transposable elements ( tes ) dispersed in the mammalian genome are on the rise , as completion of the whole human genome sequence .
contrary to our expectations , tes , thought to be  junk dna ,  were estimated to account for approximately 45% of the human genome ( international human genome sequencing consortium , 2001 ) .
tes can be categorized by various features such as the capacity to mobilize themselves , dna / rna intermediation , and the existence of a long terminal repeat ( ltr ) .
endogenous retroviruses ( ervs ) and long interspersed elements ( lines ) have gene - encoding enzymatic machinery that could mobilize to other genomic regions as autonomous elements . however , nonautonomous elements , especially alu elements , which are classified as short interspersed elements ( sine ) and sine - variable number tandem repeat - alu - like sequences ( sva ) , mobilize using the reverse transcriptase of the line .
all of the tes described above are integrated into the host genome by their rnas , but the integration of dna transposons is mediated by dna .
ervs and ltr retrotransposons have ltrs , but sines and lines are non - ltr elements [ 13 ] .
one group of retrotransposon - like elements , mammalian apparent ltr retrotransposons ( malrs ) , accounts for approximately 3.65% and 4.82% , including solitary ltrs , of the human and mouse genome , respectively .
. typical ervs consist of an internal region , gag , pol , and env with ltrs on both sides .
ltrs are very important for integration and transcription because they function as a promoter element , transcription start site , and polyadenylation signal and site .
compared to ervs , malrs have short internal sequences and ltrs at the 5 and 3 ends , but they do not have a transcription initiation site , reverse transcriptase , and primer binding site .
recent studies have shown that tes contributed to primate genome evolution through gene structure variation and regulation by integrating into an intron , gene - flanking region , and an exon [ 7 , 8 ] .
additionally , tes that integrate into introns could make alternative exons or cassette exons in a process termed  exonization  within human and mouse protein - coding genes ; in contrast , tes can make new introns in a process called  intronization . 
these events could help with the understanding of transcriptome and proteome complexities [ 911 ] .
the exonization of the primate - specific alu element is well established in the divergence of human and chimpanzee genomes as a major mechanism of exon creation [ 12 , 13 ] .
( 2003 ) reported that the alu consensus sequence could provide a splice site , leading to exonization . in the case of ltrs ,
2007 ) reported that 256 of 1057 ltr retrotransposons related to human genes were observed in protein - coding regions , and 50 protein - coding exons of 45 genes were derived wholly from ltr retrotransposons .
furthermore , they analyzed alternative exons of the interleukin 22 receptor , the alpha 2 gene ( il22ra2 ) , which was derived from malrs that provided splice sites , indicating the important roles of tes in human evolution ; this result coincided with those of previous studies [ 1 , 5 ] . importantly ,
the supt16h gene , also known as factp140 , is a component of the facilitates chromatin transcription ( fact ) complex . in eukaryotic cells ,
the transcription of genes requires rna polymerase ii and transcription factors with altered chromatin structure . in order to initiate transcription , dna needs to be in an open structure that is moderated by the alteration of chromatin with accessory factors , facilitating access to the dna .
as described above , the supt16h gene has an original transcript consisting of 26 exons .
however , the integration of an malr created a new exon that was expressed transcriptionally ( bx333035 ) .
furthermore , we found a new alternative transcript , including the malr - derived exon .
we assumed the time of integration of the malr into the genome by pcr amplification .
all of the primates assayed had the malr in the supt16h gene , which was a natural result of its conservation in the mammalian genome .
the expression patterns of the 2 transcripts in several tissues of humans , crab - eating monkey , marmoset , and squirrel monkey were ubiquitous with no significant tissue specificity or species specificity .
structurally , the 2 transcripts included the exon - derived malr , but they adopted different splice sites , leading to exons of different sizes . taken together , we showed that the malr could regulate gene expression at the transcriptional level , that is , tes could provide transcriptome diversity , resulting in the proteome 's capacity for diversification , which potentially occupies an important role in the host genome .
