BACKGROUND
consistent with pervasive transcription of the genome  <cit> , many noncoding rnas  have recently been discovered.
in addition to abundant micrornas , an increasing number of long noncoding rnas  have been identified, and their crucial functions have been experimentally confirmed.
one key aspect of their functions is tissue-specific genome modification  <cit> .
after a cell is fully differentiated, many genes are specifically silenced by polycomb proteins and lncrna-mediated histone methylation rather than by a large army of negative transcriptional factors  <cit> .
another important aspect is genomic imprinting  <cit> .
one typical example is the xist-mediated inactivation of the whole x chromosome.
because diverse tissue-specific histone methylation and gene silencing are performed by only a handful of polycomb proteins  <cit> , the great enigma of genome modification is how a few polycomb proteins dynamically and accurately target specific dna sequences.
the discovery of a large amount of lncrnas should provide key information.
the most studied case of genome modification is x inactivation, the silencing of the majority of genes on one of the x chromosomes in somatic cells to balance gene copy number during mammalian embryogenesis  <cit> .
x inactivation is mediated by the lncrna xist  <cit> , and its details have recently been elucidated  <cit> .
regarding gene silencing and dosage compensation apart from the x chromosome, several lncrnas, including hotair, play essential roles.
hotair is co-expressed with the hoxc genes, interacts with polycomb proteins and functions in trans to repress hoxd expression  <cit> .
in addition to creating and maintaining spatiotemporally patterned hoxd expression in multiple tissues during embryogenesis, hotair is also involved in aberrant gene expression in cancers  <cit> .
the recently discovered functions of xist, hotair and other lncrnas suggest the hypothesis that numerous lncrnas should exist to bridge the limited number of polycomb proteins and the diverse tissue-specific genome modification.
moreover, many of them should be evolutionarily conserved.
tiling arrays are widely used to discover new transcripts, especially new ncrnas  <cit> .
although this method is convenient and powerful, it can only uncover noncoding transcripts expressed at particular times in particular cells.
if functional domains of an lncrna, such as xist or hotair, interact with polycomb proteins, they are likely to be conserved in animals and possibly shared by other lncrnas.
this pattern of conservation allows computational genome analysis to be used to identify new lncrnas and their functional domains, as has been successfully performed for micrornas  <cit> .
the origin and evolution of these lncrnas are also of great importance and interest, but they have so far hardly been addressed, except for xist  <cit> .
in this study, we computationally investigated hotair, the first lncrna shown to function in trans.
specifically, we investigated the following questions:  whether hotair exists in all mammals or vertebrates,  whether it has functional domains shared by other known or potential ncrnas and whether they are evolutionarily conserved,  the evolutionary features of hotair, and  the possible structures of its functional domains.
we addressed the first question using infernal, a structure-based rna homology search program  <cit> , to search the genomes of  <dig> vertebrates with exons of hotair.
we addressed the second question by thoroughly evaluating all of the hits in five animals.
we addressed the third question using paml and evonc to analyze sequences orthologous to hotair exons  <cit> .
finally, we addressed the fourth question using pmmulti and mfold to predict the structures of hotair exons and the full hotair sequences  <cit> .
our results indicated that orthologues of hotair existed only in mammals and that hotair has evolved faster than the neighbouring hoxc genes.
moreover, hotair exons showed discrete evolutionary dynamics, with some having evolved significantly faster in primates.
hits of exons as a whole, with high and low scores, were poorly conserved in animals, except between closely related species.
many hits fell within introns of, or were antisense to, protein coding genes.
a comparison of all the predicted  <dig> dimensional  structures of exon <dig> and the two conserved domains of exon <dig> revealed two invariable fragments in these structures.
these results uncovered multiple facets of hotair and the implications of our results within the wide range of lncrna evolution and function are discussed.
methods
data
the sequence of human hotair was obtained from the national center for biotechnology information  database .
the unmasked genome data  of human , chimpanzee , rhesus monkey , gorilla , cow , horse , dolphin , dog , mouse , rat , platypus , chicken , and zebrafish  were downloaded from ensembl.
the sequences corresponding to the rat orthologue of hotair exon <dig>  and the sequences corresponding to the short exon of human hoxc <dig> aligned by multiz against  <dig> mammals  were obtained from the ucsc genome browser database  <cit> .
obtaining sequences orthologous to the hotair exons
each of the  <dig> exons of human hotair was used as a query to search the genomes of rhesus monkey  and dog  in ensembl using blat  <cit> .
the sequences orthologous  to each exon, except exon <dig>  in human, rhesus monkey and dog  were aligned using pmmulti ; the sequences orthologous to exon <dig> were aligned using locarna .
with the  <dig> alignment results,  <dig> queries  were built using the cmbuild and cmcalibrate functions of infernal  and then used to search the whole genomes of  <dig> vertebrates using the cmsearch function of infernal.
two short domains of exon <dig>  the ~ <dig> bp domain a and the ~ <dig> bp domain b, were identified in orthologues of exon <dig> in all  <dig> mammals.
for the orthologous sequences of the two domains in human, rhesus monkey and dog, two additional queries  were built using the cmbuild and cmcalibrate functions of infernal.
they were then used to search the whole genomes of the  <dig> vertebrates using the cmsearch function of infernal.
sequence alignment and structure prediction
sequences of the  <dig> mammals that were orthologous to hotair exon <dig> were aligned using locarna, and sequences orthologous to all of the other hotair exons from the  <dig> mammals were aligned using pmmulti for phylogenetic analysis.
structures were predicted for the orthologues of exon <dig>  exon <dig> domain a and exon <dig> domain b using pmmulti and mfold , and the predicted structures were displayed using either mfold or pseudoviewer .
in all cases, default parameters were used unless otherwise indicated.
phylogenetic analysis
using orthologous sequences of hotair exon <dig> and the concatenated homogeneous sequences of exon <dig>  exon <dig>  exon <dig> and exon <dig> in the  <dig> mammals, two phylogenetic trees were built using the dnadist and kitsch functions of phylip .
phylogenetic analysis of the two trees was performed using the baseml function of paml .
fixed parameters included model =  <dig> ; fix_kappa =  <dig> and kappa = 2; fix_alpha =  <dig> and alpha =  <dig> ; ncatg =  <dig>  fix_rho =  <dig> and rho = 0; and cleandata =  <dig>  the parameters kappa , alpha , local clock and rates of substitution were estimated under different conditions  <cit> .
the evolution of the two conserved domains of exon <dig> and the short exon of the neighbouring hoxc <dig> gene was analyzed in  <dig> mammals using evonc  <cit> .
RESULTS
the sequences of orthologous hotair exons are poorly conserved
lncrnas that function in cis, including xist, air and kcnq1ot <dig>  <cit> , should evolve closely with their nearby target genes.
hotair is the first lncrna that has been found to function in trans to regulate remote gene expression; it is co-expressed with the hoxc genes on chromosome  <dig> and down-regulates hoxd genes on chromosome  <dig> in particular tissues in human  <cit> .
because this regulation occurs between distant genomic loci, whether it exists in other mammals and non-mammalian vertebrates is of great interest.
we first searched several mammalian genomes in the ucsc and ensembl databases for matches to hotair exons using blastn and blat  <cit> .
close matches were found in primates but not in other mammals.
for example, although the whole sequence of hotair showed apparent conservation among mammalian orthologues , individually, the five short exons  returned few hits from mammalian genomes.
using blat, only exons  <dig>   <dig> and  <dig> produced hits in dog and only exon <dig> produced hits in cow between hoxc <dig> and hoxc <dig>  and all of these hits had poor scores.
this finding suggests that if hotair has orthologues in mammals and other vertebrates, they may show low sequence conservation.
this lack of conservation is not a surprise because compensatory mutations occur widely in ncrnas, and many ncrnas are conserved in structure but not in sequence  <cit> .
hotair exists only in mammals
because ncrnas are characterised by divergent sequences and conserved structures, to further address the question of whether hotair exists in mammals and other non-mammalian vertebrates, we used infernal to search whole genomes for matches to hotair exons.
infernal is a local rna alignment and search tool based on structure conservation  <cit> .
to make the covariance model necessarily representative, we chose rhesus monkey, a primate that is more distantly related to human than chimpanzee, and dog, a mammal that produced several hits in the blat search.
first, we identified sequences orthologous to hotair exons in rhesus monkey and dog; these sequences were blat search hits with high scores and successive locations between hoxc <dig> and hoxc <dig>  second, we used the sequences of six human hotair exons and their orthologues in rhesus monkey and dog to build six queries using the cmbuild and cmcalibrate functions of infernal.
using these queries, we searched the genomes of  <dig> placental mammals , the ancestral mammal platypus, and  <dig> other vertebrates .
orthologues of the hotair exons  were obtained in all of the placental mammals but not in platypus or the other vertebrates .
notably, each query produced just one high-scoring hit in the mammalian genomes.
these hits were located between hoxc10/hoxc <dig> and hoxc12/hoxc <dig> , and all of the other hits had low scores.
query <dig> did not produce any high-scoring hits in dog, mouse or rat.
moreover, query <dig> produced good matches in primates but poor matches in other mammals, especially in mouse and rat .
these results suggest that hotair exists only in mammals and that, after some evolutionary process, it became highly conserved in primates.
the downloaded genome data were released in ensembl and ucsc at the same time, except that the platypus data were released in ensembl in dec  <dig> and in ucsc in mar  <dig>  to check if different assemblies affect genome search result, we downloaded the platypus genome data from ucsc and repeated the infernal search.
the obtained results were basically the same as those obtained from the platypus data in ensembl.
fragments of hotair exons are widely found in mammalian and non-mammalian vertebrate genomes
except for one high-scoring hit located between hoxc <dig> and hoxc <dig>  low-scoring hits of short queries  were widely obtained in mammalian and other vertebrate genomes.
these hits matched a fraction of a hotair exon and it is unclear whether they contained any functional element.
however, the  <dig>  bp query <dig> produced few low-scoring hits in mammals and other vertebrates.
because the best hit was less conserved in non-primate mammals and much shorter in mouse and rat , we inferred that the functional domain conserved in mammals should be much shorter than  <dig>  bp.
further searches addressed this issue.
the rat orthologue of exon <dig> was only  <dig> bp, which was separated in the middle by an unmatched gap of  <dig> bp.
in mouse, there was a similar gap of  <dig> bp at the same position.
this gap, therefore, divided the highly conserved initial  <dig> bp of query <dig> into two domains .
we extracted the two domains from the human, rhesus monkey and dog genomes and built query6a and query6b, respectively, as described above.
as expected, searches of the  <dig> genomes with query6a and query6b produced more hits, but no new high-scoring hits were obtained.
this result suggests that the two domains of exon <dig>  which could be the backbone of hotair, are not shared by other lncrnas.
while orthologues of domains a and b of exon <dig> were equally conserved in primates, orthologues of domain a were much less conserved in other mammals, especially in rodents .
thus, the two domains may undergo different evolutionary processes or dynamics.
query6a and query6b also produced some hits with moderate or low scores.
many of these hits matched to either two specific fragments in query6a  or a specific fragment in query6b .
whether these fragments are essential parts of the two domains and whether they are functional in vertebrates are unclear.
using lifeover in ucsc, we checked whether hits show syntenic relationships among animals, and found that the coordinates of many hits, possibly in non-annotated regions, cannot be converted.
hits of queries show distinct distributions in genomes
experimental studies have revealed that both hotair and xist bind to ezh <dig>  <cit>  and that hotair contains at least two functional domains.
the 5' domain binds suz <dig>  a component of polycomb repressive complex  <dig> , whereas the 3' domain binds lsd <dig>  <cit> .
because these proteins, especially the components of prc <dig>  are bound by many lncrnas, we speculated that not all of the low-scoring hits were functionally irrelevant.
a popular method to roughly determine whether a dna sequence is functional is to evaluate its conserved context  <cit> .
we examined the hits of all of the queries in human, chimpanzee, mouse, rat and platypus.
to evaluate the distribution of hits in the genomes and determine if they were flanked by genes with the same annotation, for each query and each genome, we counted the following:  the total number of hits,  the number of hits within introns,  the number of hits within novel transcripts or known ncrnas,  the number of hits in exons of protein coding genes,  the number of hits antisense to a gene,  the number of hits in intergenic regions, and  the number of hits in the 3'utr or 5'utr of genes .
essentially, no hits fell within exons of protein coding genes, and a majority of the hits were intergenic.
nevertheless, a few observations should be noted.
first, no hits were found within xist.
second, some hits fell within novel transcripts or known ncrnas, highlighting the possibility that there could be functional elements in these transcripts or ncrnas.
third, many hits fell within introns of protein coding genes.
finally, although query <dig> was the same length as query <dig>  query <dig> and query <dig> and query <dig> was even shorter, in all mammals, query <dig> and query <dig> produced significantly fewer hits.
this high variance cannot be accounted for simply by random hits.
one potential explanation is that exon <dig>  exon <dig> and exon <dig> may contain functional elements that are shared by other ncrnas and/or distributed more widely.
a: the total number of hits, b: the number of hits in introns, c: the number of hits in novel transcripts or known ncrnas, d: the number of hits in exons of protein coding genes, e: the number of hits antisense to a gene, f: the number of intergenic hits, g: the number of hits in the 3'utr or 5'utr of genes.
flanking genes in different animals often reflect the evolutionary conservation of a dna sequence.
we specifically examined the flanking genes of each hit of query <dig>  query6a and query6b.
query <dig> had  <dig> hits flanked by the same genes in human and chimpanzee but just  <dig> hit flanked by the same gene in mouse and rat.
moreover, no hit of query <dig> was flanked by the same genes in all four mammals.
query6a had  <dig> hits flanked by the same genes in human and chimpanzee, but none in mouse and rat.
consistent with high conservation, query6b had  <dig> hits flanked by the same genes in human and chimpanzee but none in mouse and rat.
as mouse and rat have an evolutionary distance  at least  <dig> times that of human and chimpanzee  <cit> , these results indicate that hits of these queries have moderately conserved distributions in mammalian genomes.
orthologous sequences of hotair exons show different evolutionary dynamics
most protein coding genes are produced by gene duplication followed by neofunctionalisation and/or subfunctionalisation.
because an increasing number of ncrnas have been identified, the mechanisms through which these ncrnas form and evolve are of great interest.
hotair comprises five short and one long exon.
although its origin remains obscure, some exons are apparently less conserved than others.
we therefore analyzed the molecular evolution of the hotair exons.
using the concatenated sequences orthologues to exon <dig>  exon <dig>  exon <dig> and exon <dig> and the sequences orthologous to exon <dig>  we built two phylogenetic trees using phylip  <cit>  .
assuming that nucleotide substitutions followed the hky <dig> model  <cit>  and rates of nucleotide substitutions varied among sites, we analyzed sequences of hotair exons using paml.
we first compared the two trees under multiple conditions.
under all of the conditions examined, nearly the same log-likelihood  and other parameters were obtained for the two trees.
for example, if nucleotide substitution rates were variable among sites and the molecular clock was allowed to vary from branch to branch, tree a produced a slightly larger log-likelihood  for exon <dig> but slightly smaller log-likelihoods for the other exons .
because exon <dig> is the main body of hotair, we then chose tree b to perform evolutionary analysis.
we examined whether nucleotide substitution rates varied among sites in the exons using the log-likelihood ratio test, a statistical test for comparing two models  <cit> .
the smallest 2Δlnl = 2-) =  <dig>  was obtained from orthologous sequences of exon <dig> .
the probability distribution of the test can be approximated by a chi-square distribution with one degree of freedom, with  =  <dig> , supporting the model of disparate nucleotide substitution rates.
further analysis revealed that the sequences of the orthologues of different exons had different transition/transversion rate ratio , different shape parameter of the gamma distribution , and different nucleotide substitution rates between clades .
because exon <dig>  exon <dig> and exon <dig> had significant α> <dig>  most sites in these exons should have moderate substitution rates, but a few sites had fast or slow rates of substitution.
because exons  <dig>   <dig> and  <dig> all had α <  <dig>  most sites in these exons should have low substitution rates.
in addition, the values of α in exon <dig>  indicated that domain b was more conserved than domain a, which agrees with the infernal results in which the scores of the hits to query6a in non-primate mammals were lower than the scores of hits to query6b .
these results indicate asynchronous evolution of orthologous sequences of hotair exons in mammals.
to obtain stable local clock estimations, only two local clocks were specified in each run  and the remaining species had rate r <dig> =  <dig>  *: a large unstable value.
#: r2c was not stable because the clade  was too close to the root.
to examine hotair evolution in more detail, we also investigated whether nucleotide substitution rates varied among clades.
first, we performed a log-likelihood ratio test to determine whether the hky <dig> model would fit the data better with or without a global clock.
the smallest 2Δlnl = 2-) =  <dig> , with =  <dig>  , was obtained from orthologous sequences of exon <dig> .
this result clearly disproved the global clock hypothesis.
then, we set two local clocks to investigate whether the exons evolved at different rates in mammals .
for exon <dig>  exon <dig>  exon <dig> and domain b of exon <dig>  the substitution rates in primates were significantly higher than those in the other group of mammals; for exon <dig>  exon <dig> and domain a of exon <dig>  the substitution rates were not much different between the two groups .
as the 5' domain of hotair has been found to bind to suz <dig> and the 3' domain to lsd <dig>  <cit> , whether the accelerated evolution of exon <dig>  exon <dig> and domain b of exon <dig> in primates has relationship with their protein binding function awaits further investigation.
in addition, the frequencies of nucleotide substitutions  varied significantly for different branches and at different nodes.
taken together, these results suggest that hotair may be a relatively new gene, with some exons having recently undergone an accelerated evolution.
hotair evolves faster than its neighbouring hoxc genes in mammals
hoxc genes exist widely in vertebrates; hotair, in contrast, exists only in mammals.
it was therefore interesting to determine whether hotair evolved faster than the neighbouring hoxc genes.
because hoxc <dig> is absent in rat, dolphin and platypus and the long exon of human hoxc <dig> is absent in some mammals, we compared the evolution of the short exon of hoxc <dig> with the evolution of the main part of exon <dig> of hotair in  <dig> mammals .
sequences from the ucsc database that were aligned by multiz and evonc, a program for detecting selection in noncoding regions of nucleotide sequences, were used  <cit> .
for protein coding sequences, the rate of nonsynonymous/synonymous substitution was used to detect selection pressure and positive/negative selection.
to apply such detection to noncoding sequences, the rate of substitution relative to the rate of synonymous substitution in coding sequences can be modelled by the parameter δ. δ =  <dig> indicates that a site in a noncoding sequence evolved neutrally, whereas δ <  <dig> and δ >  <dig> suggest positive and negative selection, respectively  <cit> .
we concatenated the aligned orthologous region of hotair and the aligned short exon of hoxc <dig> and analyzed the concatenated sequences.
the results are shown in table  <dig>  the log-likelihood test clearly rejected the null hypothesis that the hotair region evolved neutrally, and the value of  <dig>  found for δ <dig> in the three-category case strongly suggested that the hotair region was under positive selection and evolved faster than hoxc <dig>  the exact driving force behind this positive selection remains to be elucidated.
structure prediction reveals two domains with invariable sequences and structures
as many lncrnas, including xist and hotair, can interact with both polycomb proteins and dna sequences, it is important to identify the sequences and structures of their functional domains  <cit> .
an lncrna may have a conserved backbone and/or functional domains but have varied structure in different species, making the determination of the accurate structure of the full lncrna difficult and sometimes unnecessary.
so, instead of attempting to predict the structure of the full hotair, we focused on determining the sequence and structure of possible functional domains in its exons.
because the orthologous sequences of each hotair exon were obtained using structure-based genome searches, they had the same structures as the queries built by pmmulti and infernal.
because each query produced only one high-scoring hit located between hoxc <dig> and hoxc <dig>  the structures of queries determined by pmmulti and infernal should be reasonable.
to facilitate the determination of the sequence and structure of possible functional domains, two constraints were used.
first, in the consensus structure of each query determined by pmmulti and used by infernal, functional domains should be occupied by sequences conserved in the  <dig> mammals.
second, in all of the possible structures of an exon's orthologue in a mammal predicted by other tools, functional domains should have invariant sequences and structures.
because a 5' domain of hotair binds prc <dig>  <cit> , we assumed the 5' domain should be conserved in mammals and query <dig> did not produce any high-scoring hits in dog, mouse or rat, we tried to identify the functional domain in predicted structures of exon <dig>  pseudoviewer shows that the consensus structure for exon <dig> consists of one big arc and three substructures .
the bottom substructure contains three small loops in some mammals, but is a large loop in cow, dolphin, mouse and rat; therefore it is unlikely to be a functional domain.
the middle substructure contains three tiny loops and the top substructure contains a hairpin at its end in all animals, which indicates that they could be or contain the functional domain.
to obtain more results to aid in the determination, mfold was used to predict structures of exon1's orthologous sequence in each mammal  <cit> .
mfold predicted  <dig> structures in human,  <dig> in chimpanzee,  <dig> in rhesus monkey,  <dig> in gorilla,  <dig> in cow,  <dig> in dog,  <dig> in dolphin,  <dig> in horse,  <dig> in mouse and  <dig> in rat .
notably, the hairpin was found at the same position in  <dig> of the  <dig> predicted structures in human, and in the other  <dig> cases its sequence was embedded within neighbouring sequences .
similar results were obtained from other animals.
these mfold predicted structures provide valuable and complementary information for determining the possible position and structure of the functional domain in exon <dig>
according to an experimental study, a 3' domain of hotair, located from approximately  <dig> bp to  <dig> bp, binds lsd <dig>  <cit> .
however, infernal produced short sequences for the exon <dig> orthologues in mouse and rat , which did not include the 3' end reported in the human sequence.
postulating that the 3' functional domain should be conserved in mammals and might not be as long as  <dig> bp, we analyzed the structures of domain a  and domain b  of exon <dig> in the  <dig> mammals.
as stated previously, the structure determined by pmmulti and used by infernal was compared with all of the structures predicted by mfold.
we first examined domain b. mfold predicted  <dig> structures for domain b in human,  <dig> in chimpanzee,  <dig> in rhesus monkey,  <dig> in gorilla,  <dig> in cow,  <dig> in dog,  <dig> in dolphin,  <dig> in horse,  <dig> in mouse and  <dig> in rat.
we first checked those in human and rat, the two mammals with the greatest evolutionary distance, and found that a conserved gc-rich paired fragment existed in structures predicted for all  <dig> mammals and that it closely matched the marked part  in the structure predicted by pmmulti .
we then examined all  <dig> structures of domain b predicted by mfold for the  <dig> mammals and found that in  <dig> cases, the gc-rich paired fragment had the structure shown in figure 4a , in  <dig> cases it had the structure shown in figure 4b , in  <dig> cases it had the structure shown in figure 4c , and in  <dig> cases it had the structure shown in figure 4d .
compared with the predicted functional domain in exon <dig>  this specific structure existed more obviously in domain b. in contrast, domain a of exon <dig> was much less conserved  and was gc poor , without a clear consensus substructure in structures predicted using mfold.
the sequence and structure of the two domains occur nearly invariably in structures of full hotair
although focusing on the sequence and structure of conserved  domains is reasonable, the structure of a piece of rna can be very different from that when it is embedded by long sequences.
to validate the sequence and structure of the two conserved fragments in exon <dig> and domain b of exon <dig>  we predicted structures of the full hotair in all the mammals.
the predicted sequence and structure of the fragment in exon <dig> occurs in many structures of full hotair; remarkably, the predicted sequence and structure of the fragment in domain b of exon <dig> occurs in most structures of full hotair.
for example, mfold produced  <dig> and  <dig> structures for human and rat full hotair respectively.
in humans, the predicted structure of the fragment in exon <dig> occurs in  <dig> of  <dig> full hotair structures, and the predicted structure of the fragment in domain b of exon <dig> occurs in  <dig> of  <dig> full hotair structures.
in rats, the predicted structure of the fragment in exon <dig> occurs in  <dig> of  <dig> full hotair structures, and the predicted structure of the fragment in domain b of exon <dig> occurs in  <dig> of  <dig> full hotair structures .
given the length of hotair and the number of its predicted structures, these results strongly support the predicted functional fragment in domain b of exon <dig>  the next step should be to experimentally validate these structures and their functions.
discussion
except for xist, the origin, evolution, structure and phylogenetic distribution of lncrnas have barely been investigated.
because blat failed to find orthologous sequences of hotair exons in mammals, some exons are missing in some mammals and gaps exist in many mammals in the sequences of exon orthologues identified using the rna homology search software infernal, hotair is likely to have conserved structures but divergent sequences.
this feature should be common to lncrnas rather than being unique to hotair .
for example, xist contains both rapid evolving sequences and highly conserved domains  <cit> .
what constrains lncrnas evolution is poorly understood.
as they interact with both the prc <dig> complex and specific dna sequences, co-evolution with specific dna sequences should be an important aspect.
compared with lncrnas functioning in cis to regulate local genes, the evolutionary constraints of hotair that function in tran is more intriguing.
because the infernal search produced just one high-scoring hit in each placental mammal, where this was located between hoxc <dig> and hoxc <dig>  it can be inferred that hotair exists in eutherians and that it is younger than its neighbouring hox genes.
how hotair originated remains unclear.
phylogenetic analysis revealed that within the relatively young gene, hotair exons had asynchronous evolutionary dynamics and some exons had undergone an accelerated evolutionary process in primates.
these results indicate positive selection during hotair's evolution.
accelerated evolution is also supported by the comparison between hotair exon <dig> and the short exon of hoxc <dig>  which clearly showed that the hotair exon evolved significantly faster than the hoxc <dig> exon.
structure prediction for the orthologous sequences in  <dig> mammals showed two fragments in exon <dig> and domain b of exon <dig> with invariant base pairing and 2d structure .
these fragments, located at the 5' end and close to the 3' end of hotair, respectively, could be functional domains of hotair.
one query based on a hotair exon produced only one high-scoring hit in the genome of each mammal, where this was located between hoxc <dig> and hoxc <dig>  the location of hotair in the human genome.
however, many low-scoring hits were found in other places in mammalian and other vertebrate genomes.
because a considerable number of lncrnas are believed to interact with polycomb proteins to conduct tissue-specific genome modification, we anticipated, for several reasons, that the infernal search would identify some consensus sequences for polycomb protein binding in genomes, like the tata box in promoters and the homeobox in hox genes, that are shared by other lncrnas.
first, the four families of hox genes have demonstrated complex cross regulation and compensation during embryogenesis  <cit> , which suggests that multiple hotair-like lncrnas may be needed to mediate negative feedback and dosage balances among hox genes.
second, hox genes participate in diverse cell fate determination and reprogramming  <cit> , which suggests that hox-related lncrnas may mediate genome modification at multiple loci.
third, a recent study revealed that both hotair and the repa ncrna within xist bind to the prc <dig> complex  <cit> , although it is unclear whether the binding domains are similar.
moreover, because multiple important proteins, such as nanog, oct <dig> and sox <dig>  also interact with xist  <cit> , the scope of lncrna functions should be large.
these facts make it theoretically plausible that there should be many lncrnas that share the same or similar functional domains with hotair.
however, except for one high-scoring hit, no hits with moderate scores were obtained.
to what extent lncrnas maintain conserved function with evolved sequences is poorly understood.
it is unlikely that all of the low-scoring hits are random hits, because query <dig> and query <dig>  which are equal to and shorter than query <dig>  produced much fewer hits.
in addition, some low-scoring hits fell within novel transcripts and unknown ncrnas, and many were within introns of or antisense to protein coding genes , which is consistent with the findings that many lncrnas  are antisense to protein coding genes  <cit> .
global transcriptome analysis has revealed that a large proportion of the genome can produce transcripts from both strands  <cit>  and antisense transcription is believed to have roles in gene regulation.
meanwhile, more than  <dig>  completely intronic noncoding rnas have been found to be transcribed from the introns of 74% of all unique refseq genes, which indicates that rnas transcribed from intronic regions of genes have distinct regulatory roles and are involved in a number of processes  <cit> .
to carefully compare all hits with cdna libraries should produce more information.
the evolution of lncrna sequences, including those within the vertebrate hox clusters, has been examined recently.
these studies reveal that the evolution of many lncrnas is not consistent with the neutral evolution model, and purifying selection has acted on their promoters and some conserved sites  <cit> .
however, except for xist  <cit> , the evolution of specific ncrna genes has not been examined.
compared with ancestral regions and general intergenic sequences, lncrna sequences have been shown to exhibit lower rates of nucleotide substitution, insertion, and deletion, which can be interpreted to indicate that they have undergone purifying selection  <cit> .
our analysis of orthologues of hotair in  <dig> mammals covering multiple eutherian orders suggests that hotair exons have discrete evolutionary dynamics, and that some exons evolved significantly faster in primates than in non-primate mammals.
the analysis of orthologous sequences of hotair exon <dig> and a hoxc <dig> exon in  <dig> mammals indicates that hotair may have evolved faster than its neighbouring hoxc genes.
these results suggest that hotair may have undergone an accelerated evolution in eutherians under positive selection.
in general, a gene with important function should evolve slowly due to strong functional constraints; however the opposite is often true when the gene is young .
for example, young proteins experience more variable selection pressures than established proteins  <cit> .
that hotair is not found in non-mammalian vertebrates and that it has evolved faster than nearby hoxc genes both indicate that it is a young gene that formed after the two rounds of whole genome duplication.
given that most lncrnas, including xist, have so far only been found in mammals, it is interesting to ask when and why these lncrnas emerge in higher vertebrates to mediate genome modification.
because hotair exists in mammals, it evolves faster than hoxc <dig>  and its exons have discrete evolutionary dynamics, we postulate that hotair may have formed ab initio, possibly via the activity of transposons.
hotair is involved in the prc2-mediated silencing of chromatin at hoxd loci, but its main effect in the regulation of hox gene expression may be dosage compensation.
in this sense it is similar to xist.
the lower effectiveness of dosage compensation in birds than in mammals and the lack of general dosage compensation for sex-linked genes in chickens  <cit>  may explain why hotair, like xist, is found only in eutherians.
in this study, the structures of hotair exons were predicted using two programs.
without any experimental data for structure prediction  <cit> , we adopted a comparative computational approach to predict the sequence and structure of conserved functional domains of hotair rather than the structure of the full hotair sequence in mammals.
pmmulti and mfold were used to predict multiple potential structures for orthologues of each exon.
for example, for exon <dig>  mfold predicted  <dig> structures in human,  <dig> in chimpanzee,  <dig> in rhesus monkey,  <dig> in gorilla,  <dig> in cow,  <dig> in dog,  <dig> in dolphin,  <dig> in horse,  <dig> in mouse and  <dig> in rat.
if invariant sequence base pairing and 2d structure are found in all of the structures predicted using mfold and in the consensus structure predicted using pmmulti, it is highly likely that the sequence and structure represents a functional domain.
because to produce experimental data to determine structures of lncrnas is sophisticated and time-consuming, the results of our structure prediction should be valuable for further experimental studies of hotair, and the methods should be applicable to studies of other lncrnas.
CONCLUSIONS
the lncrna hotair has poorly conserved sequences and considerably conserved structures in  <dig> examined mammals .
it shows distinct evolutionary features and has evolved faster than nearby hoxc genes.
given that exons 1- <dig> are very short, exon <dig> and a domain of exon <dig>  are absent in kangaroo, and exon <dig> is absent in mouse, rat and kangaroo, a highly conserved  <dig> bp domain in exon <dig>  initially appeared in kangaroo, should be the backbone of hotair.
these findings suggest the ab initio generation of hotair in marsupials.
structure prediction identifies two fragments, in the 5' end exon <dig> and the 3' end domain b of exon <dig> respectively, with their sequence and structure invariably occurring in various predicted structures of exon <dig>  the domain b of exon <dig> and the full hotair.
these are supported by experimental findings.
to compare the origin and evolutionary features of hotair with xist suggests that many lncrnas, may first form in marsupials and then have undergone a rapid evolution in eutherians.
an interesting question is whether their origin and evolution is intrinsically associated.
note added in proof
during the review of the manuscript, we downloaded, searched and analyzed opossum and kangaroo genome data  and opossum  in oct 2010).
whole genome search of opossum did not produce high-scoring hits.
searching the kangaroo genome with query <dig>  query <dig>  query <dig> and query <dig> each produced a high-scoring hit, which have successive addresses in genescaffold_ <dig> .
query6's hit matches query <dig> from  <dig> bp to  <dig> bp, exactly as that in rat.
what is interesting is that exon <dig> and exon <dig> were absent, domain b of exon <dig> produced a high-scoring hit, and domain a of exon <dig> was not identified.
these results, together with the phylogenetic analysis, lead to two suggestions.
first, hotair, like other lncrnas, first formed in some marsupials and underwent a rapid evolution in eutherians.
second, domain b of exon <dig> may be the backbone of hotair, because it is the only relatively long piece conserved in marsupial and eutherians.
monotremes have multiple x chromosomes but it is not clear whether they undergo dosage compensation; marsupials show dosage compensation but they lack xist  <cit> .
it is found that female marsupials may use an ancestral dosage compensation mechanism that differs from, but share common properties with, the xist based in eutherians  <cit> .
since protein-coding genes that flank the eutherian xic are well-conserved in m. domestica and vertebrates and there is a surprising break in synteny with eutherian mammals and other vertebrates, it is suggested that during the evolution of the marsupial x chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes  <cit> .
the situation of hotair, which is flanked by hoxc <dig> and hoxc <dig>  not found in vertebrates and initially occurs in marsupials as revealed in this study, seems quite similar to xist.
this raises the interesting question of whether hotair and xist, and possibly also some other lncrnas, have undergone the same evolutionary process.
abbreviations
ncrna: noncoding rna; lncrna: long noncoding rna; prc2: polycomb repressive complex  <dig>
authors' contributions
sh performed genome search, results analysis and structure prediction; sl participated in genome search; hz conceived the project, performed phylogenetic analysis, structure prediction and wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
this file contains table s <dig>  figure s <dig>  figure s <dig>  and figure s <dig>
click here for file
additional file 2
this file contains orthologues of hotair exons and their coordinates in mammals.
click here for file
additional file 3
this file contains predicted structures of full hotair in human and rat.
click here for file
acknowledgements
we are grateful to eric nawrocki at the hhmi janelia farm research campus and wendy wong at cornell university for their help in using infernal and evonc.
we also thank two anonymous reviewers for their valuable comments.
h.z.
was supported by guangdong province foundation for returned scholars and national natural science foundation of china .

 consistent with pervasive transcription of the genome, many noncoding rnas have recently been discovered. in addition to abundant micrornas, an increasing number of long noncoding rnas have been identified, and their crucial functions have been experimentally confirmed. one key aspect of their functions is tissue-specific genome modification. after a cell is fully differentiated, many genes are specifically silenced by polycomb proteins and lncrna-mediated histone methylation rather than by a large army of negative transcriptional factors. another important aspect is genomic imprinting. one typical example is the xist-mediated inactivation of the whole x chromosome. because diverse tissue-specific histone methylation and gene silencing are performed by only a handful of polycomb proteins, the great enigma of genome modification is how a few polycomb proteins dynamically and accurately target specific dna sequences. the discovery of a large amount of lncrnas should provide key information. the most studied case of genome modification is x inactivation, the silencing of the majority of genes on one of the x chromosomes in somatic cells to balance gene copy number during mammalian embryogenesis. x inactivation is mediated by the lncrna xist, and its details have recently been elucidated. regarding gene silencing and dosage compensation apart from the x chromosome, several lncrnas, including hotair, play essential roles. hotair is co-expressed with the hoxc genes, interacts with polycomb proteins and functions in trans to repress hoxd expression. in addition to creating and maintaining spatiotemporally patterned hoxd expression in multiple tissues during embryogenesis, hotair is also involved in aberrant gene expression in cancers. the recently discovered functions of xist, hotair and other lncrnas suggest the hypothesis that numerous lncrnas should exist to bridge the limited number of polycomb proteins and the diverse tissue-specific genome modification. moreover, many of them should be evolutionarily conserved. tiling arrays are widely used to discover new transcripts, especially new ncrnas. although this method is convenient and powerful, it can only uncover noncoding transcripts expressed at particular times in particular cells. if functional domains of an lncrna, such as xist or hotair, interact with polycomb proteins, they are likely to be conserved in animals and possibly shared by other lncrnas. this pattern of conservation allows computational genome analysis to be used to identify new lncrnas and their functional domains, as has been successfully performed for micrornas. the origin and evolution of these lncrnas are also of great importance and interest, but they have so far hardly been addressed, except for xist. in this study, we computationally investigated hotair, the first lncrna shown to function in trans. specifically, we investigated the following questions: whether hotair exists in all mammals or vertebrates, whether it has functional domains shared by other known or potential ncrnas and whether they are evolutionarily conserved, the evolutionary features of hotair, and the possible structures of its functional domains. we addressed the first question using infernal, a structure-based rna homology search program, to search the genomes of vertebrates with exons of hotair. we addressed the second question by thoroughly evaluating all of the hits in five animals. we addressed the third question using paml and evonc to analyze sequences orthologous to hotair exons. finally, we addressed the fourth question using pmmulti and mfold to predict the structures of hotair exons and the full hotair sequences. our results indicated that orthologues of hotair existed only in mammals and that hotair has evolved faster than the neighbouring hoxc genes. moreover, hotair exons showed discrete evolutionary dynamics, with some having evolved significantly faster in primates. hits of exons as a whole, with high and low scores, were poorly conserved in animals, except between closely related species. many hits fell within introns of, or were antisense to, protein coding genes. a comparison of all the predicted dimensional structures of exon and the two conserved domains of exon revealed two invariable fragments in these structures. these results uncovered multiple facets of hotair and the implications of our results within the wide range of lncrna evolution and function are discussed. methods data the sequence of human hotair was obtained from the national center for biotechnology information database. the unmasked genome data of human, chimpanzee, rhesus monkey, gorilla, cow, horse, dolphin, dog, mouse, rat, platypus, chicken, and zebrafish were downloaded from ensembl. the sequences corresponding to the rat orthologue of hotair exon and the sequences corresponding to the short exon of human hoxc aligned by multiz against mammals were obtained from the ucsc genome browser database. obtaining sequences orthologous to the hotair exons each of the exons of human hotair was used as a query to search the genomes of rhesus monkey and dog in ensembl using blat. the sequences orthologous to each exon, except exon in human, rhesus monkey and dog were aligned using pmmulti; the sequences orthologous to exon were aligned using locarna. with the alignment results, queries were built using the cmbuild and cmcalibrate functions of infernal and then used to search the whole genomes of vertebrates using the cmsearch function of infernal. two short domains of exon the ~ bp domain a and the ~ bp domain b, were identified in orthologues of exon in all mammals. for the orthologous sequences of the two domains in human, rhesus monkey and dog, two additional queries were built using the cmbuild and cmcalibrate functions of infernal. they were then used to search the whole genomes of the vertebrates using the cmsearch function of infernal. sequence alignment and structure prediction sequences of the mammals that were orthologous to hotair exon were aligned using locarna, and sequences orthologous to all of the other hotair exons from the mammals were aligned using pmmulti for phylogenetic analysis. structures were predicted for the orthologues of exon exon domain a and exon domain b using pmmulti and mfold, and the predicted structures were displayed using either mfold or pseudoviewer. in all cases, default parameters were used unless otherwise indicated. phylogenetic analysis using orthologous sequences of hotair exon and the concatenated homogeneous sequences of exon exon exon and exon in the mammals, two phylogenetic trees were built using the dnadist and kitsch functions of phylip. phylogenetic analysis of the two trees was performed using the baseml function of paml. fixed parameters included model =; fix_kappa = and kappa = 2; fix_alpha = and alpha =; ncatg = fix_rho = and rho = 0; and cleandata = the parameters kappa, alpha, local clock and rates of substitution were estimated under different conditions. the evolution of the two conserved domains of exon and the short exon of the neighbouring hoxc gene was analyzed in mammals using evonc.  the sequences of orthologous hotair exons are poorly conserved lncrnas that function in cis, including xist, air and kcnq1ot, should evolve closely with their nearby target genes. hotair is the first lncrna that has been found to function in trans to regulate remote gene expression; it is co-expressed with the hoxc genes on chromosome and down-regulates hoxd genes on chromosome in particular tissues in human. because this regulation occurs between distant genomic loci, whether it exists in other mammals and non-mammalian vertebrates is of great interest. we first searched several mammalian genomes in the ucsc and ensembl databases for matches to hotair exons using blastn and blat. close matches were found in primates but not in other mammals. for example, although the whole sequence of hotair showed apparent conservation among mammalian orthologues, individually, the five short exons returned few hits from mammalian genomes. using blat, only exons and produced hits in dog and only exon produced hits in cow between hoxc and hoxc and all of these hits had poor scores. this finding suggests that if hotair has orthologues in mammals and other vertebrates, they may show low sequence conservation. this lack of conservation is not a surprise because compensatory mutations occur widely in ncrnas, and many ncrnas are conserved in structure but not in sequence. hotair exists only in mammals because ncrnas are characterised by divergent sequences and conserved structures, to further address the question of whether hotair exists in mammals and other non-mammalian vertebrates, we used infernal to search whole genomes for matches to hotair exons. infernal is a local rna alignment and search tool based on structure conservation. to make the covariance model necessarily representative, we chose rhesus monkey, a primate that is more distantly related to human than chimpanzee, and dog, a mammal that produced several hits in the blat search. first, we identified sequences orthologous to hotair exons in rhesus monkey and dog; these sequences were blat search hits with high scores and successive locations between hoxc and hoxc second, we used the sequences of six human hotair exons and their orthologues in rhesus monkey and dog to build six queries using the cmbuild and cmcalibrate functions of infernal. using these queries, we searched the genomes of placental mammals, the ancestral mammal platypus, and other vertebrates. orthologues of the hotair exons were obtained in all of the placental mammals but not in platypus or the other vertebrates. notably, each query produced just one high-scoring hit in the mammalian genomes. these hits were located between hoxc10/hoxc and hoxc12/hoxc, and all of the other hits had low scores. query did not produce any high-scoring hits in dog, mouse or rat. moreover, query produced good matches in primates but poor matches in other mammals, especially in mouse and rat. these results suggest that hotair exists only in mammals and that, after some evolutionary process, it became highly conserved in primates. the downloaded genome data were released in ensembl and ucsc at the same time, except that the platypus data were released in ensembl in dec and in ucsc in mar to check if different assemblies affect genome search result, we downloaded the platypus genome data from ucsc and repeated the infernal search. the obtained results were basically the same as those obtained from the platypus data in ensembl. fragments of hotair exons are widely found in mammalian and non-mammalian vertebrate genomes except for one high-scoring hit located between hoxc and hoxc low-scoring hits of short queries were widely obtained in mammalian and other vertebrate genomes. these hits matched a fraction of a hotair exon and it is unclear whether they contained any functional element. however, the bp query produced few low-scoring hits in mammals and other vertebrates. because the best hit was less conserved in non-primate mammals and much shorter in mouse and rat, we inferred that the functional domain conserved in mammals should be much shorter than bp. further searches addressed this issue. the rat orthologue of exon was only bp, which was separated in the middle by an unmatched gap of bp. in mouse, there was a similar gap of bp at the same position. this gap, therefore, divided the highly conserved initial bp of query into two domains. we extracted the two domains from the human, rhesus monkey and dog genomes and built query6a and query6b, respectively, as described above. as expected, searches of the genomes with query6a and query6b produced more hits, but no new high-scoring hits were obtained. this result suggests that the two domains of exon which could be the backbone of hotair, are not shared by other lncrnas. while orthologues of domains a and b of exon were equally conserved in primates, orthologues of domain a were much less conserved in other mammals, especially in rodents. thus, the two domains may undergo different evolutionary processes or dynamics. query6a and query6b also produced some hits with moderate or low scores. many of these hits matched to either two specific fragments in query6a or a specific fragment in query6b. whether these fragments are essential parts of the two domains and whether they are functional in vertebrates are unclear. using lifeover in ucsc, we checked whether hits show syntenic relationships among animals, and found that the coordinates of many hits, possibly in non-annotated regions, cannot be converted. hits of queries show distinct distributions in genomes experimental studies have revealed that both hotair and xist bind to ezh and that hotair contains at least two functional domains. the 5' domain binds suz a component of polycomb repressive complex, whereas the 3' domain binds lsd. because these proteins, especially the components of prc are bound by many lncrnas, we speculated that not all of the low-scoring hits were functionally irrelevant. a popular method to roughly determine whether a dna sequence is functional is to evaluate its conserved context. we examined the hits of all of the queries in human, chimpanzee, mouse, rat and platypus. to evaluate the distribution of hits in the genomes and determine if they were flanked by genes with the same annotation, for each query and each genome, we counted the following: the total number of hits, the number of hits within introns, the number of hits within novel transcripts or known ncrnas, the number of hits in exons of protein coding genes, the number of hits antisense to a gene, the number of hits in intergenic regions, and the number of hits in the 3'utr or 5'utr of genes. essentially, no hits fell within exons of protein coding genes, and a majority of the hits were intergenic. nevertheless, a few observations should be noted. first, no hits were found within xist. second, some hits fell within novel transcripts or known ncrnas, highlighting the possibility that there could be functional elements in these transcripts or ncrnas. third, many hits fell within introns of protein coding genes. finally, although query was the same length as query query and query and query was even shorter, in all mammals, query and query produced significantly fewer hits. this high variance cannot be accounted for simply by random hits. one potential explanation is that exon exon and exon may contain functional elements that are shared by other ncrnas and/or distributed more widely. a: the total number of hits, b: the number of hits in introns, c: the number of hits in novel transcripts or known ncrnas, d: the number of hits in exons of protein coding genes, e: the number of hits antisense to a gene, f: the number of intergenic hits, g: the number of hits in the 3'utr or 5'utr of genes. flanking genes in different animals often reflect the evolutionary conservation of a dna sequence. we specifically examined the flanking genes of each hit of query query6a and query6b. query had hits flanked by the same genes in human and chimpanzee but just hit flanked by the same gene in mouse and rat. moreover, no hit of query was flanked by the same genes in all four mammals. query6a had hits flanked by the same genes in human and chimpanzee, but none in mouse and rat. consistent with high conservation, query6b had hits flanked by the same genes in human and chimpanzee but none in mouse and rat. as mouse and rat have an evolutionary distance at least times that of human and chimpanzee, these results indicate that hits of these queries have moderately conserved distributions in mammalian genomes. orthologous sequences of hotair exons show different evolutionary dynamics most protein coding genes are produced by gene duplication followed by neofunctionalisation and/or subfunctionalisation. because an increasing number of ncrnas have been identified, the mechanisms through which these ncrnas form and evolve are of great interest. hotair comprises five short and one long exon. although its origin remains obscure, some exons are apparently less conserved than others. we therefore analyzed the molecular evolution of the hotair exons. using the concatenated sequences orthologues to exon exon exon and exon and the sequences orthologous to exon we built two phylogenetic trees using phylip. assuming that nucleotide substitutions followed the hky model and rates of nucleotide substitutions varied among sites, we analyzed sequences of hotair exons using paml. we first compared the two trees under multiple conditions. under all of the conditions examined, nearly the same log-likelihood and other parameters were obtained for the two trees. for example, if nucleotide substitution rates were variable among sites and the molecular clock was allowed to vary from branch to branch, tree a produced a slightly larger log-likelihood for exon but slightly smaller log-likelihoods for the other exons. because exon is the main body of hotair, we then chose tree b to perform evolutionary analysis. we examined whether nucleotide substitution rates varied among sites in the exons using the log-likelihood ratio test, a statistical test for comparing two models. the smallest 2Δlnl = 2-) = was obtained from orthologous sequences of exon. the probability distribution of the test can be approximated by a chi-square distribution with one degree of freedom, with =, supporting the model of disparate nucleotide substitution rates. further analysis revealed that the sequences of the orthologues of different exons had different transition/transversion rate ratio, different shape parameter of the gamma distribution, and different nucleotide substitution rates between clades. because exon exon and exon had significant α> most sites in these exons should have moderate substitution rates, but a few sites had fast or slow rates of substitution. because exons and all had α < most sites in these exons should have low substitution rates. in addition, the values of α in exon indicated that domain b was more conserved than domain a, which agrees with the infernal results in which the scores of the hits to query6a in non-primate mammals were lower than the scores of hits to query6b. these results indicate asynchronous evolution of orthologous sequences of hotair exons in mammals. to obtain stable local clock estimations, only two local clocks were specified in each run and the remaining species had rate r = *: a large unstable value. #: r2c was not stable because the clade was too close to the root. to examine hotair evolution in more detail, we also investigated whether nucleotide substitution rates varied among clades. first, we performed a log-likelihood ratio test to determine whether the hky model would fit the data better with or without a global clock. the smallest 2Δlnl = 2-) =, with =, was obtained from orthologous sequences of exon. this result clearly disproved the global clock hypothesis. then, we set two local clocks to investigate whether the exons evolved at different rates in mammals. for exon exon exon and domain b of exon the substitution rates in primates were significantly higher than those in the other group of mammals; for exon exon and domain a of exon the substitution rates were not much different between the two groups. as the 5' domain of hotair has been found to bind to suz and the 3' domain to lsd, whether the accelerated evolution of exon exon and domain b of exon in primates has relationship with their protein binding function awaits further investigation. in addition, the frequencies of nucleotide substitutions varied significantly for different branches and at different nodes. taken together, these results suggest that hotair may be a relatively new gene, with some exons having recently undergone an accelerated evolution. hotair evolves faster than its neighbouring hoxc genes in mammals hoxc genes exist widely in vertebrates; hotair, in contrast, exists only in mammals. it was therefore interesting to determine whether hotair evolved faster than the neighbouring hoxc genes. because hoxc is absent in rat, dolphin and platypus and the long exon of human hoxc is absent in some mammals, we compared the evolution of the short exon of hoxc with the evolution of the main part of exon of hotair in mammals. sequences from the ucsc database that were aligned by multiz and evonc, a program for detecting selection in noncoding regions of nucleotide sequences, were used. for protein coding sequences, the rate of nonsynonymous/synonymous substitution was used to detect selection pressure and positive/negative selection. to apply such detection to noncoding sequences, the rate of substitution relative to the rate of synonymous substitution in coding sequences can be modelled by the parameter δ. δ = indicates that a site in a noncoding sequence evolved neutrally, whereas δ < and δ > suggest positive and negative selection, respectively. we concatenated the aligned orthologous region of hotair and the aligned short exon of hoxc and analyzed the concatenated sequences. the results are shown in table the log-likelihood test clearly rejected the null hypothesis that the hotair region evolved neutrally, and the value of found for δ in the three-category case strongly suggested that the hotair region was under positive selection and evolved faster than hoxc the exact driving force behind this positive selection remains to be elucidated. structure prediction reveals two domains with invariable sequences and structures as many lncrnas, including xist and hotair, can interact with both polycomb proteins and dna sequences, it is important to identify the sequences and structures of their functional domains. an lncrna may have a conserved backbone and/or functional domains but have varied structure in different species, making the determination of the accurate structure of the full lncrna difficult and sometimes unnecessary. so, instead of attempting to predict the structure of the full hotair, we focused on determining the sequence and structure of possible functional domains in its exons. because the orthologous sequences of each hotair exon were obtained using structure-based genome searches, they had the same structures as the queries built by pmmulti and infernal. because each query produced only one high-scoring hit located between hoxc and hoxc the structures of queries determined by pmmulti and infernal should be reasonable. to facilitate the determination of the sequence and structure of possible functional domains, two constraints were used. first, in the consensus structure of each query determined by pmmulti and used by infernal, functional domains should be occupied by sequences conserved in the mammals. second, in all of the possible structures of an exon's orthologue in a mammal predicted by other tools, functional domains should have invariant sequences and structures. because a 5' domain of hotair binds prc, we assumed the 5' domain should be conserved in mammals and query did not produce any high-scoring hits in dog, mouse or rat, we tried to identify the functional domain in predicted structures of exon pseudoviewer shows that the consensus structure for exon consists of one big arc and three substructures. the bottom substructure contains three small loops in some mammals, but is a large loop in cow, dolphin, mouse and rat; therefore it is unlikely to be a functional domain. the middle substructure contains three tiny loops and the top substructure contains a hairpin at its end in all animals, which indicates that they could be or contain the functional domain. to obtain more results to aid in the determination, mfold was used to predict structures of exon1's orthologous sequence in each mammal. mfold predicted structures in human, in chimpanzee, in rhesus monkey, in gorilla, in cow, in dog, in dolphin, in horse, in mouse and in rat. notably, the hairpin was found at the same position in of the predicted structures in human, and in the other cases its sequence was embedded within neighbouring sequences. similar results were obtained from other animals. these mfold predicted structures provide valuable and complementary information for determining the possible position and structure of the functional domain in exon according to an experimental study, a 3' domain of hotair, located from approximately bp to bp, binds lsd. however, infernal produced short sequences for the exon orthologues in mouse and rat, which did not include the 3' end reported in the human sequence. postulating that the 3' functional domain should be conserved in mammals and might not be as long as bp, we analyzed the structures of domain a and domain b of exon in the mammals. as stated previously, the structure determined by pmmulti and used by infernal was compared with all of the structures predicted by mfold. we first examined domain b. mfold predicted structures for domain b in human, in chimpanzee, in rhesus monkey, in gorilla, in cow, in dog, in dolphin, in horse, in mouse and in rat. we first checked those in human and rat, the two mammals with the greatest evolutionary distance, and found that a conserved gc-rich paired fragment existed in structures predicted for all mammals and that it closely matched the marked part in the structure predicted by pmmulti. we then examined all structures of domain b predicted by mfold for the mammals and found that in cases, the gc-rich paired fragment had the structure shown in figure 4a, in cases it had the structure shown in figure 4b, in cases it had the structure shown in figure 4c, and in cases it had the structure shown in figure 4d. compared with the predicted functional domain in exon this specific structure existed more obviously in domain b. in contrast, domain a of exon was much less conserved and was gc poor, without a clear consensus substructure in structures predicted using mfold. the sequence and structure of the two domains occur nearly invariably in structures of full hotair although focusing on the sequence and structure of conserved domains is reasonable, the structure of a piece of rna can be very different from that when it is embedded by long sequences. to validate the sequence and structure of the two conserved fragments in exon and domain b of exon we predicted structures of the full hotair in all the mammals. the predicted sequence and structure of the fragment in exon occurs in many structures of full hotair; remarkably, the predicted sequence and structure of the fragment in domain b of exon occurs in most structures of full hotair. for example, mfold produced and structures for human and rat full hotair respectively. in humans, the predicted structure of the fragment in exon occurs in of full hotair structures, and the predicted structure of the fragment in domain b of exon occurs in of full hotair structures. in rats, the predicted structure of the fragment in exon occurs in of full hotair structures, and the predicted structure of the fragment in domain b of exon occurs in of full hotair structures. given the length of hotair and the number of its predicted structures, these results strongly support the predicted functional fragment in domain b of exon the next step should be to experimentally validate these structures and their functions. discussion except for xist, the origin, evolution, structure and phylogenetic distribution of lncrnas have barely been investigated. because blat failed to find orthologous sequences of hotair exons in mammals, some exons are missing in some mammals and gaps exist in many mammals in the sequences of exon orthologues identified using the rna homology search software infernal, hotair is likely to have conserved structures but divergent sequences. this feature should be common to lncrnas rather than being unique to hotair. for example, xist contains both rapid evolving sequences and highly conserved domains. what constrains lncrnas evolution is poorly understood. as they interact with both the prc complex and specific dna sequences, co-evolution with specific dna sequences should be an important aspect. compared with lncrnas functioning in cis to regulate local genes, the evolutionary constraints of hotair that function in tran is more intriguing. because the infernal search produced just one high-scoring hit in each placental mammal, where this was located between hoxc and hoxc it can be inferred that hotair exists in eutherians and that it is younger than its neighbouring hox genes. how hotair originated remains unclear. phylogenetic analysis revealed that within the relatively young gene, hotair exons had asynchronous evolutionary dynamics and some exons had undergone an accelerated evolutionary process in primates. these results indicate positive selection during hotair's evolution. accelerated evolution is also supported by the comparison between hotair exon and the short exon of hoxc which clearly showed that the hotair exon evolved significantly faster than the hoxc exon. structure prediction for the orthologous sequences in mammals showed two fragments in exon and domain b of exon with invariant base pairing and 2d structure. these fragments, located at the 5' end and close to the 3' end of hotair, respectively, could be functional domains of hotair. one query based on a hotair exon produced only one high-scoring hit in the genome of each mammal, where this was located between hoxc and hoxc the location of hotair in the human genome. however, many low-scoring hits were found in other places in mammalian and other vertebrate genomes. because a considerable number of lncrnas are believed to interact with polycomb proteins to conduct tissue-specific genome modification, we anticipated, for several reasons, that the infernal search would identify some consensus sequences for polycomb protein binding in genomes, like the tata box in promoters and the homeobox in hox genes, that are shared by other lncrnas. first, the four families of hox genes have demonstrated complex cross regulation and compensation during embryogenesis, which suggests that multiple hotair-like lncrnas may be needed to mediate negative feedback and dosage balances among hox genes. second, hox genes participate in diverse cell fate determination and reprogramming, which suggests that hox-related lncrnas may mediate genome modification at multiple loci. third, a recent study revealed that both hotair and the repa ncrna within xist bind to the prc complex, although it is unclear whether the binding domains are similar. moreover, because multiple important proteins, such as nanog, oct and sox also interact with xist, the scope of lncrna functions should be large. these facts make it theoretically plausible that there should be many lncrnas that share the same or similar functional domains with hotair. however, except for one high-scoring hit, no hits with moderate scores were obtained. to what extent lncrnas maintain conserved function with evolved sequences is poorly understood. it is unlikely that all of the low-scoring hits are random hits, because query and query which are equal to and shorter than query produced much fewer hits. in addition, some low-scoring hits fell within novel transcripts and unknown ncrnas, and many were within introns of or antisense to protein coding genes, which is consistent with the findings that many lncrnas are antisense to protein coding genes. global transcriptome analysis has revealed that a large proportion of the genome can produce transcripts from both strands and antisense transcription is believed to have roles in gene regulation. meanwhile, more than completely intronic noncoding rnas have been found to be transcribed from the introns of 74% of all unique refseq genes, which indicates that rnas transcribed from intronic regions of genes have distinct regulatory roles and are involved in a number of processes. to carefully compare all hits with cdna libraries should produce more information. the evolution of lncrna sequences, including those within the vertebrate hox clusters, has been examined recently. these studies reveal that the evolution of many lncrnas is not consistent with the neutral evolution model, and purifying selection has acted on their promoters and some conserved sites. however, except for xist, the evolution of specific ncrna genes has not been examined. compared with ancestral regions and general intergenic sequences, lncrna sequences have been shown to exhibit lower rates of nucleotide substitution, insertion, and deletion, which can be interpreted to indicate that they have undergone purifying selection. our analysis of orthologues of hotair in mammals covering multiple eutherian orders suggests that hotair exons have discrete evolutionary dynamics, and that some exons evolved significantly faster in primates than in non-primate mammals. the analysis of orthologous sequences of hotair exon and a hoxc exon in mammals indicates that hotair may have evolved faster than its neighbouring hoxc genes. these results suggest that hotair may have undergone an accelerated evolution in eutherians under positive selection. in general, a gene with important function should evolve slowly due to strong functional constraints; however the opposite is often true when the gene is young. for example, young proteins experience more variable selection pressures than established proteins. that hotair is not found in non-mammalian vertebrates and that it has evolved faster than nearby hoxc genes both indicate that it is a young gene that formed after the two rounds of whole genome duplication. given that most lncrnas, including xist, have so far only been found in mammals, it is interesting to ask when and why these lncrnas emerge in higher vertebrates to mediate genome modification. because hotair exists in mammals, it evolves faster than hoxc and its exons have discrete evolutionary dynamics, we postulate that hotair may have formed ab initio, possibly via the activity of transposons. hotair is involved in the prc2-mediated silencing of chromatin at hoxd loci, but its main effect in the regulation of hox gene expression may be dosage compensation. in this sense it is similar to xist. the lower effectiveness of dosage compensation in birds than in mammals and the lack of general dosage compensation for sex-linked genes in chickens may explain why hotair, like xist, is found only in eutherians. in this study, the structures of hotair exons were predicted using two programs. without any experimental data for structure prediction, we adopted a comparative computational approach to predict the sequence and structure of conserved functional domains of hotair rather than the structure of the full hotair sequence in mammals. pmmulti and mfold were used to predict multiple potential structures for orthologues of each exon. for example, for exon mfold predicted structures in human, in chimpanzee, in rhesus monkey, in gorilla, in cow, in dog, in dolphin, in horse, in mouse and in rat. if invariant sequence base pairing and 2d structure are found in all of the structures predicted using mfold and in the consensus structure predicted using pmmulti, it is highly likely that the sequence and structure represents a functional domain. because to produce experimental data to determine structures of lncrnas is sophisticated and time-consuming, the results of our structure prediction should be valuable for further experimental studies of hotair, and the methods should be applicable to studies of other lncrnas.  the lncrna hotair has poorly conserved sequences and considerably conserved structures in examined mammals. it shows distinct evolutionary features and has evolved faster than nearby hoxc genes. given that exons 1- are very short, exon and a domain of exon are absent in kangaroo, and exon is absent in mouse, rat and kangaroo, a highly conserved bp domain in exon initially appeared in kangaroo, should be the backbone of hotair. these findings suggest the ab initio generation of hotair in marsupials. structure prediction identifies two fragments, in the 5' end exon and the 3' end domain b of exon respectively, with their sequence and structure invariably occurring in various predicted structures of exon the domain b of exon and the full hotair. these are supported by experimental findings. to compare the origin and evolutionary features of hotair with xist suggests that many lncrnas, may first form in marsupials and then have undergone a rapid evolution in eutherians. an interesting question is whether their origin and evolution is intrinsically associated. note added in proof during the review of the manuscript, we downloaded, searched and analyzed opossum and kangaroo genome data and opossum in oct 2010). whole genome search of opossum did not produce high-scoring hits. searching the kangaroo genome with query query query and query each produced a high-scoring hit, which have successive addresses in genescaffold_. query6's hit matches query from bp to bp, exactly as that in rat. what is interesting is that exon and exon were absent, domain b of exon produced a high-scoring hit, and domain a of exon was not identified. these results, together with the phylogenetic analysis, lead to two suggestions. first, hotair, like other lncrnas, first formed in some marsupials and underwent a rapid evolution in eutherians. second, domain b of exon may be the backbone of hotair, because it is the only relatively long piece conserved in marsupial and eutherians. monotremes have multiple x chromosomes but it is not clear whether they undergo dosage compensation; marsupials show dosage compensation but they lack xist. it is found that female marsupials may use an ancestral dosage compensation mechanism that differs from, but share common properties with, the xist based in eutherians. since protein-coding genes that flank the eutherian xic are well-conserved in m. domestica and vertebrates and there is a surprising break in synteny with eutherian mammals and other vertebrates, it is suggested that during the evolution of the marsupial x chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. the situation of hotair, which is flanked by hoxc and hoxc not found in vertebrates and initially occurs in marsupials as revealed in this study, seems quite similar to xist. this raises the interesting question of whether hotair and xist, and possibly also some other lncrnas, have undergone the same evolutionary process. abbreviations ncrna: noncoding rna; lncrna: long noncoding rna; prc2: polycomb repressive complex authors' contributions sh performed genome search, results analysis and structure prediction; sl participated in genome search; hz conceived the project, performed phylogenetic analysis, structure prediction and wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 this file contains table s figure s figure s and figure s click here for file additional file 2 this file contains orthologues of hotair exons and their coordinates in mammals. click here for file additional file 3 this file contains predicted structures of full hotair in human and rat. click here for file acknowledgements we are grateful to eric nawrocki at the hhmi janelia farm research campus and wendy wong at cornell university for their help in using infernal and evonc. we also thank two anonymous reviewers for their valuable comments. h.z. was supported by guangdong province foundation for returned scholars and national natural science foundation of china.
structures were predicted for exon two domains of exon and the full hotair sequence. phylogenetic analysis revealed discrete evolutionary dynamics for orthologous sequences of hotair exons. hotair exists in mammals, has poorly conserved sequences and considerably conserved structures, and has evolved faster than nearby hoxc genes. exons of hotair show distinct evolutionary features, and a bp domain in the bp exon is especially conserved. like the well-characterised lncrna xist, hotair binds to polycomb proteins to methylate histones at multiple hoxd loci, but unlike xist, many details of its structure and function, as well as the trans regulation, remain unclear. to identify conserved domains in hotair and study the phylogenetic distribution of this lncrna, we searched the genomes of mammalian and non-mammalian vertebrates for matches to its exons and the two conserved domains within the bp exon using infernal. there was just one high-scoring hit for each mammal, but many low-scoring hits were found in both mammals and non-mammalian vertebrates. these hits and their flanking genes in four placental mammals and platypus were examined to determine whether hotair contained elements shared by other lncrnas. structure prediction identifies two fragments in the 5' end exon and the 3' end domain b of exon with sequence and structure invariably occurring in various predicted structures of exon the domain b of exon and the full hotair. the sequence and structure of two fragments, in exon and the domain b of exon respectively, were identified to robustly occur in predicted structures of exon domain b of exon and the full hotair in mammals. different from all the others that function in cis to regulate local gene expression, the newly identified hotair is located between hoxc and hoxc in the human genome and regulates hoxd expression in multiple tissues. these features, together with the absence of some exons and sequences in mouse, rat and kangaroo, suggest ab initio generation of hotair in marsupials.
 an increasing number of long noncoding rnas have been identified recently. different from all the others that function in cis to regulate local gene expression, the newly identified hotair is located between hoxc and hoxc in the human genome and regulates hoxd expression in multiple tissues. like the well-characterised lncrna xist, hotair binds to polycomb proteins to methylate histones at multiple hoxd loci, but unlike xist, many details of its structure and function, as well as the trans regulation, remain unclear. moreover, hotair is involved in the aberrant regulation of gene expression in cancer.  to identify conserved domains in hotair and study the phylogenetic distribution of this lncrna, we searched the genomes of mammalian and non-mammalian vertebrates for matches to its exons and the two conserved domains within the bp exon using infernal. there was just one high-scoring hit for each mammal, but many low-scoring hits were found in both mammals and non-mammalian vertebrates. these hits and their flanking genes in four placental mammals and platypus were examined to determine whether hotair contained elements shared by other lncrnas. several of the hits were within unknown transcripts or ncrnas, many were within introns of, or antisense to, protein-coding genes, and conservation of the flanking genes was observed only between human and chimpanzee. phylogenetic analysis revealed discrete evolutionary dynamics for orthologous sequences of hotair exons. exon at the 5' end and a domain in exon near the 3' end, which contain domains that bind to multiple proteins, have evolved faster in primates than in other mammals. structures were predicted for exon two domains of exon and the full hotair sequence. the sequence and structure of two fragments, in exon and the domain b of exon respectively, were identified to robustly occur in predicted structures of exon domain b of exon and the full hotair in mammals.  hotair exists in mammals, has poorly conserved sequences and considerably conserved structures, and has evolved faster than nearby hoxc genes. exons of hotair show distinct evolutionary features, and a bp domain in the bp exon is especially conserved. these features, together with the absence of some exons and sequences in mouse, rat and kangaroo, suggest ab initio generation of hotair in marsupials. structure prediction identifies two fragments in the 5' end exon and the 3' end domain b of exon with sequence and structure invariably occurring in various predicted structures of exon the domain b of exon and the full hotair. 