BACKGROUND
x-chromosome inactivation in female placental mammals, which causes the products of genes on the x chromosome to have equal dosages in males and females, is controlled by a set of long non-coding rna  genes, including xist, tsix, jpx, and tsx .
the imprinted expression of some mammalian genes is also controlled by lncrnas .
in addition to x-inactivation and imprinted gene expression, tissue-specific dna and chromatin methylation occurs widely in animal somatic cells because in differentiated cells, most genes are silenced by dna and chromatin methylation.
because mammalian genomes contain only a few genes encoding dna methyltransferases and polycomb repressive complexes for dna and chromatin methylation and because these proteins lack sequence-specific dna-binding subunits, how these proteins are guided to different genomic sites has long been a puzzle.
recently, more than  <dig>  lncrnas have been identified in humans  <cit> , and many can bind to both polycomb proteins/dna methyltransferases and specific dna sequences  <cit> .
as scaffolds bridging protein-dna interactions, lncrnas are key players in dynamic and tissue-specific genome modification  <cit>  .
because dysregulated genome modification can cause diverse diseases, especially cancers, lncrnas have triggered immense research interest in multiple fields.
while lncrnas are thought to be associated with x-chromosome inactivation and imprinted gene expression in mammals and most lncrnas were first identified in humans  <cit> , lncrnas have also been identified recently in multiple non-mammalian organisms  <cit> .
thus, like micrornas, lncrnas may be clade- or species-specific, although the details of these patterns remain unknown.
based upon their locations, lncrnas can be classified into two groups.
many lncrnas are located antisense to the genes they regulate; typical examples include airn, h <dig>  and kcnq1ot <dig>  which control the imprinted expression of igf <dig>  igf2r, and kcnq <dig>  <cit> .
many other lncrnas are located far from their target genes; for example, hotair lies between hoxc <dig> and hoxc <dig> and regulates the expression of hoxd genes in humans  <cit> .
it is relatively easy to experimentally identify antisense lncrnas; nevertheless, few in-depth analyses have been performed.
the recently identified anril/cdkn2b-as on human chromosome 9p <dig> is antisense to and regulates the expression of three cyclin-dependent kinase  inhibitors: cdkn2a , arf , and cdkn2b   <cit> .
the expression of anril can induce cdkn2b silencing in cis and in trans through heterochromatin formation  <cit> .
the silencing of cdkn2b occurs via prc <dig> recruitment by anril, which makes the cdkn2b locus h3k27-trimethylated.
depletion of anril increases the expression of cdkn2b  <cit> .
the vital roles of these cdk inhibitors in cell-cycle control make anril an important molecule in multiple cancers.
it is estimated that the genomic region containing anril and cdkn2a/arf/cdkn2b is altered in 30–40% of human tumours  <cit> .
understanding the origin and evolution of anril will help to clarify its functions and decipher the evolution of many other lncrnas.
studies of lncrna evolution face three challenges.
first, most protein-coding genes in mammals were generated by genome and chromosome duplications that immediately produced all of the gene’s exons.
generated after two rounds of whole-genome duplication  <cit> , lncrnas are relatively young, and little is known about how and when they obtained multiple exons.
second, like other ncrnas, lncrnas exhibit conserved structures but divergent sequences due to compensatory mutations  <cit> .
thus, specific genome-search techniques that are more powerful than blast/blat are needed to search for homologous lncrna genes in different organisms.
third, lncrnas may be clade- or even species-specific.
when a particular lncrna is absent in a given organism, it may be difficult to determine whether the gene was never present or underwent a birth-and-death process.
the anril gene is not only important but also quite unusual in that it contains  <dig> exons, making its origin and evolution particularly intriguing.
to decipher its evolutionary history, we searched the genomes of  <dig> organisms, including non-mammalian vertebrates , non-placental mammals, non-primate placental mammals , and primates, to obtain sequences homologous to the exons of the human anril gene.
in-depth analyses of these sequences yielded several interesting conclusions.
anril originated in the eutherian ancestor and initially contained only a few exons and splicing signals.
later, it underwent clade-specific evolution, obtaining additional exons in some mammals but gradually losing exons in rodents.
notably, its genomic sequence expanded significantly in simians  through the insertion of multiple transposons.
some transposons were inserted into selective sites within the exons, and some transposons were transformed into the exons.
these transposons not only modified the sequence and structure of anril but also caused the gene to become highly conserved.
a large-scale survey of lncrnas in the database http://www.lncrnadb.org reveals that many lncrnas contain transposons.
these findings indicate for the first time that transposons have contributed significantly to lncrna evolution in simians.
this phenomenon is a remarkable aspect of lncrna evolution in primates.