to identify the sequences related to the malr , we scanned the human reference genome sequence , human ests , and human refseq mrnas . using the malr consensus sequence as a query
, we screened nonredundant databases through blast version 2.2.26 + to identify the novel hybrid transcripts of the malr and supt16h .
then , we aligned the transcripts of the supt16h gene to identify the precise splicing patterns .
tes included in the supt16h transcripts and their genomic loci were identified using the repeatmasker program ( http://www.repeatmasker.org/ ) , referencing a library containing the consensus sequences from repbase update . using the est and refseq mrnas , we accurately reconstructed the supt16h gene structure . to analyze the sequences of the various supt16h transcripts , we aligned them using the clustalw program . using the trizol reagent ( invitrogen ) , we extracted the total rna from the cerebrum , cerebellum , pituitary gland , heart , lung , spleen , liver , pancreas , kidney , and urinary bladder of the crab - eating monkey ; the heart , lung , brain , stomach , liver , kidney , pancreas , colon , spleen , and small intestine of the marmoset ; and the heart , esophagus , diencephalon , small intestine , lung , pancreas , cerebrum , colon , and stomach of the squirrel monkey .
the total rna from human tissues , including the adrenal gland , cerebellum , whole brain , heart , kidney , liver , lung , testis , trachea , bone marrow , fetal brain , fetal liver , placenta , prostate , salivary gland , skeletal muscle , spinal cord , thymus , thyroid , and uterus , were purchased from clontech .
we isolated the mrna from the total rna by using polyatract mrna isolation systems ( promega ) .
we used the genomic dna of the following species : ( 1 ) hominoids : common chimpanzee ( pan troglodytes ) , gorilla ( gorilla gorilla ) , orangutan ( pongo pygmaeus ) , and gibbon ( hylobates agilis ) ; ( 2 ) old world monkeys : japanese monkey ( macaca fuscata ) , rhesus macaque ( m. mulatta ) , bonnet macaque ( m. radiata ) , mandrill ( mandrillus sphinx ) , mangabey ( cercocebus agilis ) , african green monkey ( chlorocebus aethiops ) , colobus ( colobus guereza ) , and langur ( trachypithecus cristatus ) ; ( 3 ) new world monkeys : night monkey ( aotus trivirgatus ) , squirrel monkey ( saimiri sciureus ) , and common marmoset ( callithrix jacchus ) ; and ( 4 ) prosimian : ring - tailed lemur ( lemur catta ) and a galago ( otolemur crassicaudatus ) .
these genomic dna were isolated from the heparinized blood samples according to a standard protocol . after anticoagulant ,
and then , proteinase k and phenol equilibrated with tris - cl were treated for extraction .
the viscous aqueous was transferred and ammonium acetate and ethanol were added to form a precipitate .
lastly , the dna precipitate was washed , dried , and dissolved in te ( tris and edta ) .
takenaka ( primate research institute , kyoto university ) kindly provided genomic dna of primates after conducting this procedure .   using the genomic dna of humans and 17 primates , the malr in the supt16h gene
m - mlv reverse transcriptase ( promega ) with an annealing temperature of 42c and an rnase inhibitor ( promega ) were used for the reverse transcriptions of the transcripts .
the supt16h transcripts were amplified with a primer pair from genbank ( accession number nm001033560 ) consisting of s1 ( 5-aattggcggaaaggagaag-3 ) and as1 ( 5-gaggcagtcattccagctct-3 ) .
the supt16h transcripts related to the malr were amplified with a primer pair from genbank ( bx_333035 ) consisting of s2 ( 5-tccagttctctgttctgcca-3 ) and as2 ( 5-tgtgtggagcttcaagcaag-3 ) .
the pcr samples were subjected to an initial denaturation step of 4  min at 94c , followed by 30 cycles of pcr at 40  s of denaturation at 95c , 40  s of annealing at 55c , and 90  s of extension at 72c , followed by a final extension step of 7  min at 72c . as a standard control ,
gapdh was amplified with a primer pair consisting of gph - s ( 5-gagccccagccttctccatg-3 ) and gph - as ( 5-gaaatcccatcaccatcttccagg-3 ) from human gapdh ( genbank accession number nm002046 ) .
