  <cit>  and to emergence of complex or sporadic diseases in humans  <cit> .
to reconcile apparently conflicting data, we previously proposed that, in parallel to single nucleotide mutations that confer alterations in the gene expression patterns or amino acids sequences, genomic rearrangements may have played an important role during primate evolution, providing creation of novel but rare regulatory modules as well as protein coding and/or non-coding genes  <cit> .
indeed, combination of exon shuffling, retrotransposition and gene promoter fusion have led to genes harbouring completely new structures and expression patterns selectively in the primate lineage .
these rare events would have been nevertheless particularly important in shaping human genes found expressed in reproductive organs, as exemplified by the chimeric pote-actin genes  <cit> , or involved in hominoid brain neurotransmission, as exemplified by the glud <dig> gene  <cit> .
the study of primate-specific gene creation and early evolution requires the discovery of genes that have retained characteristic features of their youth  <cit> .
the pmchl system, which combines the retroposition/exon shuffling and the segmental duplication models, has been one of the first hominoid-specific gene creation model described  <cit> .
we have shown that these genes have been created in the hominoid lineage through i) retroposition at the ancestral chromosome 5p <dig> locus in catarrhini of an antisense pro-melanin-concentrating hormone  gene transcript, ii) local rearrangement leading to a truncated version of the retrogene, iii) sequence remodelling  and iv) final duplication at the ancestral 5q <dig> locus in hominids.
furthermore, processed and unprocessed transcripts were characterized in a human fetal brain library  <cit>  as well as in developing human brain  <cit> .
these mrnas were found to encode a putative nuclear protein of  <dig> kd, named vmch-p <dig>  that was only identified using in vitro translation systems or transfected cell models  <cit> .
however, many questions remained unsolved regarding the region and time of insertion of the retrogene, the fine structure of both genes , their expression patterns  and their protein coding potential in human cells.
in this paper, we established the structure of pmchl1/pmchl <dig> genes and demonstrated that alternatively spliced transcripts encompassing exons  <dig> to  <dig> are mostly expressed in human testis.
we established further the evolutionary history and regional organization of pmchl genes at both loci on human chromosome  <dig> and proposed that a single retroposition event followed by point mutations provided novel exonic sequences in transcriptional sense direction.
several short open reading frames  were found encoded within the spliced pmchl rnas but most of them were not conserved in the primate lineage.
this suggests a lack of overt functionality of these orfs, even though the existence of a small species-specific protein cannot be ruled out.
finally, we attempted to identify proteins generated from pmchl genes in macaque and human tissues with an antiserum directed against vmch-p <dig> and its variants but we failed to detect them.
therefore, spliced transcripts from the primate-specific pmchl1/pmchl <dig> genes would likely represent mrna-like non-protein coding rnas .
RESULTS
<dig>  structure and distribution of spliced pmchl transcripts in human brain and testis
in our previous study  <cit> , we characterized several alternatively spliced transcripts harbouring exons  <dig> to  <dig> of the original pmchl1/pmchl <dig> genes.
six transcripts corresponding to pmchl <dig> spliced rna were found in testis and/or fetal brain.
two pmchl <dig> spliced rna were reported only in testis.
in order to further precise the exon/intron structure of pmchl genes and to further investigate the tissue distribution of spliced pmchl transcripts, we examined the presence of additional rnas in human testis and cortex marathon cdna libraries, as well as in a human fetal prefrontal cortex sample.
for this, we performed pcr experiments  using primer pairs designed to amplify  transcripts encompassing the most distant known exons, previously named exons  <dig> and  <dig>  sub-cloning of the pcr products and sequencing of individual clones allowed the discovery of a novel exon, located between exon  <dig> and former exon  <dig>  it was named exon  <dig> and previously named exons  <dig>   <dig> and  <dig> are now renamed exons  <dig>   <dig> and  <dig> .
we identified six pmchl <dig> spliced variants in adult testicular marathon cdna library , two of which  had already been identified in the same cdna library .
however, transcript  bears one a to g mutation within exon  <dig>  resulting in an arginine to glycine mutation in orf <dig>  noteworthy, transcript  corresponds also to two testis ests , and was also found in a human fetal brain marathon cdna library in our previous study  <cit> .
this apparently abundant transcript harbours exons 1-2b and 6a, like most spliced transcripts in testis which were obtained in a single round of pcr.
in contrast, transcripts harbouring exon 6b were identified after two rounds of pcr and never contained exon 6a.
transcripts containing exon 2a were never observed in the present study.
we also identified four novel alternative pmchl <dig> splicings in adult testis, which all contained a partial exon  <dig>  shorter than the original exon  <dig>  observed in a pmchl <dig> transcript .
sequence analysis  revealed that mutations in the pmchl <dig> sequence created a novel gene-specific splice donor site  which is systematically used in the present pmchl <dig> transcripts.
we further identified one novel pmchl <dig> splice variant in a fetal prefrontal cortex sample .
this transcript was the only one to harbour an alternative splice donor in exon  <dig>  which was never observed in testis rnas.
in our previous study  <cit> , we reported two alternative acceptor sites in exon 5b  separated by only four nucleotides and with apparent testis- and brain-specificities .
our present results show that most testis transcripts use the exon 5a splice acceptor site, and two use the alternative 5bt site.
however, the pmchl <dig> transcripts identified in fetal brain also use the alternative 5bt site indicating that it could not be considered anymore as a testis-specific splice acceptor site.
the alternative 5bb site, previously reported in a fetal brain transcript was not found in our present study.
in contrast to human testis and fetal brain, we could not detect any spliced pmchl rna harbouring exons  <dig> and 6a in the human adult cortex marathon cdna library using the 3–7/3– <dig> primer pair.
this primer pair was further used in rt-pcr experiments combined with southern blot to determine the tissue distribution of the spliced transcripts in testis, prefrontal cortex and cerebellum in adult human and macaque .
in agreement with our results using the marathon cdna libraries, we detected spliced pmchl rna harbouring exons  <dig> and 6a in human adult testis, but not in adult prefrontal cortex and cerebellum.
thus, pmchl <dig> transcripts are found in testis and fetal brain and are more abundant than pmchl <dig> transcripts that are observed only in testis.
in addition, pmchl <dig> gene expression was reported in ht <dig> cells subjected to rage , in which an est  encompassing exons 2a and  <dig> of pmchl <dig> has been identified.
in macaque, no spliced transcripts were identified by southern blot  in agreement with our sequence analysis indicating that the macaque pmchl <dig> gene lacks the exon 6a acceptor splice site .
taken together, our findings indicate that pmchl <dig> and pmchl <dig> genes: i) give rise to a complex pattern of alternative splicings, ii) are subject to distinct tissue-specific expressions and iii) are developmentally regulated .
the finding that a rather high diversity of spliced transcripts are present in testis is not surprising, because of the permissive chromatin environment present in gonads, allowing high transcriptional activity even from weak tissue-specific promoters  <cit> .
thus, most retroposons evolve into non-functional pseudogenes that are transcribed only in the testis  <cit> .
however, the abrupt emergence of a new chimeric gene in primates could potentially contribute to reproductive barriers and thus play a role in speciation  <cit> .
in this regard the hominoid-specific oncogene tre <dig> appears expressed only in testis while the two parental genes usp <dig> and tbc1d <dig>  that fused to generate the tre <dig> gene, are expressed in a broad range of human tissues  <cit> .
in addition, the presence of spliced pmchl transcripts in fetal brain, is rather suggestive of a functional role during human brain development.
this would imply that the retroposon acquired an active promoter and has been subjected to selection pressure.
whether these spliced pmchl transcripts actually play a functional role in testis and fetal brain is an obvious question, which we further addressed below.
<dig>  evolutionary history of the pmchl genes
retroposition-driven creation of pmchl <dig> gene occurred 30– <dig> mya in primate lineage
to gain further insights into the evolutionary history of the pmchl gene family, we extended our previously initiated sequence analysis of pmchl and pmch genes.
for this, genomic dnas were pcr amplified with the pmchl- or pmch-specific primer pairs indicated in table  <dig> and the pcr products were sequenced.
novel sequences were submitted to genbank under the following accession numbers.
for pmchl1:  ,  ,  ,  ,  ,  ,  .
for pmch:  ,  ,  ,  ,  ,  ,  ,  ].
annealing temperature  that was used for pcr and gene-specificity are indicated for each oligonucleotide pair.
we first carried out the phylogenetic analysis of the exon/intron boundaries of the pmchl genes .
consensus gt/ag splice donor and acceptor sites are present in all species bearing the retroposon, i.e.
all catarrhines of this study, with the exception of the splice acceptor site of exon 6a which is present only in hominoids.
moreover, the exon 5c donor site and the 6a and 6b splice acceptor sites were not consensual in the platyrrhines analysed here, i. e. before the retroposition event, suggesting that canonical splicing could not occur in this ancestral region before the arrival of the retroposed sequence.
our sequence analysis showed that these splice sites were created de novo through single nucleotide mutations.
thus, the splicing between exons  <dig> and  <dig> corresponds to de novo exonisation and not to an alu-driven exonisation mechanism  <cit> , exon  <dig> being absent in platyrrhines and then conserved through selection pressure.
in contrast, splice sites of exons  <dig> and  <dig> pre-dated the retroposition event, indicating that a fusion of the pmch-derived exons with pre-existing exons is likely to have occurred.
whether these exons were expressed before the retroposition remains to be determined, but no expressed sequence tag corresponding to exons  <dig> and  <dig> alone could be identified in mammalian est databases.
furthermore, chimeric transcripts formed by transcription of two consecutive genes into a single rna can occur quite frequently in human cells  <cit> .
a similar mechanism may be involved in the production of fused transcripts encompassing either exon  <dig> or  <dig> of pmchl1/pmchl <dig> genes.
however, further characterization of putative promoters inside the pmchl genes needs to be done before involving such rna domain accretion process on regulation of these genes.
a nucleotidic phylogenetic analysis  was performed to date more precisely the retroposition event.
as pmchl genes encompass part of the pmch gene sequence , it was possible to align primate sequences of these specific parts of the pmchl <dig> and pmch genes.
the phylogenetic analysis was performed using the parsimony, maximum-likelihood and neighbour-joining methods and with the rat and mouse pmch sequences as outgroups .
all phylogenetic methods led to congruent data, with high bootstrap values with the neighbour joining method.
the tree showed an apparent aberration relative to our present knowledge concerning the relationship between species and the creation of the pmchl <dig> gene .
we expected the pmch sequences of cebus capucinus and saguinus oedipus to be grouped with the pmch sequence of tarsius syrichta rather than with the pmchl sequences.
however, a noteworthy low bootstrap value  was found for this branching.
the position of the pmchl sequences as well as the uncertainty for positioning the cebus capucinus and saguinus oedipus pmch sequences suggest that the retroposition event leading to the pmchl <dig> gene occurred very shortly  after the split of platyrrhini/cathyrrhini, i.e.
30– <dig> mya  <cit> .
when focusing on the pmchl genes, in the regions encompassing exons 1-2b  and exons 5– <dig>  the nucleotidic phylogenetic trees fully correspond to accepted species trees, indicating that no particular and global  evolutionary event interfered.
however, we observed a difference in the pan troglodytes/pan paniscus/homo sapiens positioning between the two phylogenetic trees.
this simply corresponds to an inherent irresolution in the hominidae speciation, which could be inferred  from a complex speciation with interbreeding before final separation of chimpanzees, gorillas and humans  <cit> .
all three species should probably be grouped under the pan or homo clade as previously suggested  <cit> .
we previously reported the presence of a complete alu-sq sequence element within pmchl exon  <dig>  <cit> , but the insertion event into pmchl <dig> could not be dated precisely.
it is worth noting that this sequence appears to be an alusg, and not an alu sq element.
our present sequence analysis reveals that all primate species carrying the pmchl <dig> exon  <dig> harbour the alusg sequence .
thus, the insertion was likely concomitant to the retroposition, occurring after the divergence of cebus species , and before the divergence of the cercopithecoids, approximately between 30– <dig> mya.
a  <dig> kb element encompassing pmchl <dig> and adjacent intronic/exonic sequence of cdh <dig> on 5p <dig> duplicated to create pmchl <dig> on 5q <dig> at the time of hominid divergence
we previously proposed  <cit>  that pmchl <dig> was created from a duplication of a large, but undefined in size, genomic dna fragment comprising pmchl <dig>  "jumping" from ancestral hominid chromosome 5p <dig> to 5q <dig>  here, we precisely determined the limits of the duplicon by similarity using a blast search  <cit> .
it appeared that a fragment of  <dig> kb, encompassing  <dig> kb upstream and  <dig> kb downstream of the  <dig> kb of pmchl <dig> was duplicated .
this  <dig> kb duplicon corresponds to a large part  of the 5' portion of intron  <dig> of the cdh <dig> gene encoding brain cadherin , as well as cdh <dig> exon  <dig> and the last  <dig> kb of its intron  <dig>  no other exons  than those derived from the pmch gene were found in the duplicon.
the percentages of identity between the 5p and 5q elements were equivalent all along the duplicon , and are close to 98%, in agreement with a very recent duplication event.
we have therefore precisely mapped the limits of the pmchl1/pmchl <dig> duplicon.
a question concerning the creation of novel genes, particularly in the case of segmental duplication, is relative to the acceptor sites.
which kind of sequences allows invasion by foreign sequences?
which kind of sequences can we find at the segmental duplication boundaries?
no clear boundaries specificities are described for segmental duplication except for a significant enrichment in short interspersed elements  such as young alu y and alu s sequences and other repeats similar to these involved in ig heavy chain recombination in pericentromeric and interstitial segmental duplications  <cit> .
alu mediated dna duplications have exceptionally been reported in eukaryotes  <cit> .
these duplications appeared however to affect mainly hyper-recombinogenic chromosomal regions, and particularly for secondary duplications  <cit> .
long interspersed elements  like line  <dig> elements were also directly  implicated in exon recombination and have been proposed to mediate exon shuffling  <cit> , but none of the previously described human chimeric genes  <cit>  harbour this kind of element at its boundaries.
in the case of pmchl1/l <dig>  no particular sines or lines sequences could be found at the boundaries, neither at the first insertion site of the pmch antisense retroposon  gene at the 5p <dig> locus), nor at the 5q <dig> locus when creating pmchl <dig>  recently, a duplication-driven model for dna transposition has been put forth by eichler's group  <cit>  suggesting that the probability for a dna element to be duplicated correlated with the degree of proximity to so-called core duplicons.
in this context, a core duplicon named glu5– <dig> and corresponding to a truncated version of the gusb gene, has been found in close vicinity to pmchl <dig> and pmchl <dig> genes on 5p <dig> and 5q <dig> respectively  <cit> .
whether the proximity with this duplicon was determinant for emergence of the pmchl <dig> gene remains at this stage a matter of speculation but the timing of both glu5– <dig> duplicon expansion and intrachromosomal duplication of pmchl <dig> in primates fits very well.
these duplication events are also congruent with the global surge in intrachromosomal duplications at the time of hominid divergence, as previously predicted  <cit>  and experimentally proven  <cit> .
<dig>  analysis of the protein coding potential of pmchl transcripts
we next addressed the protein coding potential of the spliced pmchl rnas.
we examined the sequences of all pmchl transcripts reported in the present and in our previous study  <cit>  to identify orfs longer than  <dig> bp.
ten short orfs of less than  <dig> bp were found.
pmchl transcripts harbouring exons 5-6a or 5-6b present all together seven orfs that are  <dig> to  <dig> bp in length , i.e.
they would encode proteins of  <dig> to  <dig> amino acids.
orfs of less than  <dig> bp  are often assumed not to be translated.
however, many well known functional proteins of less than  <dig> amino acids in length have been reported, including the small inducible cytokine families ccl and cxcl  <cit> , and the xenobiotic defensin and defensin-related cryptidin factors  <cit> .
furthermore, a recent study has shown that among the  <dig>  predicted proteins encoded by the  <dig>  fantom mouse full-length cdna sequences, 12% of the proteins  are less than  <dig> amino acids in length  <cit> .
this suggests that there might be up to  <dig> times more small proteins than the  <dig> entries present for mus musculus to date in the swissprot protein database .
interestingly, most of the small proteins with known function are evolutionarily conserved  <cit>  or present conserved sequence motifs  <cit> .
notably, a recent report  <cit>  indicates that orfs <  <dig> bp in length, that are not evolutionarily conserved, are unlikely to be translated into functional proteins.
given that the pmchl orfs present on exons 5-6a and 5-6b are not conserved among homo sapiens, pan troglodytes and pongo pygmaeus, due to frameshift-causing insertions/deletions , we propose that these orfs are most likely non-functional.
the longest orf identified on pmchl transcripts is  <dig> bp  long and locates within the alu sequence in exon 2a.
four transcripts harbouring exon 2a were identified in testis in our previous study  <cit> .
however, the corresponding putative protein is not conserved due to a single nucleotide insertion in the human pmchl <dig> sequence causing a frameshift in the middle of the orf.
therefore, this orf is also spurious according to the criteria of clamp and colleagues  <cit> .
pmchl transcripts encompassing exons 1-2a and 1-2b harbour two orfs, named orf1a and orf1b, respectively .
even though these orfs are also less than  <dig> bp in length, they present the same lengths in homo sapiens, pan troglodytes and pongo pygmaeus, and share > 90% sequence identity.
in macaca fascicularis, the orf is shortened and runs only on exon  <dig> due to the presence of an early stop cordon.
these orfs are of particular interest because they share a large part of sequence identity with orf <dig> present on unspliced pmchl rna, and with the pro-mch precursor because it mainly locates in exon  <dig>  i.e.
in the region derived from exon  <dig> of the ancestor pmch gene.
the putative  <dig> kd protein corresponding to orf <dig>  previously named vmch-p <dig>  presents a putative nuclear localisation signal  at the n-terminus , and is among the longest orfs, encoding  <dig> amino acids .
in a previous study  <cit> , we examined the protein coding potential of orf <dig> carrying out in vitro translation experiments and cos- <dig> cell transfections with the flag epitope-tagged orf <dig>  the results indicated a weak protein-coding potential, depending on particular plasmid constructions, providing mrna stabilising elements and enhanced promoter activity  <cit> .
in the present study, we used a vmch-p <dig> antiserum directed against the thirteen n-terminal vmch-p <dig> amino acids, comprising the putative nls .
this allows the determination of the expression of orf <dig>  as well as the orf1a and orf1b variants .
the reactivity of the vmch-p <dig> antiserum was demonstrated in western blot experiments using a recombinant gst-vmch-p <dig> protein produced in bacteria.
vmch-p <dig> antiserum recognized the gst-vmch-p <dig> protein, migrating at about  <dig> kd, whereas the preimmune serum did not .
next, we used the vmch-p <dig> antiserum to examine expression of orf <dig> and its variants in human and macaque tissues .
we tested human adult testis, hippocampus and prefrontal cortex extracts from a new-born and a foetus, as well as four macaca fascicularis cerebral areas .
these tissues and cerebral areas were chosen for the presence of orf1-bearing pmchl transcripts in rt-pcr experiments .
in our western blot experiments, no signal could be detected in all human and macaque tissues tested, at the expected size of 8– <dig> kd for the putative vmch-p <dig> protein and its variants.
this strongly suggests that these putative proteins are not translated in vivo in the human and macaque tissues that we tested.
we further carried out western blot and immunoprecipitation experiments on hek <dig> cells transfected with pmchl1/ <dig> sequences bearing orf <dig> to detect low levels of vmch-p <dig> protein.
even though high levels of orf1-bearing pmchl1/ <dig> transcripts were detected by rt-pcr, no signal corresponding to the vmch-p <dig> protein could be detected .
one explanation for the lack of protein detection, that we cannot exclude, is a very low protein expression level below our detection threshold.
also, for the putative macaque protein, we further cannot exclude an altered epitope-recognition of the antibody due to a lysine to glutamic acid mutation within the epitope.
assuming that the failure to detect the vmch-p <dig> protein or its variants is not due to these technical limitations, the lack of translation of orf <dig> like-bearing mrnas could reside in the moderate consensus with the optimal sequence for translation initiation described by kozak  <cit> .
actually, only the consensual adenine at position - <dig> is present.
what might be the role of the large variety of spliced pmchl mrnas in human testis and fetal brain?
it is tempting to propose that these pmchl transcripts work mainly as an mrna-like non-protein coding rna .
since the realization that 98% of the transcriptional output in mammals consists of ncrnas, the enthusiasm for this class of rnas has grown tremendously  <cit>  and has been granted its own noncode database  <cit> .
numerous classes of ncrnas have been reported, most of which are small ncrnas , but also long ncrnas  such as xist and the antisense tsix transcripts involved in × inactivation in mammals  <cit> , or the air rna that appears to be responsible for imprinted repression of nearby genes  through an antisense-mediated mechanism  <cit> .
several mrna-like ncrnas that are transcribed by rna polymerase ii, spliced and polyadenylated have also been reported  <cit> , including in human  <cit> .
interestingly, many small ncrnas are located in introns of coding or non-coding mrnas  <cit> .
the functional roles of ncrna are diverse, corresponding mainly to adaptor functions targeting nucleic acids to various enzymatic complexes  and gene expression regulation/silencing .
do the pmchl transcripts host small ncrnas in their introns, and/or do the pmchl transcripts control the expression of neighbouring genes in cis  or in trans through rna-rna duplexes  genes)?
we are now addressing these intriguing questions.
CONCLUSIONS
we provide here new data concerning spliced pmchl transcripts, further precising the pmchl gene structure.
sequencing data of the pmchl genes in several non-human primates offered a substantial improvement of the creation model proposed previously  <cit> .
in particular, we proposed that the initial retroposition occurred within an intron of the cdh <dig> gene soon after platyrrhine/catarrhine divergence and was concomitant with the insertion of an alu sg element.
our sequence analysis further points to the presence of short orfs that present little or no evolutionary conservation, suggesting that spliced pmchl transcripts are non-protein coding rnas.
this proposal is further supported by our expression analysis of the most relevant pmchl orfs in human and macaque tissues, which failed to detect any corresponding protein.

 there is an ancient but still active debate in the molecular biologist community about the relative contribution of structural genomic modifications that could account for the phenotypic differences observed between primate species, particularly in the emergence of new brain structure and functions. very debated results were found when determining the ka/ks ratio, a tentative indicator of positive darwinian selection, in the coding region of genes expressed in the mammalian brain. however, genome-wide comparative studies of mammalian promoters suggested an accelerated evolution of primate promoters during the last million years. recently, divergence between human and chimpanzee sequences have been re-evaluated to almost 5%, resulting mainly from indel events and copy number variants that strongly contributed in shaping primate genomes, offering therefore a wide variety of sites at which primate lineage-specific genetic novelty could happen. indeed, recent segmental duplications are particularly enriched in genes that display expression differences between humans and chimpanzees. in addition, an accelerated rate of contraction or expansion in gene families, including brain-expressed genes, operated in primates when compared with other mammals. on a larger scale, cnvs contributed significantly to diverse expression phenotypes in primates and to emergence of complex or sporadic diseases in humans. to reconcile apparently conflicting data, we previously proposed that, in parallel to single nucleotide mutations that confer alterations in the gene expression patterns or amino acids sequences, genomic rearrangements may have played an important role during primate evolution, providing creation of novel but rare regulatory modules as well as protein coding and/or non-coding genes. indeed, combination of exon shuffling, retrotransposition and gene promoter fusion have led to genes harbouring completely new structures and expression patterns selectively in the primate lineage. these rare events would have been nevertheless particularly important in shaping human genes found expressed in reproductive organs, as exemplified by the chimeric pote-actin genes, or involved in hominoid brain neurotransmission, as exemplified by the glud gene. the study of primate-specific gene creation and early evolution requires the discovery of genes that have retained characteristic features of their youth. the pmchl system, which combines the retroposition/exon shuffling and the segmental duplication models, has been one of the first hominoid-specific gene creation model described. we have shown that these genes have been created in the hominoid lineage through i) retroposition at the ancestral chromosome 5p locus in catarrhini of an antisense pro-melanin-concentrating hormone gene transcript, ii) local rearrangement leading to a truncated version of the retrogene, iii) sequence remodelling and iv) final duplication at the ancestral 5q locus in hominids. furthermore, processed and unprocessed transcripts were characterized in a human fetal brain library as well as in developing human brain. these mrnas were found to encode a putative nuclear protein of kd, named vmch-p that was only identified using in vitro translation systems or transfected cell models. however, many questions remained unsolved regarding the region and time of insertion of the retrogene, the fine structure of both genes, their expression patterns and their protein coding potential in human cells. in this paper, we established the structure of pmchl1/pmchl genes and demonstrated that alternatively spliced transcripts encompassing exons to are mostly expressed in human testis. we established further the evolutionary history and regional organization of pmchl genes at both loci on human chromosome and proposed that a single retroposition event followed by point mutations provided novel exonic sequences in transcriptional sense direction. several short open reading frames were found encoded within the spliced pmchl rnas but most of them were not conserved in the primate lineage. this suggests a lack of overt functionality of these orfs, even though the existence of a small species-specific protein cannot be ruled out. finally, we attempted to identify proteins generated from pmchl genes in macaque and human tissues with an antiserum directed against vmch-p and its variants but we failed to detect them. therefore, spliced transcripts from the primate-specific pmchl1/pmchl genes would likely represent mrna-like non-protein coding rnas.  structure and distribution of spliced pmchl transcripts in human brain and testis in our previous study, we characterized several alternatively spliced transcripts harbouring exons to of the original pmchl1/pmchl genes. six transcripts corresponding to pmchl spliced rna were found in testis and/or fetal brain. two pmchl spliced rna were reported only in testis. in order to further precise the exon/intron structure of pmchl genes and to further investigate the tissue distribution of spliced pmchl transcripts, we examined the presence of additional rnas in human testis and cortex marathon cdna libraries, as well as in a human fetal prefrontal cortex sample. for this, we performed pcr experiments using primer pairs designed to amplify transcripts encompassing the most distant known exons, previously named exons and sub-cloning of the pcr products and sequencing of individual clones allowed the discovery of a novel exon, located between exon and former exon it was named exon and previously named exons and are now renamed exons and. we identified six pmchl spliced variants in adult testicular marathon cdna library, two of which had already been identified in the same cdna library. however, transcript bears one a to g mutation within exon resulting in an arginine to glycine mutation in orf noteworthy, transcript corresponds also to two testis ests, and was also found in a human fetal brain marathon cdna library in our previous study. this apparently abundant transcript harbours exons 1-2b and 6a, like most spliced transcripts in testis which were obtained in a single round of pcr. in contrast, transcripts harbouring exon 6b were identified after two rounds of pcr and never contained exon 6a. transcripts containing exon 2a were never observed in the present study. we also identified four novel alternative pmchl splicings in adult testis, which all contained a partial exon shorter than the original exon observed in a pmchl transcript. sequence analysis revealed that mutations in the pmchl sequence created a novel gene-specific splice donor site which is systematically used in the present pmchl transcripts. we further identified one novel pmchl splice variant in a fetal prefrontal cortex sample. this transcript was the only one to harbour an alternative splice donor in exon which was never observed in testis rnas. in our previous study, we reported two alternative acceptor sites in exon 5b separated by only four nucleotides and with apparent testis- and brain-specificities. our present results show that most testis transcripts use the exon 5a splice acceptor site, and two use the alternative 5bt site. however, the pmchl transcripts identified in fetal brain also use the alternative 5bt site indicating that it could not be considered anymore as a testis-specific splice acceptor site. the alternative 5bb site, previously reported in a fetal brain transcript was not found in our present study. in contrast to human testis and fetal brain, we could not detect any spliced pmchl rna harbouring exons and 6a in the human adult cortex marathon cdna library using the 3–7/3– primer pair. this primer pair was further used in rt-pcr experiments combined with southern blot to determine the tissue distribution of the spliced transcripts in testis, prefrontal cortex and cerebellum in adult human and macaque. in agreement with our results using the marathon cdna libraries, we detected spliced pmchl rna harbouring exons and 6a in human adult testis, but not in adult prefrontal cortex and cerebellum. thus, pmchl transcripts are found in testis and fetal brain and are more abundant than pmchl transcripts that are observed only in testis. in addition, pmchl gene expression was reported in ht cells subjected to rage, in which an est encompassing exons 2a and of pmchl has been identified. in macaque, no spliced transcripts were identified by southern blot in agreement with our sequence analysis indicating that the macaque pmchl gene lacks the exon 6a acceptor splice site. taken together, our findings indicate that pmchl and pmchl genes: i) give rise to a complex pattern of alternative splicings, ii) are subject to distinct tissue-specific expressions and iii) are developmentally regulated. the finding that a rather high diversity of spliced transcripts are present in testis is not surprising, because of the permissive chromatin environment present in gonads, allowing high transcriptional activity even from weak tissue-specific promoters. thus, most retroposons evolve into non-functional pseudogenes that are transcribed only in the testis. however, the abrupt emergence of a new chimeric gene in primates could potentially contribute to reproductive barriers and thus play a role in speciation. in this regard the hominoid-specific oncogene tre appears expressed only in testis while the two parental genes usp and tbc1d that fused to generate the tre gene, are expressed in a broad range of human tissues. in addition, the presence of spliced pmchl transcripts in fetal brain, is rather suggestive of a functional role during human brain development. this would imply that the retroposon acquired an active promoter and has been subjected to selection pressure. whether these spliced pmchl transcripts actually play a functional role in testis and fetal brain is an obvious question, which we further addressed below. evolutionary history of the pmchl genes retroposition-driven creation of pmchl gene occurred 30– mya in primate lineage to gain further insights into the evolutionary history of the pmchl gene family, we extended our previously initiated sequence analysis of pmchl and pmch genes. for this, genomic dnas were pcr amplified with the pmchl- or pmch-specific primer pairs indicated in table and the pcr products were sequenced. novel sequences were submitted to genbank under the following accession numbers. for pmchl1:,,,,,,. for pmch:,,,,,,, ]. annealing temperature that was used for pcr and gene-specificity are indicated for each oligonucleotide pair. we first carried out the phylogenetic analysis of the exon/intron boundaries of the pmchl genes. consensus gt/ag splice donor and acceptor sites are present in all species bearing the retroposon, i.e. all catarrhines of this study, with the exception of the splice acceptor site of exon 6a which is present only in hominoids. moreover, the exon 5c donor site and the 6a and 6b splice acceptor sites were not consensual in the platyrrhines analysed here, i. e. before the retroposition event, suggesting that canonical splicing could not occur in this ancestral region before the arrival of the retroposed sequence. our sequence analysis showed that these splice sites were created de novo through single nucleotide mutations. thus, the splicing between exons and corresponds to de novo exonisation and not to an alu-driven exonisation mechanism, exon being absent in platyrrhines and then conserved through selection pressure. in contrast, splice sites of exons and pre-dated the retroposition event, indicating that a fusion of the pmch-derived exons with pre-existing exons is likely to have occurred. whether these exons were expressed before the retroposition remains to be determined, but no expressed sequence tag corresponding to exons and alone could be identified in mammalian est databases. furthermore, chimeric transcripts formed by transcription of two consecutive genes into a single rna can occur quite frequently in human cells. a similar mechanism may be involved in the production of fused transcripts encompassing either exon or of pmchl1/pmchl genes. however, further characterization of putative promoters inside the pmchl genes needs to be done before involving such rna domain accretion process on regulation of these genes. a nucleotidic phylogenetic analysis was performed to date more precisely the retroposition event. as pmchl genes encompass part of the pmch gene sequence, it was possible to align primate sequences of these specific parts of the pmchl and pmch genes. the phylogenetic analysis was performed using the parsimony, maximum-likelihood and neighbour-joining methods and with the rat and mouse pmch sequences as outgroups. all phylogenetic methods led to congruent data, with high bootstrap values with the neighbour joining method. the tree showed an apparent aberration relative to our present knowledge concerning the relationship between species and the creation of the pmchl gene. we expected the pmch sequences of cebus capucinus and saguinus oedipus to be grouped with the pmch sequence of tarsius syrichta rather than with the pmchl sequences. however, a noteworthy low bootstrap value was found for this branching. the position of the pmchl sequences as well as the uncertainty for positioning the cebus capucinus and saguinus oedipus pmch sequences suggest that the retroposition event leading to the pmchl gene occurred very shortly after the split of platyrrhini/cathyrrhini, i.e. 30– mya. when focusing on the pmchl genes, in the regions encompassing exons 1-2b and exons 5– the nucleotidic phylogenetic trees fully correspond to accepted species trees, indicating that no particular and global evolutionary event interfered. however, we observed a difference in the pan troglodytes/pan paniscus/homo sapiens positioning between the two phylogenetic trees. this simply corresponds to an inherent irresolution in the hominidae speciation, which could be inferred from a complex speciation with interbreeding before final separation of chimpanzees, gorillas and humans. all three species should probably be grouped under the pan or homo clade as previously suggested. we previously reported the presence of a complete alu-sq sequence element within pmchl exon, but the insertion event into pmchl could not be dated precisely. it is worth noting that this sequence appears to be an alusg, and not an alu sq element. our present sequence analysis reveals that all primate species carrying the pmchl exon harbour the alusg sequence. thus, the insertion was likely concomitant to the retroposition, occurring after the divergence of cebus species, and before the divergence of the cercopithecoids, approximately between 30– mya. a kb element encompassing pmchl and adjacent intronic/exonic sequence of cdh on 5p duplicated to create pmchl on 5q at the time of hominid divergence we previously proposed that pmchl was created from a duplication of a large, but undefined in size, genomic dna fragment comprising pmchl "jumping" from ancestral hominid chromosome 5p to 5q here, we precisely determined the limits of the duplicon by similarity using a blast search. it appeared that a fragment of kb, encompassing kb upstream and kb downstream of the kb of pmchl was duplicated. this kb duplicon corresponds to a large part of the 5' portion of intron of the cdh gene encoding brain cadherin, as well as cdh exon and the last kb of its intron no other exons than those derived from the pmch gene were found in the duplicon. the percentages of identity between the 5p and 5q elements were equivalent all along the duplicon, and are close to 98%, in agreement with a very recent duplication event. we have therefore precisely mapped the limits of the pmchl1/pmchl duplicon. a question concerning the creation of novel genes, particularly in the case of segmental duplication, is relative to the acceptor sites. which kind of sequences allows invasion by foreign sequences? which kind of sequences can we find at the segmental duplication boundaries? no clear boundaries specificities are described for segmental duplication except for a significant enrichment in short interspersed elements such as young alu y and alu s sequences and other repeats similar to these involved in ig heavy chain recombination in pericentromeric and interstitial segmental duplications. alu mediated dna duplications have exceptionally been reported in eukaryotes. these duplications appeared however to affect mainly hyper-recombinogenic chromosomal regions, and particularly for secondary duplications. long interspersed elements like line elements were also directly implicated in exon recombination and have been proposed to mediate exon shuffling, but none of the previously described human chimeric genes harbour this kind of element at its boundaries. in the case of pmchl1/l no particular sines or lines sequences could be found at the boundaries, neither at the first insertion site of the pmch antisense retroposon gene at the 5p locus), nor at the 5q locus when creating pmchl recently, a duplication-driven model for dna transposition has been put forth by eichler's group suggesting that the probability for a dna element to be duplicated correlated with the degree of proximity to so-called core duplicons. in this context, a core duplicon named glu5– and corresponding to a truncated version of the gusb gene, has been found in close vicinity to pmchl and pmchl genes on 5p and 5q respectively. whether the proximity with this duplicon was determinant for emergence of the pmchl gene remains at this stage a matter of speculation but the timing of both glu5– duplicon expansion and intrachromosomal duplication of pmchl in primates fits very well. these duplication events are also congruent with the global surge in intrachromosomal duplications at the time of hominid divergence, as previously predicted and experimentally proven. analysis of the protein coding potential of pmchl transcripts we next addressed the protein coding potential of the spliced pmchl rnas. we examined the sequences of all pmchl transcripts reported in the present and in our previous study to identify orfs longer than bp. ten short orfs of less than bp were found. pmchl transcripts harbouring exons 5-6a or 5-6b present all together seven orfs that are to bp in length, i.e. they would encode proteins of to amino acids. orfs of less than bp are often assumed not to be translated. however, many well known functional proteins of less than amino acids in length have been reported, including the small inducible cytokine families ccl and cxcl, and the xenobiotic defensin and defensin-related cryptidin factors. furthermore, a recent study has shown that among the predicted proteins encoded by the fantom mouse full-length cdna sequences, 12% of the proteins are less than amino acids in length. this suggests that there might be up to times more small proteins than the entries present for mus musculus to date in the swissprot protein database. interestingly, most of the small proteins with known function are evolutionarily conserved or present conserved sequence motifs. notably, a recent report indicates that orfs < bp in length, that are not evolutionarily conserved, are unlikely to be translated into functional proteins. given that the pmchl orfs present on exons 5-6a and 5-6b are not conserved among homo sapiens, pan troglodytes and pongo pygmaeus, due to frameshift-causing insertions/deletions, we propose that these orfs are most likely non-functional. the longest orf identified on pmchl transcripts is bp long and locates within the alu sequence in exon 2a. four transcripts harbouring exon 2a were identified in testis in our previous study. however, the corresponding putative protein is not conserved due to a single nucleotide insertion in the human pmchl sequence causing a frameshift in the middle of the orf. therefore, this orf is also spurious according to the criteria of clamp and colleagues. pmchl transcripts encompassing exons 1-2a and 1-2b harbour two orfs, named orf1a and orf1b, respectively. even though these orfs are also less than bp in length, they present the same lengths in homo sapiens, pan troglodytes and pongo pygmaeus, and share > 90% sequence identity. in macaca fascicularis, the orf is shortened and runs only on exon due to the presence of an early stop cordon. these orfs are of particular interest because they share a large part of sequence identity with orf present on unspliced pmchl rna, and with the pro-mch precursor because it mainly locates in exon i.e. in the region derived from exon of the ancestor pmch gene. the putative kd protein corresponding to orf previously named vmch-p presents a putative nuclear localisation signal at the n-terminus, and is among the longest orfs, encoding amino acids. in a previous study, we examined the protein coding potential of orf carrying out in vitro translation experiments and cos- cell transfections with the flag epitope-tagged orf the results indicated a weak protein-coding potential, depending on particular plasmid constructions, providing mrna stabilising elements and enhanced promoter activity. in the present study, we used a vmch-p antiserum directed against the thirteen n-terminal vmch-p amino acids, comprising the putative nls. this allows the determination of the expression of orf as well as the orf1a and orf1b variants. the reactivity of the vmch-p antiserum was demonstrated in western blot experiments using a recombinant gst-vmch-p protein produced in bacteria. vmch-p antiserum recognized the gst-vmch-p protein, migrating at about kd, whereas the preimmune serum did not. next, we used the vmch-p antiserum to examine expression of orf and its variants in human and macaque tissues. we tested human adult testis, hippocampus and prefrontal cortex extracts from a new-born and a foetus, as well as four macaca fascicularis cerebral areas. these tissues and cerebral areas were chosen for the presence of orf1-bearing pmchl transcripts in rt-pcr experiments. in our western blot experiments, no signal could be detected in all human and macaque tissues tested, at the expected size of 8– kd for the putative vmch-p protein and its variants. this strongly suggests that these putative proteins are not translated in vivo in the human and macaque tissues that we tested. we further carried out western blot and immunoprecipitation experiments on hek cells transfected with pmchl1/ sequences bearing orf to detect low levels of vmch-p protein. even though high levels of orf1-bearing pmchl1/ transcripts were detected by rt-pcr, no signal corresponding to the vmch-p protein could be detected. one explanation for the lack of protein detection, that we cannot exclude, is a very low protein expression level below our detection threshold. also, for the putative macaque protein, we further cannot exclude an altered epitope-recognition of the antibody due to a lysine to glutamic acid mutation within the epitope. assuming that the failure to detect the vmch-p protein or its variants is not due to these technical limitations, the lack of translation of orf like-bearing mrnas could reside in the moderate consensus with the optimal sequence for translation initiation described by kozak. actually, only the consensual adenine at position - is present. what might be the role of the large variety of spliced pmchl mrnas in human testis and fetal brain? it is tempting to propose that these pmchl transcripts work mainly as an mrna-like non-protein coding rna. since the realization that 98% of the transcriptional output in mammals consists of ncrnas, the enthusiasm for this class of rnas has grown tremendously and has been granted its own noncode database. numerous classes of ncrnas have been reported, most of which are small ncrnas, but also long ncrnas such as xist and the antisense tsix transcripts involved in × inactivation in mammals, or the air rna that appears to be responsible for imprinted repression of nearby genes through an antisense-mediated mechanism. several mrna-like ncrnas that are transcribed by rna polymerase ii, spliced and polyadenylated have also been reported, including in human. interestingly, many small ncrnas are located in introns of coding or non-coding mrnas. the functional roles of ncrna are diverse, corresponding mainly to adaptor functions targeting nucleic acids to various enzymatic complexes and gene expression regulation/silencing. do the pmchl transcripts host small ncrnas in their introns, and/or do the pmchl transcripts control the expression of neighbouring genes in cis or in trans through rna-rna duplexes genes)? we are now addressing these intriguing questions.  we provide here new data concerning spliced pmchl transcripts, further precising the pmchl gene structure. sequencing data of the pmchl genes in several non-human primates offered a substantial improvement of the creation model proposed previously. in particular, we proposed that the initial retroposition occurred within an intron of the cdh gene soon after platyrrhine/catarrhine divergence and was concomitant with the insertion of an alu sg element. our sequence analysis further points to the presence of short orfs that present little or no evolutionary conservation, suggesting that spliced pmchl transcripts are non-protein coding rnas. this proposal is further supported by our expression analysis of the most relevant pmchl orfs in human and macaque tissues, which failed to detect any corresponding protein. 
our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric pmchl genes. several novel spliced pmchl transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. sequence analysis of the spliced pmchl transcripts identified only short orfs of less than bp, with low or no evolutionary conservation. sequencing of pmchl genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin gene, soon after platyrrhine/catarrhine divergence, i.e. western blot analyses of human and macaque tissues expressing pmchl rna failed to reveal any protein corresponding to vmch-p and protein variants encoded by spliced transcripts. mutations before divergence of hylobatidae led to creation of new exons and finally pmchl duplicated in an ancestor of hominids to generate pmchl at the human chromosome 5q a complex pattern of spliced and unspliced pmchl rnas were found in human brain and testis. brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in homo sapiens.
 brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in homo sapiens. pmchl arose from retroposition of a pro-melanin-concentrating hormone antisense mrna on the ancestral human chromosome 5p when platyrrhines and catarrhines diverged. mutations before divergence of hylobatidae led to creation of new exons and finally pmchl duplicated in an ancestor of hominids to generate pmchl at the human chromosome 5q a complex pattern of spliced and unspliced pmchl rnas were found in human brain and testis.  several novel spliced pmchl transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. sequencing of pmchl genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin gene, soon after platyrrhine/catarrhine divergence, i.e. 30– mya, and was concomitant with the insertion of an alusg element. sequence analysis of the spliced pmchl transcripts identified only short orfs of less than bp, with low or no evolutionary conservation. western blot analyses of human and macaque tissues expressing pmchl rna failed to reveal any protein corresponding to vmch-p and protein variants encoded by spliced transcripts.  our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric pmchl genes. these genes produce multiple spliced transcripts, bearing short, non-conserved and apparently non-translated orfs that may function as mrna-like non-coding rnas. 