BACKGROUND
despite their presence in organisms as diverse as fungi, plants and animals, the functions and evolution of makorin  proteins in eukaryotes remain poorly understood.
makorins are zinc finger proteins with a typical c3hc <dig> motif called the ring domain.
this protein-protein interaction motif is found in most e <dig> ubiquitin ligases, a category of enzymes mediating the transfer of ubiquitin from an e <dig> ubiquitin-conjugating enzyme to target protein substrates.
the ring domain is responsible for the ubiquitin ligase activity, leading to mono-ubiquitination and/or to synthesis of poly-ubiquitin chains on lysine residues .
accordingly, some makorin proteins work as e <dig> ubiquitin ligases  <cit> .
in makorin proteins, the ring domain is associated with typical arrays of one to four c3h domains, a type of zinc finger found in a variety of ribonucleoproteins  <cit> .
another motif rich in cys and his residues, with so far unknown function, is also generally present in mkrn proteins  <cit> .
three functional mkrn genes, mkrn <dig>   <dig> and  <dig>  have been described so far in vertebrates, with mkrn <dig> being specific to therian mammals.
makorin <dig> has been well studied in mammals.
its described functions are linked to e <dig> ubiquitin ligase activity, for example ubiquitination-mediated degradation of capsid proteins as a defence mechanism against virus infection  <cit> .
mkrn <dig> is also involved in the control of cell cycle arrest and apoptosis through ubiquitination and proteasome-dependent degradation of proteins p <dig> and p <dig>  <cit> .
mkrn <dig> targets human telomerase catalytic subunit  for proteasome processing during differentiation or cell cycle arrest  <cit> .
makorin <dig> might also have transcriptional activity and regulates rna polymerase ii-dependent transcription independently from e <dig> ligase activity, with either negative or positive effects on gene expression  <cit> .
changes in mkrn <dig> expression have been associated with different types of cancer and other diseases, as observed for other ring proteins with e <dig> ligase activity  <cit> .
a close relative to mkrn <dig> is mkrn <dig> , which has been detected only in therian mammals.
mkrn <dig> has been first identified in the prader-willi syndrome critical region on 15q <dig>  in the human genome  <cit> , but with no obvious role in the disease  <cit> .
no function has been assigned so far to mkrn <dig>  mkrn <dig> in fact corresponds to an intronless retrocopy of mkrn <dig> generated through reverse transcription of an mkrn <dig> mrna molecule.
the formation of such retrogenes is catalyzed by the reverse transcriptase encoded by autonomous retrotransposable elements.
several other retrocopies of mkrn <dig> have been identified in mammalian genomes, most of them probably corresponding to pseudogenes.
the possible involvement of such pseudogenes in the regulation of the founding source gene mkrn <dig> has been proposed and debated  <cit> .
the third functional member of the makorin family described in vertebrates is mkrn <dig>  this gene partially overlaps with the raf <dig> proto-oncogene in antisense transcriptional orientation  <cit> .
functional data have been only published for the clawed frog xenopus laevis, where mkrn <dig> is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3beta  in the phosphatidylinositol 3-kinase/akt pathway.
the third c3h zinc finger, the cys-his motif as well as the ring zinc finger are necessary for this anti-neurogenic activity  <cit> .
during the screening of a testis cdna library from the platyfish xiphophorus maculatus , we have identified a gene with gonad-specific expression representing a new divergent member of the makorin gene family in vertebrates, which was named mkrn <dig>  comparative analyses in different species revealed the evolutionary dynamics of this family of ring finger proteins, suggesting an ancestral gonad-specific function and maternal embryonic expression before duplication in vertebrates.
RESULTS
mkrn <dig> is a new member of the vertebrate makorin gene family
in order to identify new potential gonad-specific transcription factors or regulatory proteins expressed in fish gonads, we randomly sequenced clones from a testis cdna library of the platyfish xiphophorus maculatus.
in silico screening for sequences containing zinc finger domains revealed a clone with similarity to uncharacterized members of the makorin gene family  <cit>  .
since three functional mkrn genes have been described so far in vertebrates, the newly identified gene was called makorin <dig> .
we could identify sequences orthologous to platyfish mkrn <dig> in other fish species .
all fully sequenced fish genomes including zebrafish , medaka , <cit> , fugu   <cit>  and three-spined stickleback  contained mkrn <dig> as a single copy gene.
mkrn <dig> ests were also identified in databases for other fish species without available genome draft.
mkrn <dig> is present in birds  and amphibians as well as in most mammals, including human .
mkrn <dig> sequences form a clear monophyletic group within the makorin molecular phylogeny .
surprisingly, analysis of genome sequence drafts and est data failed to detect mkrn <dig> in two murid rodents, the mouse mus musculus and rat rattus norvegicus, even after targeted scrutiny of chromosomal segments syntenic to mkrn4-containing loci from other species .
partial mkrn <dig> sequences were found in other rodents such as ord's kangaroo rat , thirteen-lined ground squirrel  and guinea pig , as well as in the american pika ochotona princeps .
no mkrn4-related sequences were identified in the genome draft of another lagomorph, the rabbit oryctolagus cuniculus, and in the northern tree shrew tupaia belangeri .
for all these species, no mkrn <dig> est sequence was present in databases.
hence, these results suggest that the mkrn <dig> gene has been lost in murid rodents .
in some other rodent or lagomorph species, mkrn <dig> may be also on the way of degradation  or even might have been already eliminated, but genomic sequences of better quality will be required to confirm this hypothesis.
we noticed that mkrn <dig> was annotated as a pseudogene in the current ensembl human genome release, under the name of mkrnp <dig>  <cit> .
very similar orthologous sequences were identified in syntenic regions of the genome of chimpanzee , orangutan  and macaque , a degree of conservation surprising for a pseudogene evolving under neutral selection.
only gorilla did not show any mkrn <dig> sequence, despite the presence of a large syntenic region on chromosome  <dig> including several genes surrounding mkrn <dig> in other primates.
however, the draft sequence of this region contains many gaps, especially at the position where mkrn4/mkrnp <dig> would be expected.
hence, mkrn <dig> might be absent from draft but present in the genome of the gorilla.
in human, mkrnp <dig> was annotated as pseudogene due to a premature stop codon at position  <dig> in the ensembl transcript.
we suggest that the translation start codon was wrongly annotated in the ensembl prediction.
a start codon in frame + <dig>  using the atg at position  <dig> of the predicted transcript, leads to a full length protein without any further interruption of the open reading frame.
this atg is conserved between primate sequences and annotated as the bona fide start codon in genbank entry eax <dig>  used in sequence alignments and molecular phylogenies .
primate sequences were reannotated accordingly .
using primate sequences as query against est databases in blast analysis, no evidence of transcription could be found.
we then compared the rate of non-synonymous  to the rate of synonymous substitutions  between mkrn sequences in primates  and other vertebrates.
the average ka/ks value obtained between primate mkrn <dig> sequences was  <dig> , indicating an excess of synonymous vs. non-synonymous substitutions.
this value was similar to those obtained between primate mkrn <dig>  and mkrn <dig> .
the average ka/ks ratio for mkrn <dig> between fish sequences  was  <dig> .
taken together, these observations suggest that mkrn <dig> evolved under purifying selection in primates and fish, as observed for other makorin genes.
mkrn <dig> might be a functional protein-coding gene in primates but expressed at a very low level, in a particular type of cells or under very specific conditions.
alternatively, mkrn <dig> might have lost its expression potential after initial functionality in different primate species.
no mkrn <dig> sequence was found in the genome of more divergent species, including the elephant shark callorhinchus milii   <cit>  and the invertebrate chordates branchiostoma floridae  and ciona intestinalis/savignyi .
large-scale duplication and the evolution of the makorin gene family
mkrn <dig>  mkrn <dig> and mkrn <dig> are all present in both ray-finned fish and tetrapod lineages, indicating that these genes have been formed through gene duplications at least  <dig> million years ago early in the vertebrate lineage.
mkrn <dig> and mkrn <dig> but not mkrn <dig> were detected in the genome of elephant shark callorhinchus milii, a cartilaginous fish.
in contrast, we could identify only one makorin gene in amphioxus and sea squirts , as well as in other more divergent non-vertebrate species including yeast and rice.
in the fly drosophila melanogaster, four makorin genes were identified, but three of them clearly corresponded to retrocopies of the only bona fide intron-containing mkrn gene.
hence, the mkrn gene family observed in vertebrates is probably the result of two events of gene duplication having taken place between the emergence of vertebrates and the divergence between tetrapods and ray-finned fish.
at least the duplication having led to mkrn <dig> and mkrn <dig> occurred before the split between cartilaginous and bony fish.
in order to know if gene duplications at the origin of the mkrn family might be linked to rounds of whole genome duplications having occurred at the basis of the vertebrate lineage  <cit> , we tested if mkrn genes might be included in large ancient duplicated regions called paralogons having resulted from such events.
on human chromosome  <dig>  mkrn <dig> and the raf <dig> proto-oncogene partially overlap in antisense transcriptional orientation.
this syntenic relationship is conserved in most vertebrates but not in teleost fish, with the exception of the zebrafish, where mkrn <dig> and raf <dig> are closely linked on chromosome  <dig> .
strikingly, in medaka raf <dig> is located close to mkrn <dig> but not mkrn <dig>  we further analyzed syntenic relationships between genomic regions containing mkrn <dig> and mkrn <dig> in human, chicken, medaka and zebrafish genomes .
we could identify a set of genes with duplicated copies linked to mkrn <dig> and mkrn <dig> in at least one species .
other genes like raf <dig> or cnpb showed only one copy but either linked to mkrn <dig> in some species or to mkrn <dig> in others, suggesting differential loss of paralogous copies after duplication.
these results indicate that mkrn <dig> and mkrn <dig> are parts of ancient large duplicated regions present in distant vertebrate species.
hence, these paralogons might have been generated by one of the whole genome duplications having occurred in the ancestral vertebrate lineage, the so called 1r and 2r duplications  <cit> .
both mkrn <dig> and mkrn <dig> are called ohnologs .
on the other hand, we can not exclude the occurrence of a very large segmental gene duplication having generated these duplicated blocks.
the region containing mkrn <dig> shows little conservation between mammals and fish .
we could not find any evidence for synteny between the mkrn1-containing region and other mkrn regions.
it has been previously proposed that mkrn <dig> and mkrn <dig> are indeed the result of a single gene duplication event  <cit> .
retroposition and the evolution of the makorin gene family
in addition to mkrn <dig>  mkrn <dig> and mkrn <dig>  multiple other mkrn-like sequences are present in mammalian genomes.
some of these sequences correspond to intronless retrocopies of mkrn <dig>  their functional status concerning expression, regulation and mutual interference has been the source of controversial debate  <cit> .
in order to identify all vertebrate mkrn-related sequences, we searched over the ensembl genome browser the current genome releases of representatives of all vertebrate groups including all mammalian genomes  <cit> .
the great majority of identified sequences corresponded to retrogenes of mkrn <dig> , with the characteristic absence of introns due to reverse transcription of the mature mrna molecule  <cit> .
besides mkrn <dig>  which probably corresponds to an expressed functional retrogene present in human and mouse, as many as ten different retrogenes located on eight different chromosomes were identified in the human genome.
most of them presented inactivating mutations or severely truncated open reading frames.
almost no est corresponding to these retrogenes was detected in databases.
hence, most of these mkrn <dig> retrocopies probably correspond to pseudogenes.
six mkrn <dig> retropseudogenes located on five different chromosomes were identified in the mouse.
positions are based on human grch <dig> and mouse ncbim <dig> assemblies.
gene names marked with "*" refer to reference  <cit> .
we detected makorin <dig> and other retrocopies of makorin <dig> only in mammals and not in chicken, frog and all five fish genomes analyzed.
an mkrn <dig> sequence was detected in opossum, suggesting an event of retroposition that took place before the split of marsupials from therian mammals.
no mkrn <dig> was found in the current genome draft of the platypus.
retrocopies of mkrn <dig> were found in the mouse but neither in the human nor in non-mammalian vertebrate genomes .
these three retropseudogenic copies of mkrn <dig> are located on three different chromosomes in m. musculus.
no retrocopy of mkrn <dig> was found in all vertebrate genomes analysed, but three potentially functional retrocopies of the single intron-containing mkrn gene were identified in the fruit fly drosophila melanogaster.
taken together, these observations demonstrate an intriguing tendency of the mkrn gene family to generate duplicated copies by retroposition.
structural evolutionary dynamics in the makorin family
the evolution of the nature and number of zinc fingers in the makorin protein family was investigated .
in most plants and invertebrate species , the single makorin protein contains the ring domain flanked by three c3h zinc fingers on its n-terminal side and one c3h zinc finger on its c-terminal side.
an additional motif rich in cysteine and histidine residues  is located between the third c3h zinc finger and the ring domain .
this structure, likely to be ancestral in vertebrates, is also found in most, if not all mkrn <dig> and mkrn <dig> proteins.
in contrast, this otherwise well conserved structure was not found in mkrn <dig> proteins .
while the ring domain and the c-terminal c3h zinc finger are found in all mkrn <dig> sequences, the third c3h zinc finger and the cyshis motif are missing and have been probably lost in the ancestral mkrn <dig> gene early after duplication.
only fish and xenopus have kept the first two c3h complete domains, which are both absent from mammalian sequences.
in birds, a remnant of one of these domains has been retained in chicken and zebra finch under the form of a c2h domain, the second c3h domain having been maintained only in zebra finch but not in chicken.
these results indicate that the evolution of the mkrn <dig> protein is associated with the dynamic loss of zinc finger domains otherwise conserved in mkrn proteins.
this is consistent with a to some extent faster evolution also reflected by the longer branches in the makorin-like group in the phylogenetic tree .
another example of zinc finger domain loss is visible in mkrn3: this mkrn1-derived retrogene encodes a protein having lost the second c3h zinc finger  in human and mouse.
in the much conserved mkrn <dig> and mkrn <dig> groups, the platypus sequences present a reduced number of zinc fingers, like the opossum mkrn <dig> sequence.
this might reflect a biological reality, but might also be an artifact of local poor sequence assembly quality of the genome drafts for these species.
evolution of makorin expression in vertebrates
we compared the expression of the different vertebrate makorin genes in different species representative of the fish , amphibian  and bird  lineages  .
this might allow to assess the functional evolution of mkrn genes in vertebrates and to estimate the ancestral expression pattern and function in the primitive vertebrate before gene duplication.
in adult tissue, the whole mkrn gene family showed a clear gonad-biased expression in fish and frog, with particular high expression in ovary .
accordingly, mkrn <dig> is upregulated during ovarian maturation in flatfish, with strong expression in previtellogenic ovarian follicles  <cit> .
such a specificity of expression was not observed in birds and mammals, even if mkrn <dig> has been reported to be strongly expressed in mouse adult testis  <cit>  and mkrn <dig> showed a slightly testis-biased expression in chicken.
taken together, these results suggested that the ancestral mkrn gene was preferentially expressed in the gonads in a primitive vertebrate, with conservation of expression pattern after duplication in fish and amphibians but partial relaxation of this specificity and possible extension of functions in birds and mammals.
mkrn expression during early embryonic stages in fish largely reflected maternally stored mrna expressed during oogenesis  <cit> , since mrna levels were very high at very early stages .
rna levels then decreased throughout development, probably due to dilution effect through continuous cell division, and reached a very low level shortly after midblastula transition , the state of zygotic transcription activation.
mbt takes place around stage  <dig> in medaka  <cit>  and starts at cycle  <dig> corresponding to  <dig> cell stage in zebrafish  <cit>  .
maternal expression was observed for all three mkrn genes in both medaka and zebrafish, suggesting here again an ancestral expression pattern.
as observed in adults, no tissue-specific expression was observed in chicken embryos one day before hatching .
CONCLUSIONS
through the identification of mkrn <dig>  a new member of the makorin gene family present in tetrapods and ray-finned fishes, we were able to gain some insights into the structural and functional evolution of this e <dig> ubiquitin ligase gene family.
we could show that different evolutionary dynamics affect different mkrn genes in vertebrates.
while mkrn <dig> and mkrn <dig> were detected in all bony vertebrates studied, mkrn <dig> was found to be absent from rat and mouse, two rodent murids, as well as from rabbit, a lagomorph.
in addition, both mkrn <dig> and mkrn <dig> proteins have retained the ancestral arrangement of zinc fingers, with four c3h motifs surrounding the ring domain responsible for the ubiquitin ligase activity.
in contrast, mkrn <dig> has lost several motifs early after its formation through duplication, and subsequent lineage-specific zinc finger loss was observed.
taken together, these observations suggest that mkrn <dig> is subject to more relaxed evolutionary constraints than mkrn <dig> and mkrn <dig>  with possible development of new general and lineage-specific functions.
however, all mkrn proteins contain the ring domain with adjacent c-terminal c3h zinc finger, indicating that e <dig> ubiquitin ligase activity and interactions with nucleic acids might be properties common to all makorin proteins.
it appears therefore possible that mkrn <dig> function, if still required in rat and mouse, might be performed by mkrn <dig> or mkrn <dig>
through comparative genomics, we could find evidence that mkrn <dig> and mkrn <dig> have been duplicated together with other genes through a large-scale duplication.
these genes are embedded in paralogons, i.e.
in large syntenic chromosomal blocks of duplicated genes within a same genome.
these paralogons were found in both tetrapods and ray-finned fish, tracing back their origin to at least  <dig> million years ago.
we propose that mkrn <dig> and mkrn <dig> duplication was the result of either 1r or 2r, the two events of genome duplication having taken place at the basis of the vertebrate lineage  <cit> .
both 1r and 2r arose before the split between cartilaginous and bony fish; one might therefore expect the presence of both mkrn <dig> and mkrn <dig> in the genome of elephant shark.
however, only mkrn <dig> was detected.
this might be explained by secondary loss of mkrn <dig> or incomplete coverage of the elephant shark genome.
the molecular mechanism of formation of mkrn <dig> remains elusive.
involvement of 1r or 2r appears possible  or of a more local event of segmental duplication.
conservation of intron position between mkrn <dig>  mkrn <dig> and mkrn <dig>  suggests that retroposition was not involved.
after formation of mkrn <dig>  mkrn <dig> and mkrn <dig>  the makorin gene family was shaped by lineage-specific retroposition-mediated duplications in vertebrates.
such retrogenes are formed by reverse transcription of generally mature mrna molecules followed by integration of the formed cdna into a new location of the genome.
in mammals, retrogene formation is catalyzed by reverse transcriptase encoded by line- <dig> retrotransposons  <cit> .
retrogenes were detected for mkrn <dig> and mkrn <dig> in mammals but not in other vertebrates, and not for mkrn <dig>  most of the retrogenes are pseudogenes, but at least one of them, mkrn <dig>  is functional and conserved in therian mammals.
hence, retroposition is an evolutionary mechanism of duplication and functional diversification in the mkrn family.
there is at the moment no satisfying explanation for the strong tendency of the mkrn family to generate retrogenes.
germ cell expression, necessary for retrogene formation and transmission to the next generation, might predispose mkrn genes to duplication through retroposition.
goncalves et al.
<cit>  have proposed that the gene expression level in the germ line is correlated with the number of retropseudogenes, since abundant mrnas are more likely to be taken as a substrate by reverse-transcriptases.
however, high mkrn expression is not the only explanation, since no retrocopy was detected for mkrn genes in amphibians and fish, in which they show a strong gonadal expression.
other factors might be involved, for example the affinity of expressed reverse transcriptases for mrna molecules.
finally, comparative expression analysis showed a strongly gonad-biased expression pattern for mkrn <dig>  mkrn <dig> and mkrn <dig> in medaka and zebrafish  and amphibians, with particularly strong expression in ovary.
even if some mkrn genes showed also preferential gonadal expression in chicken and mouse, expression was much less specific in birds and mammals.
maternal rna was detected for all three genes in early medaka and zebrafish embryos.
taken together, these results suggest that the ancestral single-copy mkrn gene was gonad-specific in primitive vertebrates, with a possible role in sexual development or gonad functions.
this ancestral gene was also possibly maternally expressed and therefore potentially involved in fundamental processes such as fertilization, cell division or germ cell development  <cit> .
nothing is known concerning the functions of mkrn proteins in fish and amphibian gonads and embryos, with the exception of a described role for mkrn <dig> as a negative regulator of neurogenesis in xenopus  <cit> .
at the moment it is therefore difficult to propose a function for the ancestral vertebrate mkrn protein, but results obtained for mkrn <dig> suggest a link with the regulation of cell proliferation and apoptosis  <cit> .
an involvement of the ubiquitin pathway in testis and spermatogenesis has been proposed for different organisms and testis-specific transcripts of different ubiquitin-interacting factors have been reported .
some data indicated an involvement of the ubiquitin pathway in the maintenance and development of ovarian germ cells in the fly  <cit>  and the zebrafish  <cit>  and during maturation of the ovary in mouse  <cit> .
several examples of maternally-expressed ring finger proteins with essential functions during development have been reported  <cit> .
multispecies comparative functional analysis of mkrn genes is now required to better understand the evolution of this gene family in vertebrates.

 despite their presence in organisms as diverse as fungi, plants and animals, the functions and evolution of makorin proteins in eukaryotes remain poorly understood. makorins are zinc finger proteins with a typical c3hc motif called the ring domain. this protein-protein interaction motif is found in most e ubiquitin ligases, a category of enzymes mediating the transfer of ubiquitin from an e ubiquitin-conjugating enzyme to target protein substrates. the ring domain is responsible for the ubiquitin ligase activity, leading to mono-ubiquitination and/or to synthesis of poly-ubiquitin chains on lysine residues. accordingly, some makorin proteins work as e ubiquitin ligases. in makorin proteins, the ring domain is associated with typical arrays of one to four c3h domains, a type of zinc finger found in a variety of ribonucleoproteins. another motif rich in cys and his residues, with so far unknown function, is also generally present in mkrn proteins. three functional mkrn genes, mkrn and have been described so far in vertebrates, with mkrn being specific to therian mammals. makorin has been well studied in mammals. its described functions are linked to e ubiquitin ligase activity, for example ubiquitination-mediated degradation of capsid proteins as a defence mechanism against virus infection. mkrn is also involved in the control of cell cycle arrest and apoptosis through ubiquitination and proteasome-dependent degradation of proteins p and p. mkrn targets human telomerase catalytic subunit for proteasome processing during differentiation or cell cycle arrest. makorin might also have transcriptional activity and regulates rna polymerase ii-dependent transcription independently from e ligase activity, with either negative or positive effects on gene expression. changes in mkrn expression have been associated with different types of cancer and other diseases, as observed for other ring proteins with e ligase activity. a close relative to mkrn is mkrn, which has been detected only in therian mammals. mkrn has been first identified in the prader-willi syndrome critical region on 15q in the human genome, but with no obvious role in the disease. no function has been assigned so far to mkrn mkrn in fact corresponds to an intronless retrocopy of mkrn generated through reverse transcription of an mkrn mrna molecule. the formation of such retrogenes is catalyzed by the reverse transcriptase encoded by autonomous retrotransposable elements. several other retrocopies of mkrn have been identified in mammalian genomes, most of them probably corresponding to pseudogenes. the possible involvement of such pseudogenes in the regulation of the founding source gene mkrn has been proposed and debated. the third functional member of the makorin family described in vertebrates is mkrn this gene partially overlaps with the raf proto-oncogene in antisense transcriptional orientation. functional data have been only published for the clawed frog xenopus laevis, where mkrn is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3beta in the phosphatidylinositol 3-kinase/akt pathway. the third c3h zinc finger, the cys-his motif as well as the ring zinc finger are necessary for this anti-neurogenic activity. during the screening of a testis cdna library from the platyfish xiphophorus maculatus, we have identified a gene with gonad-specific expression representing a new divergent member of the makorin gene family in vertebrates, which was named mkrn comparative analyses in different species revealed the evolutionary dynamics of this family of ring finger proteins, suggesting an ancestral gonad-specific function and maternal embryonic expression before duplication in vertebrates.  mkrn is a new member of the vertebrate makorin gene family in order to identify new potential gonad-specific transcription factors or regulatory proteins expressed in fish gonads, we randomly sequenced clones from a testis cdna library of the platyfish xiphophorus maculatus. in silico screening for sequences containing zinc finger domains revealed a clone with similarity to uncharacterized members of the makorin gene family. since three functional mkrn genes have been described so far in vertebrates, the newly identified gene was called makorin. we could identify sequences orthologous to platyfish mkrn in other fish species. all fully sequenced fish genomes including zebrafish, medaka,, fugu and three-spined stickleback contained mkrn as a single copy gene. mkrn ests were also identified in databases for other fish species without available genome draft. mkrn is present in birds and amphibians as well as in most mammals, including human. mkrn sequences form a clear monophyletic group within the makorin molecular phylogeny. surprisingly, analysis of genome sequence drafts and est data failed to detect mkrn in two murid rodents, the mouse mus musculus and rat rattus norvegicus, even after targeted scrutiny of chromosomal segments syntenic to mkrn4-containing loci from other species. partial mkrn sequences were found in other rodents such as ord's kangaroo rat, thirteen-lined ground squirrel and guinea pig, as well as in the american pika ochotona princeps. no mkrn4-related sequences were identified in the genome draft of another lagomorph, the rabbit oryctolagus cuniculus, and in the northern tree shrew tupaia belangeri. for all these species, no mkrn est sequence was present in databases. hence, these results suggest that the mkrn gene has been lost in murid rodents. in some other rodent or lagomorph species, mkrn may be also on the way of degradation or even might have been already eliminated, but genomic sequences of better quality will be required to confirm this hypothesis. we noticed that mkrn was annotated as a pseudogene in the current ensembl human genome release, under the name of mkrnp. very similar orthologous sequences were identified in syntenic regions of the genome of chimpanzee, orangutan and macaque, a degree of conservation surprising for a pseudogene evolving under neutral selection. only gorilla did not show any mkrn sequence, despite the presence of a large syntenic region on chromosome including several genes surrounding mkrn in other primates. however, the draft sequence of this region contains many gaps, especially at the position where mkrn4/mkrnp would be expected. hence, mkrn might be absent from draft but present in the genome of the gorilla. in human, mkrnp was annotated as pseudogene due to a premature stop codon at position in the ensembl transcript. we suggest that the translation start codon was wrongly annotated in the ensembl prediction. a start codon in frame + using the atg at position of the predicted transcript, leads to a full length protein without any further interruption of the open reading frame. this atg is conserved between primate sequences and annotated as the bona fide start codon in genbank entry eax used in sequence alignments and molecular phylogenies. primate sequences were reannotated accordingly. using primate sequences as query against est databases in blast analysis, no evidence of transcription could be found. we then compared the rate of non-synonymous to the rate of synonymous substitutions between mkrn sequences in primates and other vertebrates. the average ka/ks value obtained between primate mkrn sequences was, indicating an excess of synonymous vs. non-synonymous substitutions. this value was similar to those obtained between primate mkrn and mkrn. the average ka/ks ratio for mkrn between fish sequences was. taken together, these observations suggest that mkrn evolved under purifying selection in primates and fish, as observed for other makorin genes. mkrn might be a functional protein-coding gene in primates but expressed at a very low level, in a particular type of cells or under very specific conditions. alternatively, mkrn might have lost its expression potential after initial functionality in different primate species. no mkrn sequence was found in the genome of more divergent species, including the elephant shark callorhinchus milii and the invertebrate chordates branchiostoma floridae and ciona intestinalis/savignyi. large-scale duplication and the evolution of the makorin gene family mkrn mkrn and mkrn are all present in both ray-finned fish and tetrapod lineages, indicating that these genes have been formed through gene duplications at least million years ago early in the vertebrate lineage. mkrn and mkrn but not mkrn were detected in the genome of elephant shark callorhinchus milii, a cartilaginous fish. in contrast, we could identify only one makorin gene in amphioxus and sea squirts, as well as in other more divergent non-vertebrate species including yeast and rice. in the fly drosophila melanogaster, four makorin genes were identified, but three of them clearly corresponded to retrocopies of the only bona fide intron-containing mkrn gene. hence, the mkrn gene family observed in vertebrates is probably the result of two events of gene duplication having taken place between the emergence of vertebrates and the divergence between tetrapods and ray-finned fish. at least the duplication having led to mkrn and mkrn occurred before the split between cartilaginous and bony fish. in order to know if gene duplications at the origin of the mkrn family might be linked to rounds of whole genome duplications having occurred at the basis of the vertebrate lineage, we tested if mkrn genes might be included in large ancient duplicated regions called paralogons having resulted from such events. on human chromosome mkrn and the raf proto-oncogene partially overlap in antisense transcriptional orientation. this syntenic relationship is conserved in most vertebrates but not in teleost fish, with the exception of the zebrafish, where mkrn and raf are closely linked on chromosome. strikingly, in medaka raf is located close to mkrn but not mkrn we further analyzed syntenic relationships between genomic regions containing mkrn and mkrn in human, chicken, medaka and zebrafish genomes. we could identify a set of genes with duplicated copies linked to mkrn and mkrn in at least one species. other genes like raf or cnpb showed only one copy but either linked to mkrn in some species or to mkrn in others, suggesting differential loss of paralogous copies after duplication. these results indicate that mkrn and mkrn are parts of ancient large duplicated regions present in distant vertebrate species. hence, these paralogons might have been generated by one of the whole genome duplications having occurred in the ancestral vertebrate lineage, the so called 1r and 2r duplications. both mkrn and mkrn are called ohnologs. on the other hand, we can not exclude the occurrence of a very large segmental gene duplication having generated these duplicated blocks. the region containing mkrn shows little conservation between mammals and fish. we could not find any evidence for synteny between the mkrn1-containing region and other mkrn regions. it has been previously proposed that mkrn and mkrn are indeed the result of a single gene duplication event. retroposition and the evolution of the makorin gene family in addition to mkrn mkrn and mkrn multiple other mkrn-like sequences are present in mammalian genomes. some of these sequences correspond to intronless retrocopies of mkrn their functional status concerning expression, regulation and mutual interference has been the source of controversial debate. in order to identify all vertebrate mkrn-related sequences, we searched over the ensembl genome browser the current genome releases of representatives of all vertebrate groups including all mammalian genomes. the great majority of identified sequences corresponded to retrogenes of mkrn, with the characteristic absence of introns due to reverse transcription of the mature mrna molecule. besides mkrn which probably corresponds to an expressed functional retrogene present in human and mouse, as many as ten different retrogenes located on eight different chromosomes were identified in the human genome. most of them presented inactivating mutations or severely truncated open reading frames. almost no est corresponding to these retrogenes was detected in databases. hence, most of these mkrn retrocopies probably correspond to pseudogenes. six mkrn retropseudogenes located on five different chromosomes were identified in the mouse. positions are based on human grch and mouse ncbim assemblies. gene names marked with "*" refer to reference. we detected makorin and other retrocopies of makorin only in mammals and not in chicken, frog and all five fish genomes analyzed. an mkrn sequence was detected in opossum, suggesting an event of retroposition that took place before the split of marsupials from therian mammals. no mkrn was found in the current genome draft of the platypus. retrocopies