the supt16h gene located on chromosome 14q11.2 encodes accessory factors that may facilitate access to dna by unpackaging the chromatin structure .
it contains 26 exons with several transposable elements such as malrs ( ltr ) , alus ( sine ) , and l1s ( line ) distributed in the intronic regions .
the supt16h transcript related to the malr ( malr - derived transcript ) has 3 exons , and its last exon was created by the integration of the malr ltr element in the opposite direction of the host gene ( figure 1 ) .
malrs are considered to function in the mammalian genome by providing alternative splice sites , promoters , and other cis - regulatory elements , similar to other tes that have been conserved during primate evolution .
for example , rnf19 has an alternative transcript resulting from the exonization of malr and alujo .
specifically , the malr in the first exon has promoter activity in the sense orientation in the african green monkey fibroblast cell line , cos7 , and the human colorectal carcinoma cell line , hct116 . as described above ,
malrs are known to be present in genomes of rodents to primates and humans , indicating that they were distributed before the divergence of eutherian mammals . in order to compare the integration time of the malr in the supt16h gene with that from previous reports , we conducted pcr amplification using the genomic dna of human and other primates ( figure 2 ) .
we identified an amplicon that was 481  bp long in all of the primates that we assessed , indicating that the malr in the supt16h gene integrated into the genome of a common ancestor before the divergence of the new world monkey and prosimians .
in addition , from the sequencing alignment , we also identified 40  bp and 4  bp of specific sequences in japanese macaque ( figure 2(b ) ) .
these sequences were not detected in any other primate , including the rhesus macaque , which belongs to the old world monkey family .
tes distributed in the mammalian genome have played important roles in gene regulation and evolution .
one of the typical characteristics of tes which is that they affect the transcription of cellular genes by exonization , which creates an alternatively spliced exon by integration into exonic or intronic regions , has been reported in many studies [ 24 , 25 ] .
actually , these alternative transcripts are transient and flexible according to the cellular environment and stimuli .
however , alternatively spliced variants can be stabilized and increased without selection , and exonization can lead to not only transcriptome diversity but also proteome diversity [ 2 , 10 ] . furthermore , these events occur differently or specifically according to the tissue , developmental stage , and disease ; thus , alternative transcripts are used to mine disease diagnostic markers [ 9 , 26 ] . to confirm whether the malr sequences are expressed and could regulate transcription , we conducted rt - pcr analysis of the refseq mrna ( original transcript ) and malr - derived transcript ( bx333035 ) from several human tissues .
we analyzed its expression in tissues of other primates , including the crab - eating monkey , common marmoset , and squirrel monkey ( figure 3 ) . like the expression in humans , it was ubiquitously expressed in the tissues of these primates . because we identified japanese monkey - specific sequences , we conducted rt - pcr analysis in rhesus macaque samples to compare the expression in old world monkeys .
however , we could not detect a difference in the supt16h expression between the japanese monkey and rhesus macaque ( data not shown ) .
gene expression is a process that produces rna or proteins through transcription , splicing , and translation .
the supt16h gene is a subunit of the fact complex that acts as a chromatin - specific transcription elongation factor and interacts with histone h2a / h2b to disassemble nucleosomes and promote transcription elongation .
namely , the broad expression of supt16h in human and other primate tissues indicated its essential function for gene expression .
as shown in figure 4 , a transcript related to the malr was also expressed broadly in human tissues .
this product was also detected in tissues of crab - eating monkey , common marmoset , and squirrel monkey .
there was hardly any difference in the expression of 2 transcripts which indicated malr did not affect the expression of the supt16h gene .
its product was longer than that of the original transcript because alternative splicing retained an intronic region .
the malr in the intron was not spliced and exonized ( exon 2 ) wholly between exons 2 and 3 ( figure 3 ) . comparing the new alternative transcript and the malr - derived transcript ( bx_333035 ) , we identified specific features of alternative splicing .
entire sequences of the new exon of the alternative transcript identified in rt - pcr were derived from the malr .
as shown in figure 5 , the malr provided both typical alternative splice sites , the acceptor site ( ag ) and a nucleotide ( g ) of the donor site ( gt ) .
although these splicing sites were typical sequences recognized by the spliceosome , the splicing pattern could be changed . in case of the transcript related to the malr ( bx_333035 ) , the malr in the intronic region was also exonized to form a new alternative transcript .