RESULTS
infernal searches identify putative anril exons only in placental mammals
because lncrnas exhibit conserved structures but divergent sequences, we used infernal, a structure-based rna search program  <cit> , to search for orthologous sequences of anril exons in the genomes of  <dig> organisms .
first, we used infernal to build  <dig> covariance models  based on exons  <dig> to  <dig> of the human anril gene.
we then searched these cms against the genomes of selected primates and mammals and found that for multiple cms, high-scoring hits were obtained only in simians.
to make the cms more representative, we re-built cm <dig> to cm <dig> based on the exons in human and the putative exons in macaque and used these cms to search the  <dig> genomes .
the macaque genome was chosen because it is neither so close to the human genome that the resulting cms would be overly specific nor so distant from the human genome that the structure and sequence information contained in human exons would be weakened or blurred, thus ensuring that truly putative exons could be identified in the target genomes.
still, infernal searches failed to identify hits of ≥  <dig> successive cms with high or medium scores in vertebrates and non-placental mammals.
sequences homologous to the exons of the human anril gene  first appeared in xenarthra  and afrotheria  before occurring more widely in laurasiatheria .
however, in the branch of the species tree comprising rabbit  and rodents, the number of exons decreased steadily from rabbit to kangaroo rat; neither medium-scoring hits nor hits of successive cms were found in mouse or rat.
notably, all  <dig> exons of anril were present only in simians .
thus, anril may present an impressive case of two-stage, clade-specific evolution.
our genome search results were carefully verified to ensure their reasonability and reliability.
first, we used blat at the ucsc genome browser to search the human anril exons against the  <dig> organismal genomes.
in mammals and prosimians , the hits were much shorter than the human anril exons, and successive series of putative exons were not found.
on the other hand, many hits appeared to be false positives .
in comparison, infernal produced fewer and longer hits, with two important features:  the highest-scoring hits matched the full cms in most simians and some mammals, had extremely low e-values, and were successively distributed on the dna strand antisense to cdkn2a/cdkn2b;  other hits that did not match the cms well, with medium or low scores, had very large e-values .
this comparison indicates that, as reported previously  <cit> , infernal significantly outperformed blat in reliably identifying sequences orthologous to anril exons.
second, to confirm that the failure to detect exons in non-placental mammals and vertebrates was not due to the evolutionary distance between human/macaque and these organisms, we built cms based on the identified exons in rabbit and horse and re-searched these cms against the genomes of opossum and chicken.
again, no exons were detected.
third, we examined the multiz-aligned regions of the  <dig> exons in the  <dig> organisms in the ucsc genome browser and found that no or only extremely poorly aligned sequences were present in the vertebrates and non-placental mammals .
finally, to confirm that the failure to detect exons in mouse and rat was not influenced by the human/macaque-based cms, we built cms based on the identified exons in rabbit and re-searched these cms against the mouse and rat genomes.
again, neither medium-scoring hits nor hits of successive cms were obtained.
putative anril exons in mammals lack splicing signals
because the anril gene in mammals has only a few exons, it may not be transcribed and functional.
we indirectly addressed this issue by searching for splicing signals across exon-intron boundaries.
a recent large-scale study has revealed that the vast majority of lncrna introns are flanked by canonical splicing signals  and that lncrna genes do not differ from protein-coding genes in splicing-signal usage  <cit> .
our search found that in simians,  <dig> exon-intron boundaries had splicing signals, while  <dig> did not.
in mammals ,  <dig> exon-intron boundaries had splicing signals, while  <dig> did not .
the significant difference between simians and other mammals  suggests that mammalian anril genes may not be properly transcribed or may yield only rudimentary transcripts.
this result, together with the finding that anril contains only a few exons in mammals, suggests that anril may be functional only in simians.
multiple transposons are inserted into anril exons in simians
a surprising finding of our genome search was that some cms produced abundant hits , strongly suggesting that some anril exons are or contain transposons.
we therefore used repeatmasker with repbase, an important transposon database, to scan human anril.
the scan identified  <dig> transposons and simple repeats .
because this result indicated that transposons might be present in the anril genes of other organisms, the infernal hits were closely examined to detect potential transposons in the anril exons of multiple organisms.
for convenience, hereafter we use te# to denote abundant hits matching a fixed region within cm# and e#te# to specifically denote the region within cm#.
numerous hits matched bp 81– <dig> in cm <dig> , bp 202– <dig> in cm <dig> , bp 396– <dig> in cm <dig> , bp 30– <dig> in cm <dig> , bp 249– <dig> in cm <dig> , and bp 375– <dig> in cm <dig>  .
specifically, te <dig> is much longer than the mer1a in human exon  <dig>  our examination of infernal hits revealed that some tes were simian-specific, but many were present in mammals; nearly all of them were inserted or transformed into anril exons at or before the divergence between marmoset and other simians.
in tree shrew  and in two prosimians , the anril exons showed peculiar features:  fewer exons were present in these organisms than in laurasiatheria;  in mouse lemur, the positions of exon  <dig> and exon  <dig>  were reversed; and  in tarsier, cm <dig> and cm <dig> produced many hits with scores higher than those of exon  <dig> and exon  <dig>  moreover, of the  <dig> hits for cm <dig> on chromosome  <dig> in the marmoset genome,  <dig> had higher scores than marmoset exon  <dig> itself, indicating that these regions may be closer to human exon  <dig>  these findings suggest that the anril gene evolved substantially from mammals to prosimians and from prosimians to simians and that this process was strongly influenced by transposon activity.
transposons are inserted into exons within structural contexts
as mobile elements, active transposons insert into many sites in a genome.
what drives their insertion and whether transposons selectively insert into specific sites are interesting questions with limited answers.
we examined the hits for cm <dig> and cm <dig>  including te <dig>  te8a, and te8b, across the set of genomes.
in rabbit, exon  <dig> matched bp 1– <dig> and 307– <dig> of cm <dig> but left bp 75– <dig> of cm <dig> unmatched.
meanwhile, there is a hit elsewhere that exactly matched bp 75– <dig> of cm <dig> .
if this hit were inserted into the gap in exon  <dig>  a sequence nearly identical to human exon  <dig> would be obtained.
in the genomes of other non-simian organisms, all exon  <dig> sequences contained a gap equal to or smaller than that found in rabbit exon  <dig>  and hits matching bp 80– <dig> of cm <dig> were found widely elsewhere .
note that compared to these hits , the mer1a transposon in human anril is located closer to the 3′ end of exon  <dig>
to examine the context of the insertion of te <dig>  we used meme, a program that detects potential motifs in multiple sequences, to analyse the exon  <dig> sequences.
meme identified multiple motifs within exon  <dig> in these organisms.
a sequence alignment revealed that the two initial motifs were highly conserved.
currently, we are unable to satisfactorily explain why the sequence of motif  <dig>  in some mammalian and prosimian exon  <dig> is highly similar to the sequence of motif  <dig>  in simian e3te <dig>  probably, motif  <dig> contains conserved residues surrounding the insertion site of e3te <dig> and occurs at the position in a species-specific way.
to check whether motif  <dig> , which was present in mammalian and prosimian exon  <dig> sequences but absent in simian exon  <dig> sequences, was really lost in simians, we ran meme using exon 3 + 300 bp at the 5′ end of intron  <dig> in simians.
we found that this motif was shifted from exon  <dig> in mammals and prosimians into intron  <dig> in simians .
meme revealed that the insertion of te8b into exon  <dig> also occurred in a motif-specific context .
we further analysed intron  <dig> and intron  <dig>  in human, the simian-specific mer1a transposon penerates into intron  <dig>  additionally, the the1a transposon  in human exon  <dig> was followed by another simian-specific transposon at the 5′ end of intron  <dig>  thus, simian-specific transposons also provide the gu splicing signals for the matured exon  <dig> and exon  <dig> in simians.
compared to the 3′ ends of exon  <dig> and exon  <dig> in mammals, the 3′ ends of exon  <dig> and exon  <dig> in simians appear more structured.
tes have modified the sequences and structures of anril exons
the insertion of multiple tes into anril should affect the structure and function of this gene.
because an lncrna could fold into many different structures, making structural prediction difficult, we examined how the insertion of tes modified the sequences and structures of the anril exons.
te <dig> exhibits a palindromic structure that is conserved from lizard to human, with two terminal inverted repeats flanking a short internal region .
this specific structure causes the formation of a stem structure  .
to determine whether the stem structure contains any micrornas, we searched e3te <dig> against the microrna database  and found that it matched the stem-loop structure of two mirna families with high scores  .
next, we examined exon  <dig>  exon  <dig>  and exon  <dig>  which were transformed by te <dig>  te <dig>  and te <dig>  exon  <dig> and the highest-scoring te <dig> had highly similar sequences, but exon  <dig> contained a 20-bp sequence in mammals and prosimians that was absent in simians .
to determine the impact of this 20-bp sequence on the structure of exon  <dig>  we used rnafold to predict the structures of exon  <dig> and the highest-scoring te <dig>  we found that without this 20-bp sequence, exon  <dig> formed a more stable hairpin .
in contrast, exon  <dig> contained a 40-bp sequence that was absent in the highest-scoring te <dig> .
rnafold revealed that the 40-bp addition caused more nucleotides to pair in exon  <dig> than in the highest-scoring te <dig>  only single-nucleotide differences were found between exon  <dig> and the highest-scoring te <dig>  providing no clear evidence of structural effects.
these results indicate that multiple tes may have considerably modified the sequences and structures of the anril exons, but their impact on the global structure of anril remains unclear.
anril exons became conserved after te insertions
many transposons are suggested to have evolved in a nearly neutral manner  <cit> , but some have played active roles during primate evolution  <cit> .
thus, we performed further analyses to examine whether te insertions accelerated or slowed the evolution of anril.
the evolution of the concatenated 12s and 16s mitochondrial-rrna sequences, which have been widely used in phylogenetic studies  <cit> , was used for comparison.
first, using the f <dig> model in phylip, we calculated the pairwise distances of the concatenated rrnas, exon  <dig>  e3te <dig>  the left context of the te <dig> insertion site, and exon  <dig>  and of the concatenated rrnas, exon  <dig>  e8te8a, and the ancient exon  <dig>   between human and other species.
all three exons evolved more rapidly in mammals but less rapidly in simians compared to the two rrnas.
the insertion of te <dig> into the ancient exon  <dig> and of te8b into the ancient exon  <dig> may have contributed to the conservation of exon  <dig> and exon  <dig> in simians.
we also used the maximum composite likelihood model in mega to repeat the computation and obtained the same results .
second, to examine whether insertion or transformation into anril exons affected the evolution of the tes themselves, we built phylogenetic trees for te13/exon  <dig> , te3/e3te <dig> , te7/exon  <dig> , te12/exon  <dig> , and te8a/e8te8a  .
in these trees, the inserted and transformed tes  were grouped together with high statistical support and had short terminal branches, but the free tes  were not reliably grouped together and had long terminal branches.
thus, the transposons within the anril gene in simians are most likely vertically inherited from ancestral sequences instead of being copied from elsewhere.
relative rate tests confirmed that the highest-scoring free te <dig> evolved faster than exon  <dig> in simians; more meaningfully, exon  <dig> in mammals  evolved faster than exon  <dig> in simians   <cit> .
in the maximum-likelihood and bayesian trees, the subtrees of exon  <dig> and e3te <dig> were highly consistent, but the remaining parts were not comparable, lending further support to the above conclusion.
multiple transposons may have contributed to lncrna evolution
the above results provide compelling evidence that transposons were essential to the evolution and especially to the maturation of anril.
to examine whether the tes identified in anril also occur in other lncrnas, we built cms of these tes and searched these cms against the lncrnas in http://www.lncrnadb.org <cit> .
the cms of e7te <dig>  e12te <dig>  and e19te19c produced medium- or high-scoring hits in multiple lncrnas .
to examine whether transposons generally make important contributions to lncrnas, we used repeatmasker to scan all lncrnas in the lncrna database.
we found that many lncrnas contain multiple transposons .
these results suggest that transposons are widely associated with lncrna evolution.
discussion
most protein-coding genes in mammals were generated by genome and chromosome duplication  <cit> , but when and how lncrnas obtained multiple exons remain poorly understood.
the answers to these questions will help to decipher when lncrnas obtained their functions.
the lncrna xist obtained its exons by the pseudogenisation of protein-coding genes  <cit> , but this phenomenon is unlikely to have occurred widely.
with  <dig> exons, anril provides a valuable case study for lncrna evolution.
consistent with the hypothesis that most lncrnas occur in placental mammals  <cit> , we found that anril originated in the eutherian ancestor and gradually obtained more exons during its evolution.
the evolution of anril shows several notable features.
first, mammalian anril genes lack splicing signals and thus may not be properly transcribed.
second, simian anril genes contain splicing signals and multiple transposons.
third, pairwise sequence distances, phylogenetic trees, and relative-rate tests all indicate that anril became highly conserved in simians after transposon insertion.
fourth, our analysis of intron  <dig> and intron  <dig> indicates that simian-specific transposons may also provide splicing signals for anril in simians.
finally, anril apparently gradually lost exons during rodent evolution, containing numerous exons in guinea pig but none in mouse and rat.
although human anril is known to contain transposons  <cit>  and transposons are known to have contributed significantly to human and vertebrate lncrnas  <cit> , this report provides the first in-depth analysis showing that the insertion of multiple transposons into anril in simians may have been essential for the evolution and function of anril.
we are now examining whether significant transposon insertion also occurred in other simian lncrnas.
because lncrna exons lack a codon structure, it is unclear whether the transformation of transposons into anril exons involved a typical exonisation process  <cit> .
importantly, anril is the first typical case of clade-specific evolution of lncrnas, and further studies are needed to elucidate the birth-and-death process in rodents.
we postulate that the distinct pattern of two-stage, clade-specific evolution may be a feature of many other lncrnas.
the reported level of homology between human and mouse h <dig>  is approximately 66%, while that between human and mouse xist is approximately 49%   <cit> .
because infernal shows high power to detect orthologous sequences of anril exons in mammals, the absence of high-scoring hits or medium-scoring hits for successive cms reliably indicates the absence of anril in mouse and rat.
although no anril exons were found in mouse and rat, a large region of the mouse  chromosome 9p <dig> contains single-nucleotide mutations that are associated with many diseases, including coronary artery disease  <cit> .
a recent review has summarised these mutations  <cit> , showing that nearly all of them are located in anril introns.
aligned sequences in the ucsc genome browser show that anril introns contain multiple conserved sites or sites with strong epigenomic marks, indicating the functional importance of anril intron sequences.
some transposons have specifically contributed to mammalian evolution through the rewiring of pregnancy-related gene-regulatory networks  <cit> .
during early primate evolution, many thousands of dna elements became integrated and fixed  <cit> , and numerous primate-specific sines  triggered the evolution of primate-specific functions  <cit> .
these transposon bursts in primates likely strongly influenced the structural evolution of primate genomes  <cit> , but their functional significance remains inadequately understood.
our analysis of anril reveals that transposons not only inserted or transformed into anril exons but also caused anril to become highly conserved in simians.
thus, these transposons obtained functional significance by contributing to lncrna evolution, and the evolution of significant lncrnas and transposons may be deeply co-opted.
this postulation can reasonably explain the findings that many lncrnas contain multiple transposons and that transposons contribute significantly to human and vertebrate lncrnas  <cit> .
given that approximately one-third of lncrnas appear to have arisen within the primate lineage  <cit>  and that many transposons are primate-specific, such co-option has likely had a profound impact on primate evolution and physiology  <cit> .
specifically, the absence of anril in mouse and rat and the truncated and non-functional hotair in mouse may make the epigenomic regulation of many important genes differ between humans and mice/rats  <cit> .
transposons also influence genome methylation at many sites in somatic cells.
these observations raise a critical question: to what extent do lncrnas and transposons affect the comparability of human and mouse/rat cancers?
françois jacob postulated that the emergence of novel forms and functions over time can occur via a ‘tinkering’ process through random combinations of pre-existing elements  <cit> .
more recently, it has been suggested that the pool of transposable elements can be domesticated to serve as a ‘warehouse’ for natural selection, potentially acting as a source of lineage-specific elements  <cit> .
cordaux et al.
explored an example of tinkering along the human evolutionary lineage and identified a primate-specific chimeric gene consisting of a host gene merged with a transposable element  <cit> .
subsequent reports have further examined such phenomena  <cit> .
anril provides a remarkable example because it involves the domestication of both new and ancient transposons with impressive site selectivity.
our analyses of anril also raise the intriguing question of the fate of the transposons in tree shrew and prosimians.
our results should further promote the recently renewed interest in transposon function and evolution  <cit> .
CONCLUSIONS
transposons contribute significantly to the evolution of anril and considerable other lncrnas.
anril obtained splicing signals and became conserved after transposon insertions in simians but lost all exons in some rodents, showing a two-stage and clade-specific evolutionary process.

 x-chromosome inactivation in female placental mammals, which causes the products of genes on the x chromosome to have equal dosages in males and females, is controlled by a set of long non-coding rna genes, including xist, tsix, jpx, and tsx. the imprinted expression of some mammalian genes is also controlled by lncrnas. in addition to x-inactivation and imprinted gene expression, tissue-specific dna and chromatin methylation occurs widely in animal somatic cells because in differentiated cells, most genes are silenced by dna and chromatin methylation. because mammalian genomes contain only a few genes encoding dna methyltransferases and polycomb repressive complexes for dna and chromatin methylation and because these proteins lack sequence-specific dna-binding subunits, how these proteins are guided to different genomic sites has long been a puzzle. recently, more than lncrnas have been identified in humans, and many can bind to both polycomb proteins/dna methyltransferases and specific dna sequences. as scaffolds bridging protein-dna interactions, lncrnas are key players in dynamic and tissue-specific genome modification. because dysregulated genome modification can cause diverse diseases, especially cancers, lncrnas have triggered immense research interest in multiple fields. while lncrnas are thought to be associated with x-chromosome inactivation and imprinted gene expression in mammals and most lncrnas were first identified in humans, lncrnas have also been identified recently in multiple non-mammalian organisms. thus, like micrornas, lncrnas may be clade- or species-specific, although the details of these patterns remain unknown. based upon their locations, lncrnas can be classified into two groups. many lncrnas are located antisense to the genes they regulate; typical examples include airn, h and kcnq1ot which control the imprinted expression of igf igf2r, and kcnq. many other lncrnas are located far from their target genes; for example, hotair lies between hoxc and hoxc and regulates the expression of hoxd genes in humans. it is relatively easy to experimentally identify antisense lncrnas; nevertheless, few in-depth analyses have been performed. the recently identified anril/cdkn2b-as on human chromosome 9p is antisense to and regulates the expression of three cyclin-dependent kinase inhibitors: cdkn2a, arf, and cdkn2b. the expression of anril can induce cdkn2b silencing in cis and in trans through heterochromatin formation. the silencing of cdkn2b occurs via prc recruitment by anril, which makes the cdkn2b locus h3k27-trimethylated. depletion of anril increases the expression of cdkn2b. the vital roles of these cdk inhibitors in cell-cycle control make anril an important molecule in multiple cancers. it is estimated that the genomic region containing anril and cdkn2a/arf/cdkn2b is altered in 30–40% of human tumours. understanding the origin and evolution of anril will help to clarify its functions and decipher the evolution of many other lncrnas. studies of lncrna evolution face three challenges. first, most protein-coding genes in mammals were generated by genome and chromosome duplications that immediately produced all of the gene’s exons. generated after two rounds of whole-genome duplication, lncrnas are relatively young, and little is known about how and when they obtained multiple exons. second, like other ncrnas, lncrnas exhibit conserved structures but divergent sequences due to compensatory mutations. thus, specific genome-search techniques that are more powerful than blast/blat are needed to search for homologous lncrna genes in different organisms. third, lncrnas may be clade- or even species-specific. when a particular lncrna is absent in a given organism, it may be difficult to determine whether the gene was never present or underwent a birth-and-death process. the anril gene is not only important but also quite unusual in that it contains exons, making its origin and evolution particularly intriguing. to decipher its evolutionary history, we searched the genomes of organisms, including non-mammalian vertebrates, non-placental mammals, non-primate placental mammals, and primates, to obtain sequences homologous to the exons of the human anril gene. in-depth analyses of these sequences yielded several interesting conclusions. anril originated in the eutherian ancestor and initially contained only a few exons and splicing signals. later, it underwent clade-specific evolution, obtaining additional exons in some mammals but gradually losing exons in rodents. notably, its genomic sequence expanded significantly in simians through the insertion of multiple transposons. some transposons were inserted into selective sites within the exons, and some transposons were transformed into the exons. these transposons not only modified the sequence and structure of anril but also caused the gene to become highly conserved. a large-scale survey of lncrnas in the database http://www.lncrnadb.org reveals that many lncrnas contain transposons. these findings indicate for the first time that transposons have contributed significantly to lncrna evolution in simians. this phenomenon is a remarkable aspect of lncrna evolution in primates.  infernal searches identify putative anril exons only in placental mammals because lncrnas exhibit conserved structures but divergent sequences, we used infernal, a structure-based rna search program, to search for orthologous sequences of anril exons in the genomes of organisms. first, we used infernal to build covariance models based on exons to of the human anril gene. we then searched these cms against the genomes of selected primates and mammals and found that for multiple cms, high-scoring hits were obtained only in simians. to make the cms more representative, we re-built cm to cm based on the exons in human and the putative exons in macaque and used these cms to search the genomes. the macaque genome was chosen because it is neither so close to the human genome that the resulting cms would be overly specific nor so distant from the human genome that the structure and sequence information contained in human exons would be weakened or blurred, thus ensuring that truly putative exons could be identified in the target genomes. still, infernal searches failed to identify hits of ≥  successive cms with high or medium scores in vertebrates and non-placental mammals. sequences homologous to the exons of the human anril gene first appeared in xenarthra and afrotheria before occurring more widely in laurasiatheria. however, in the branch of the species tree comprising rabbit and rodents, the number of exons decreased steadily from rabbit to kangaroo rat; neither medium-scoring hits nor hits of successive cms were found in mouse or rat. notably, all exons of anril were present only in simians. thus, anril may present an impressive case of two-stage, clade-specific evolution. our genome search results were carefully verified to ensure their reasonability and reliability. first, we used blat at the ucsc genome browser to search the human anril exons against the organismal genomes. in mammals and prosimians, the hits were much shorter than the human anril exons, and successive series of putative exons were not found. on the other hand, many hits appeared to be false positives. in comparison, infernal produced fewer and longer hits, with two important features: the highest-scoring hits matched the full cms in most simians and some mammals, had extremely low e-values, and were successively distributed on the dna strand antisense to cdkn2a/cdkn2b; other hits that did not match the cms well, with medium or low scores, had very large e-values. this comparison indicates that, as reported previously, infernal significantly outperformed blat in reliably identifying sequences orthologous to anril exons. second, to confirm that the failure to detect exons in non-placental mammals and vertebrates was not due to the evolutionary distance between human/macaque and these organisms, we built cms based on the identified exons in rabbit and horse and re-searched these cms against the genomes of opossum and chicken. again, no exons were detected. third, we examined the multiz-aligned regions of the exons in the organisms in the ucsc genome browser and found that no or only extremely poorly aligned sequences were present in the vertebrates and non-placental mammals. finally, to confirm that the failure to detect exons in mouse and rat was not influenced by the human/macaque-based cms, we built cms based on the identified exons in rabbit and re-searched these cms against the mouse and rat genomes. again, neither medium-scoring hits nor hits of successive cms were obtained. putative anril exons in mammals lack splicing signals because the anril gene in mammals has only a few exons, it may not be transcribed and functional. we indirectly addressed this issue by searching for splicing signals across exon-intron boundaries. a recent large-scale study has revealed that the vast majority of lncrna introns are flanked by canonical splicing signals and that lncrna genes do not differ from protein-coding genes in splicing-signal usage. our search found that in simians, exon-intron boundaries had splicing signals, while did not. in mammals, exon-intron boundaries had splicing signals, while did not. the significant difference between simians and other mammals suggests that mammalian anril genes may not be properly transcribed or may yield only rudimentary transcripts. this result, together with the finding that anril contains only a few exons in mammals, suggests that anril may be functional only in simians. multiple transposons are inserted into anril exons in simians a surprising finding of our genome search was that some cms produced abundant hits, strongly suggesting that some anril exons are or contain transposons. we therefore used repeatmasker with repbase, an important transposon database, to scan human anril. the scan identified transposons and simple repeats. because this result indicated that transposons might be present in the anril genes of other organisms, the infernal hits were closely examined to detect potential transposons in the anril exons of multiple organisms. for convenience, hereafter we use te# to denote abundant hits matching a fixed region within cm# and e#te# to specifically denote the region within cm#. numerous hits matched bp 81– in cm, bp 202– in cm, bp 396– in cm, bp 30– in cm, bp 249– in cm, and bp 375– in cm. specifically, te is much longer than the mer1a in human exon our examination of infernal hits revealed that some tes were simian-specific, but many were present in mammals; nearly all of them were inserted or transformed into anril exons at or before the divergence between marmoset and other simians. in tree shrew and in two prosimians, the anril exons showed peculiar features: fewer exons were present in these organisms than in laurasiatheria; in mouse lemur, the positions of exon and exon were reversed; and in tarsier, cm and cm produced many hits with scores higher than those of exon and exon moreover, of the hits for cm on chromosome in the marmoset genome, had higher scores than marmoset exon itself, indicating that these regions may be closer to human exon these findings suggest that the anril gene evolved substantially from mammals to prosimians and from prosimians to simians and that this process was strongly influenced by transposon activity. transposons are inserted into exons within structural contexts as mobile elements, active transposons insert into many sites in a genome. what drives their insertion and whether transposons selectively insert into specific sites are interesting questions with limited answers. we examined the hits for cm and cm including te te8a, and te8b, across the set of genomes. in rabbit, exon matched bp 1– and 307– of cm but left bp 75– of cm unmatched. meanwhile, there is a hit elsewhere that exactly matched bp 75– of cm. if this hit were inserted into the gap in exon a sequence nearly identical to human exon would be obtained. in the genomes of other non-simian organisms, all exon sequences contained a gap equal to or smaller than that found in rabbit exon and hits matching bp 80– of cm were found widely elsewhere. note that compared to these hits, the mer1a transposon in human anril is located closer to the 3′ end of exon to examine the context of the insertion of te we used meme, a program that detects potential motifs in multiple sequences, to analyse the exon sequences. meme identified multiple motifs within exon in these organisms. a sequence alignment revealed that the two initial motifs were highly conserved. currently, we are unable to satisfactorily explain why the sequence of motif in some mammalian and prosimian exon is highly similar to the sequence of motif in simian e3te probably, motif contains conserved residues surrounding the insertion site of e3te and occurs at the position in a species-specific way. to check whether motif, which was present in mammalian and prosimian exon sequences but absent in simian exon sequences, was really lost in simians, we ran meme using exon 3 + 300 bp at the 5′ end of intron in simians. we found that this motif was shifted from exon in mammals and prosimians into intron in simians. meme revealed that the insertion of te8b into exon also occurred in a motif-specific context. we further analysed intron and intron in human, the simian-specific mer1a transposon penerates into intron additionally, the the1a transposon in human exon was followed by another simian-specific transposon at the 5′ end of intron thus, simian-specific transposons also provide the gu splicing signals for the matured exon and exon in simians. compared to the 3′ ends of exon and exon in mammals, the 3′ ends of exon and exon in simians appear more structured. tes have modified the sequences and structures of anril exons the insertion of multiple tes into anril should affect the structure and function of this gene. because an lncrna could fold into many different structures, making structural prediction difficult, we examined how the insertion of tes modified the sequences and structures of the anril exons. te exhibits a palindromic structure that is conserved from lizard to human, with two terminal inverted repeats flanking a short internal region. this specific structure causes the formation of a stem structure. to determine whether the stem structure contains any micrornas, we searched e3te against the microrna database and found that it matched the stem-loop structure of two mirna families with high scores. next, we examined exon exon and exon which were transformed by te te and te exon and the highest-scoring te had highly similar sequences, but exon contained a 20-bp sequence in mammals and prosimians that was absent in simians. to determine the impact of this 20-bp sequence on the structure of exon we used rnafold to predict the structures of exon and the highest-scoring te we found that without this 20-bp sequence, exon formed a more stable hairpin. in contrast, exon contained a 40-bp sequence that was absent in the highest-scoring te. rnafold revealed that the 40-bp addition caused more nucleotides to pair in exon than in the highest-scoring te only single-nucleotide differences were found between exon and the highest-scoring te providing no clear evidence of structural effects. these results indicate that multiple tes may have considerably modified the sequences and structures of the anril exons, but their impact on the global structure of anril remains unclear. anril exons became conserved after te insertions many transposons are suggested to have evolved in a nearly neutral manner, but some have played active roles during primate evolution. thus, we performed further analyses to examine whether te insertions accelerated or slowed the evolution of anril. the evolution of the concatenated 12s and 16s mitochondrial-rrna sequences, which have been widely used in phylogenetic studies, was used for comparison. first, using the f model in phylip, we calculated the pairwise distances of the concatenated rrnas, exon e3te the left context of the te insertion site, and exon and of the concatenated rrnas, exon e8te8a, and the ancient exon between human and other species. all three exons evolved more rapidly in mammals but less rapidly in simians compared to the two rrnas. the insertion of te into the ancient exon and of te8b into the ancient exon may have contributed to the conservation of exon and exon in simians. we also used the maximum composite likelihood model in mega to repeat the computation and obtained the same results. second, to examine whether insertion or transformation into anril exons affected the evolution of the tes themselves, we built phylogenetic trees for te13/exon, te3/e3te, te7/exon, te12/exon, and te8a/e8te8a. in these trees, the inserted and transformed tes were grouped together with high statistical support and had short terminal branches, but the free tes were not reliably grouped together and had long terminal branches. thus, the transposons within the anril gene in simians are most likely vertically inherited from ancestral sequences instead of being copied from elsewhere. relative rate tests confirmed that the highest-scoring free te evolved faster than exon in simians; more meaningfully, exon in mammals evolved faster than exon in simians. in the maximum-likelihood and bayesian trees, the subtrees of exon and e3te were highly consistent, but the remaining parts were not comparable, lending further support to the above conclusion. multiple transposons may have contributed to lncrna evolution the above results provide compelling evidence that transposons were essential to the evolution and especially to the maturation of anril. to examine whether the tes identified in anril also occur in other lncrnas, we built cms of these tes and searched these cms against the lncrnas in http://www.lncrnadb.org. the cms of e7te e12te and e19te19c produced medium- or high-scoring hits in multiple lncrnas. to examine whether transposons generally make important contributions to lncrnas, we used repeatmasker to scan all lncrnas in the lncrna database. we found that many lncrnas contain multiple transposons. these results suggest that transposons are widely associated with lncrna evolution. discussion most protein-coding genes in mammals were generated by genome and chromosome duplication, but when and how lncrnas obtained multiple exons remain poorly understood. the answers to these questions will help to decipher when lncrnas obtained their functions. the lncrna xist obtained its exons by the pseudogenisation of protein-coding genes, but this phenomenon is unlikely to have occurred widely. with exons, anril provides a valuable case study for lncrna evolution. consistent with the hypothesis that most lncrnas occur in placental mammals, we found that anril originated in the eutherian ancestor and gradually obtained more exons during its evolution. the evolution of anril shows several notable features. first, mammalian anril genes lack splicing signals and thus may not be properly transcribed. second, simian anril genes contain splicing signals and multiple transposons. third, pairwise sequence distances, phylogenetic trees, and relative-rate tests all indicate that anril became highly conserved in simians after transposon insertion. fourth, our analysis of intron and intron indicates that simian-specific transposons may also provide splicing signals for anril in simians. finally, anril apparently gradually lost exons during rodent evolution, containing numerous exons in guinea pig but none in mouse and rat. although human anril is known to contain transposons and transposons are known to have contributed significantly to human and vertebrate lncrnas, this report provides the first in-depth analysis showing that the insertion of multiple transposons into anril in simians may have been essential for the evolution and function of anril. we are now examining whether significant transposon insertion also occurred in other simian lncrnas. because lncrna exons lack a codon structure, it is unclear whether the transformation of transposons into anril exons involved a typical exonisation process. importantly, anril is the first typical case of clade-specific evolution of lncrnas, and further studies are needed to elucidate the birth-and-death process in rodents. we postulate that the distinct pattern of two-stage, clade-specific evolution may be a feature of many other lncrnas. the reported level of homology between human and mouse h is approximately 66%, while that between human and mouse xist is approximately 49%. because infernal shows high power to detect orthologous sequences of anril exons in mammals, the absence of high-scoring hits or medium-scoring hits for successive cms reliably indicates the absence of anril in mouse and rat. although no anril exons were found in mouse and rat, a large region of the mouse chromosome 9p contains single-nucleotide mutations that are associated with many diseases, including coronary artery disease. a recent review has summarised these mutations, showing that nearly all of them are located in anril introns. aligned sequences in the ucsc genome browser show that anril introns contain multiple conserved sites or sites with strong epigenomic marks, indicating the functional importance of anril intron sequences. some transposons have specifically contributed to mammalian evolution through the rewiring of pregnancy-related gene-regulatory networks. during early primate evolution, many thousands of dna elements became integrated and fixed, and numerous primate-specific sines triggered the evolution of primate-specific functions. these transposon bursts in primates likely strongly influenced the structural evolution of primate genomes, but their functional significance remains inadequately understood. our analysis of anril reveals that transposons not only inserted or transformed into anril exons but also caused anril to become highly conserved in simians. thus, these transposons obtained functional significance by contributing to lncrna evolution, and the evolution of significant lncrnas and transposons may be deeply co-opted. this postulation can reasonably explain the findings that many lncrnas contain multiple transposons and that transposons contribute significantly to human and vertebrate lncrnas. given that approximately one-third of lncrnas appear to have arisen within the primate lineage and that many transposons are primate-specific, such co-option has likely had a profound impact on primate evolution and physiology. specifically, the absence of anril in mouse and rat and the truncated and non-functional hotair in mouse may make the epigenomic regulation of many important genes differ between humans and mice/rats. transposons also influence genome methylation at many sites in somatic cells. these observations raise a critical question: to what extent do lncrnas and transposons affect the comparability of human and mouse/rat cancers? françois jacob postulated that the emergence of novel forms and functions over time can occur via a ‘tinkering’ process through random combinations of pre-existing elements. more recently, it has been suggested that the pool of transposable elements can be domesticated to serve as a ‘warehouse’ for natural selection, potentially acting as a source of lineage-specific elements. cordaux et al. explored an example of tinkering along the human evolutionary lineage and identified a primate-specific chimeric gene consisting of a host gene merged with a transposable element. subsequent reports have further examined such phenomena. anril provides a remarkable example because it involves the domestication of both new and ancient transposons with impressive site selectivity. our analyses of anril also raise the intriguing question of the fate of the transposons in tree shrew and prosimians. our results should further promote the recently renewed interest in transposon function and evolution.  transposons contribute significantly to the evolution of anril and considerable other lncrnas. anril obtained splicing signals and became conserved after transposon insertions in simians but lost all exons in some rodents, showing a two-stage and clade-specific evolutionary process. 
in simians, multiple transposons were inserted and transformed into exons of the anril gene, after which anril became highly conserved. anril shows a two-stage, clade-specific evolutionary process and is fully developed only in simians. many long non-coding rna genes identified in mammals have multiple exons and functional domains, allowing them to bind to polycomb proteins, dna methyltransferases, and specific dna sequences to regulate genome methylation. a further survey reveals that multiple transposons exist in many lncrnas. the domestication of multiple transposons indicates an impressive pattern of “evolutionary tinkering” and is likely to be important for anril’s structure and function. anril/cdkn2b-as originated in placental mammals, obtained additional exons during mammalian evolution but gradually lost them during rodent evolution, and reached exons only in simians. anril/cdkn2b-as consists of exons on human chromosome 9p and regulates the expression of three cyclin-dependent kinase inhibitors.
 many long non-coding rna genes identified in mammals have multiple exons and functional domains, allowing them to bind to polycomb proteins, dna methyltransferases, and specific dna sequences to regulate genome methylation. little is known about the origin and evolution of lncrnas. anril/cdkn2b-as consists of exons on human chromosome 9p and regulates the expression of three cyclin-dependent kinase inhibitors.  anril/cdkn2b-as originated in placental mammals, obtained additional exons during mammalian evolution but gradually lost them during rodent evolution, and reached exons only in simians. anril lacks splicing signals in mammals. in simians, multiple transposons were inserted and transformed into exons of the anril gene, after which anril became highly conserved. a further survey reveals that multiple transposons exist in many lncrnas.  anril shows a two-stage, clade-specific evolutionary process and is fully developed only in simians. the domestication of multiple transposons indicates an impressive pattern of “evolutionary tinkering” and is likely to be important for anril’s structure and function. the evolution of lncrnas and that of transposons may be highly co-opted in primates. many lncrnas may be functional only in simians. 