of mkrn were found in the mouse but neither in the human nor in non-mammalian vertebrate genomes. these three retropseudogenic copies of mkrn are located on three different chromosomes in m. musculus. no retrocopy of mkrn was found in all vertebrate genomes analysed, but three potentially functional retrocopies of the single intron-containing mkrn gene were identified in the fruit fly drosophila melanogaster. taken together, these observations demonstrate an intriguing tendency of the mkrn gene family to generate duplicated copies by retroposition. structural evolutionary dynamics in the makorin family the evolution of the nature and number of zinc fingers in the makorin protein family was investigated. in most plants and invertebrate species, the single makorin protein contains the ring domain flanked by three c3h zinc fingers on its n-terminal side and one c3h zinc finger on its c-terminal side. an additional motif rich in cysteine and histidine residues is located between the third c3h zinc finger and the ring domain. this structure, likely to be ancestral in vertebrates, is also found in most, if not all mkrn and mkrn proteins. in contrast, this otherwise well conserved structure was not found in mkrn proteins. while the ring domain and the c-terminal c3h zinc finger are found in all mkrn sequences, the third c3h zinc finger and the cyshis motif are missing and have been probably lost in the ancestral mkrn gene early after duplication. only fish and xenopus have kept the first two c3h complete domains, which are both absent from mammalian sequences. in birds, a remnant of one of these domains has been retained in chicken and zebra finch under the form of a c2h domain, the second c3h domain having been maintained only in zebra finch but not in chicken. these results indicate that the evolution of the mkrn protein is associated with the dynamic loss of zinc finger domains otherwise conserved in mkrn proteins. this is consistent with a to some extent faster evolution also reflected by the longer branches in the makorin-like group in the phylogenetic tree. another example of zinc finger domain loss is visible in mkrn3: this mkrn1-derived retrogene encodes a protein having lost the second c3h zinc finger in human and mouse. in the much conserved mkrn and mkrn groups, the platypus sequences present a reduced number of zinc fingers, like the opossum mkrn sequence. this might reflect a biological reality, but might also be an artifact of local poor sequence assembly quality of the genome drafts for these species. evolution of makorin expression in vertebrates we compared the expression of the different vertebrate makorin genes in different species representative of the fish, amphibian and bird lineages. this might allow to assess the functional evolution of mkrn genes in vertebrates and to estimate the ancestral expression pattern and function in the primitive vertebrate before gene duplication. in adult tissue, the whole mkrn gene family showed a clear gonad-biased expression in fish and frog, with particular high expression in ovary. accordingly, mkrn is upregulated during ovarian maturation in flatfish, with strong expression in previtellogenic ovarian follicles. such a specificity of expression was not observed in birds and mammals, even if mkrn has been reported to be strongly expressed in mouse adult testis and mkrn showed a slightly testis-biased expression in chicken. taken together, these results suggested that the ancestral mkrn gene was preferentially expressed in the gonads in a primitive vertebrate, with conservation of expression pattern after duplication in fish and amphibians but partial relaxation of this specificity and possible extension of functions in birds and mammals. mkrn expression during early embryonic stages in fish largely reflected maternally stored mrna expressed during oogenesis, since mrna levels were very high at very early stages. rna levels then decreased throughout development, probably due to dilution effect through continuous cell division, and reached a very low level shortly after midblastula transition, the state of zygotic transcription activation. mbt takes place around stage in medaka and starts at cycle corresponding to cell stage in zebrafish. maternal expression was observed for all three mkrn genes in both medaka and zebrafish, suggesting here again an ancestral expression pattern. as observed in adults, no tissue-specific expression was observed in chicken embryos one day before hatching.  through the identification of mkrn a new member of the makorin gene family present in tetrapods and ray-finned fishes, we were able to gain some insights into the structural and functional evolution of this e ubiquitin ligase gene family. we could show that different evolutionary dynamics affect different mkrn genes in vertebrates. while mkrn and mkrn were detected in all bony vertebrates studied, mkrn was found to be absent from rat and mouse, two rodent murids, as well as from rabbit, a lagomorph. in addition, both mkrn and mkrn proteins have retained the ancestral arrangement of zinc fingers, with four c3h motifs surrounding the ring domain responsible for the ubiquitin ligase activity. in contrast, mkrn has lost several motifs early after its formation through duplication, and subsequent lineage-specific zinc finger loss was observed. taken together, these observations suggest that mkrn is subject to more relaxed evolutionary constraints than mkrn and mkrn with possible development of new general and lineage-specific functions. however, all mkrn proteins contain the ring domain with adjacent c-terminal c3h zinc finger, indicating that e ubiquitin ligase activity and interactions with nucleic acids might be properties common to all makorin proteins. it appears therefore possible that mkrn function, if still required in rat and mouse, might be performed by mkrn or mkrn through comparative genomics, we could find evidence that mkrn and mkrn have been duplicated together with other genes through a large-scale duplication. these genes are embedded in paralogons, i.e. in large syntenic chromosomal blocks of duplicated genes within a same genome. these paralogons were found in both tetrapods and ray-finned fish, tracing back their origin to at least million years ago. we propose that mkrn and mkrn duplication was the result of either 1r or 2r, the two events of genome duplication having taken place at the basis of the vertebrate lineage. both 1r and 2r arose before the split between cartilaginous and bony fish; one might therefore expect the presence of both mkrn and mkrn in the genome of elephant shark. however, only mkrn was detected. this might be explained by secondary loss of mkrn or incomplete coverage of the elephant shark genome. the molecular mechanism of formation of mkrn remains elusive. involvement of 1r or 2r appears possible or of a more local event of segmental duplication. conservation of intron position between mkrn mkrn and mkrn suggests that retroposition was not involved. after formation of mkrn mkrn and mkrn the makorin gene family was shaped by lineage-specific retroposition-mediated duplications in vertebrates. such retrogenes are formed by reverse transcription of generally mature mrna molecules followed by integration of the formed cdna into a new location of the genome. in mammals, retrogene formation is catalyzed by reverse transcriptase encoded by line- retrotransposons. retrogenes were detected for mkrn and mkrn in mammals but not in other vertebrates, and not for mkrn most of the retrogenes are pseudogenes, but at least one of them, mkrn is functional and conserved in therian mammals. hence, retroposition is an evolutionary mechanism of duplication and functional diversification in the mkrn family. there is at the moment no satisfying explanation for the strong tendency of the mkrn family to generate retrogenes. germ cell expression, necessary for retrogene formation and transmission to the next generation, might predispose mkrn genes to duplication through retroposition. goncalves et al. have proposed that the gene expression level in the germ line is correlated with the number of retropseudogenes, since abundant mrnas are more likely to be taken as a substrate by reverse-transcriptases. however, high mkrn expression is not the only explanation, since no retrocopy was detected for mkrn genes in amphibians and fish, in which they show a strong gonadal expression. other factors might be involved, for example the affinity of expressed reverse transcriptases for mrna molecules. finally, comparative expression analysis showed a strongly gonad-biased expression pattern for mkrn mkrn and mkrn in medaka and zebrafish and amphibians, with particularly strong expression in ovary. even if some mkrn genes showed also preferential gonadal expression in chicken and mouse, expression was much less specific in birds and mammals. maternal rna was detected for all three genes in early medaka and zebrafish embryos. taken together, these results suggest that the ancestral single-copy mkrn gene was gonad-specific in primitive vertebrates, with a possible role in sexual development or gonad functions. this ancestral gene was also possibly maternally expressed and therefore potentially involved in fundamental processes such as fertilization, cell division or germ cell development. nothing is known concerning the functions of mkrn proteins in fish and amphibian gonads and embryos, with the exception of a described role for mkrn as a negative regulator of neurogenesis in xenopus. at the moment it is therefore difficult to propose a function for the ancestral vertebrate mkrn protein, but results obtained for mkrn suggest a link with the regulation of cell proliferation and apoptosis. an involvement of the ubiquitin pathway in testis and spermatogenesis has been proposed for different organisms and testis-specific transcripts of different ubiquitin-interacting factors have been reported. some data indicated an involvement of the ubiquitin pathway in the maintenance and development of ovarian germ cells in the fly and the zebrafish and during maturation of the ovary in mouse. several examples of maternally-expressed ring finger proteins with essential functions during development have been reported. multispecies comparative functional analysis of mkrn genes is now required to better understand the evolution of this gene family in vertebrates. 
via partial sequencing of a testis cdna library from the poeciliid fish xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn in addition to the already described mkrn and mkrn mkrn is the third example of a makorin gene present in both tetrapods and ray-finned fish. rt-qpcr analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish and amphibians, but a strong relaxation of this specificity in birds and mammals. our analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. mkrn and mkrn are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution. all three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos. from the three major vertebrate mkrn genes, mkrn shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. comparative expression analysis strongly suggests that the ancestral e ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos. the nature and number of zinc fingers were found to be conserved in mkrn and mkrn but much more variable in mkrn with lineage-specific differences. however, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids.
 members of the makorin gene family encode ring/c3h zinc finger proteins with u ubiquitin ligase activity. although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution.  via partial sequencing of a testis cdna library from the poeciliid fish xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn in addition to the already described mkrn and mkrn mkrn is the third example of a makorin gene present in both tetrapods and ray-finned fish. however, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. mkrn and mkrn are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. intriguingly, many mkrn and mkrn intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. the nature and number of zinc fingers were found to be conserved in mkrn and mkrn but much more variable in mkrn with lineage-specific differences. rt-qpcr analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish and amphibians, but a strong relaxation of this specificity in birds and mammals. all three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos.  our analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. from the three major vertebrate mkrn genes, mkrn shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. comparative expression analysis strongly suggests that the ancestral e ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos. 