. only 1 site , the acceptor site , was recognized by the spliceosome , and the donor site was skipped .
additionally , the elongated exon included malr - derived sequences and sequences of intron and exon 3 of the original and new alternative transcript .
in spite of the different splicing pattern , the new alternative transcript was expressed broadly in tissues of humans and other primates , similar to the original and malr - related transcripts ( figures 3 and 4 ) .
alternative splicing is a common mechanism by which most genes are processed [ 27 , 28 ] . in a previous report
alternative splicing can be controlled by genetic mutation and epigenetic alteration [ 30 , 31 ] .
however , the spliceosome can recognize splicing sites created by mutation and cryptic splicing sites , leading to alternative splicing .
in addition to point mutations , the integration of transposable elements can induce alternative splicing as genetic mutations [ 2 , 25 ] .
if the 2 transcripts showed different and tissue - specific expression patterns , we could assume that tissue - specific factors contributed to alternative splicing ; however , both transcripts were expressed broadly in tissues of humans and primates .
various mechanisms can be considered to understand the differences between the 2 transcripts . compared to original transcript ,
however , the new exon includes intronic sequences by the typical splice site , ag - gt ( 35 ) , in the new alternative transcript , and ag ( 3 ) in the malr - derived transcript existing in the intron .
if so , we could guess that the acceptor site and donor site of the new exon could be marked as a weak splice site by epigenetic regulation , such as histone modifications and dna methylationan , although it was the typical splice site . in a previous study ,
histone modification was reported as important factor for alternative splicing . among histone modifications such as h3k36me3 , h3k79me1 , h2bk5me1 , h3k27me1 , h3k27me2 , and
h3k27me3 enriched in exon , the donor site of the new exon could be recognized weakly by the spliceosome with h3k36me3 which is found in weakly expressed exon [ 35 , 36 ] .
additionally , the dna methylation and the cg content in the new exonic region could affect the expression of the 2 transcripts .
zhou et al . ( 2012 ) reported that exclusive exons have lower levels of cg and methylated cg , whereas retained intron has higher levels .
we suggest that the malr integrated into an intron of the supt16h gene before the divergence of new world monkeys and prosimians , and it played a biological role in the evolution of the supt16h gene during primate evolution by providing an alternative splice site .
although further analyses are required to conclusively elucidate the functions of the transcriptional variants of the supt16h gene in primate genomes , the results of this study will help us understand the characteristics of the transposable element insertion driving the transcript diversification and its effect on the evolution of the host gene .
 the supt16h gene known as factp140 is required for the transcription of other genes . for transcription , 
 genes need to be complexed with accessory factors , including transcription factors and rna polymerase ii . 
 one such factor , fact , interacts with histones h2a 
 / h2b for nucleosome disassembly and transcription elongation . 
 the supt16h gene has a transcript and many expressed sequence tags ( ests ) . 
 we were especially interested in an malr - derived transcript ( est , bx333035 ) that included a new exon introduced by a transposable element , a mammalian apparent ltr retrotransposon ( malr ) . 
 the malr was detected ranging from humans to galagos , indicating the malr in the supt16h gene is integrated into the primate ancestor genome . 
 a new exon was created by alternative donor site provided by the malr . 
 the original transcript and the malr - derived transcript were expressed in various human , rhesus monkey , and other primate tissues . 
 additionally , we identified a new alternative transcript that included the malr , but there was no significant difference in the expression of the original transcript and the malr - derived transcript . 
 interestingly , the new alternative transcript and the malr - derived transcript had the malr sequence in the new exon , but they had different structures by adopting different 3 splice sites . from this study , we verified transposable elements that contributed to transcriptome diversity . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion