BACKGROUND
the latest estimates indicate that the human genome comprises only  <dig>  protein coding genes  <cit> .
this number seems surprisingly low, considering the phenotypic complexity of humans.
adding another layer of complexity, it is now appreciated that fusion transcripts -- which are derived of exons from two or more distinct genes -- can exponentially increase the protein coding/functional capacity of the human genome  <cit> .
there is now a body of evidence to indicate that numerous genes within the human genome are transcribed as fusion transcripts .
notably, some fusion transcripts are more tissue specific than non-fusion transcripts, and are translated into proteins  <cit> .
here, we use prostate  cells as a model to study fusion transcription given extensive studies that indicate that the prostate readily expresses fusion transcripts.
for example, the most studied fusion in prostate cancer is formed between the tmprss <dig> and erg genes, resulting in erg transcription being driven by the androgen-responsive tmprss <dig> promoter .
this fusion is observed in ~50 % of primary prostate tumors, and ~41 % of lymph node metastatic tumors  <cit> .
hundreds of novel fusion genes that are formed by chromosomal rearrangements have since been discovered in prostate cancer genomes  <cit> .
interestingly, some of these chromosomal rearrangement fusion genes can produce fusion transcripts comprising exons from more than two genes  <cit> .
the chinnaiyan laboratory extended their seminal tmprss2-erg study by using rnaseq to identify  <dig> other fusion transcripts that are not produced by chromosomal alterations   <cit> .
later studies using rnaseq estimated that there may be as many as  <dig> transcription-mediated fusion transcripts that are expressed in the prostate  <cit> .
importantly, maher and colleagues revealed that some transcription-mediated fusion transcripts such as slc45a3-elk <dig> are more highly expressed in metastatic prostate cancers compared to benign cells  <cit> .
other studies  <cit>  have since correlated slc45a3-elk <dig> expression with an unfavorable prostate cancer prognosis, resulting in a growing interest in fusion transcription in the prostate cancer biomarker field  <cit> .
a recent study of  <dig> diverse cancer cases has identified  <dig> fusion transcripts, some of which comprise kinase genes that have great potential to be targeted therapeutically  <cit> .
additionally, a more recent extensive study of  <dig> rnaseq libraries discovered  <dig> transcription-mediated fusion transcripts, many of which are expressed in the prostate and/or associated with various types of cancer  <cit> .
interestingly, fusion transcripts have also been found to be formed between mitochondrial dna with nuclear dna, occurring at a similar frequency as fusion transcripts that comprise solely of nuclear dna  <cit> .
in this study we characterized the genomic sequences flanking fusion transcripts to better understand the mechanisms that mediate fusion transcription, using prostate  as a model given the aforementioned extensive studies in this tissue.
indeed, a study in prostate  cells reveals that the ctcf transcription factor mediates changes in chromosomal conformation that results in the possible formation of at least  <dig> fusion transcripts {qin,  <dig> #33}.
here, we reveal that the sequences flanking fusion loci are similar to non-fusion loci, indicating that the mechanisms adopted by fusion transcription are likely to be similar to non-fusion transcription and intron splicing.
RESULTS
identification of fusion transcripts in prostate cancer
a recent study indicates that the number of protein coding genes in the human genome is similar to lower vertebrates  <cit> .
thus, there has been a growing interest in fusion transcription as a mechanism to account for some of the phenotypic complexities of humans  <cit> .
here, we used the fusionmap program to first identify fusion transcripts in prostate  rna-seq data sets as this program offers one of the best compromises between sensitivity and false positives  <cit> .
predicted fusion transcripts were then searched against the genome using the blat function of the ucsc genome browser, and manually inspected to filter out predicted fusion transcripts that map to other regions of the genome .
this resulted in the detection of a total of  <dig> high-confidence fusion transcripts from ren and colleagues  and our  dataset of clinical prostate cancers and their adjacent non-cancer prostate cells, and from our dataset of cultured lncap cells treated with androgens  and anti-androgens  .
the majority of these fusion transcripts  are derived from genes that are located next to each other in the genome, otherwise referred to as “read-through transcripts”  <cit> , or transcription induced chimeras  <cit>  .
this observation is supported by a recent study in prostate cancer cells that indicates that a high percentage of fusion transcripts involve neighbouring genes {qin,  <dig> #33}.
of the other fusion transcripts,  <dig>  are derived from genes that are located on different chromosomes, and  <dig>  are derived from non-adjacent genes that are on the same chromosome .
notably, a majority of fusion transcripts were solely detected in either ren and colleagues  or the lncap  datasets , and some fusion transcripts were exclusively detected in lncap cells that were treated with either bicalutamide , enzalutamide , or dht  .
this highlights the importance of using many different biological data sets to identify fusion transcripts.fig.
<dig>
a circos plot from rnaseq data of fusion transcripts from the ren et.
al.
dataset  <cit> , from our clinical prostate cancers and adjacent non-cancer prostate cells , and from lncap prostate cancer cells that were treated with either  <dig> nm androgen  or 10 μm anti-androgen .
b venn diagram detailing how many unique fusion transcripts were detected between the different rnaseq datasets.
c venn diagram detailing how many unique fusion transcripts were detected between androgen or anti-androgen treated lncap cells
most fusion transcripts are formed at annotated exon junctions, use classical gt-ag intron donor-acceptor motifs, and preserve the open reading frame
previous studies have characterized fusion transcript splice sites at the exon usage level  <cit>  or at the rna sequence level  <cit> , but to our knowledge, there has been no attempt to characterize the genomic sequence surrounding fusion junctions.
thus, we have characterized the nucleotide sequences flanking fusion junctions at the genes that fusion transcripts are derived  to better understand the mechanisms that mediate fusion expression.
an analysis of fusion junctions revealed that most fusion transcripts  detected in our fusionmap analysis are formed at the exon borders of either one or both parental genes , and that most  use the canonical gt-ag intron donor-acceptor splice sites .
interestingly, this observation also applied to fusion transcripts that are the result of chromosomal rearrangements such as tmprss2-erg.
thus, fusion transcripts that are located within genomic regions that undergo genomic rearrangements still use the same transcriptional machinery as non-fusion loci, unless chromosomal breakpoints occur within exons.
notably, 50 %  and 70 %  of fusion transcripts that use the classical gt-ag intron donor-acceptor sites correspond to fusion junctions that were located at neither or only one exon boundary of the parental genes, respectively .
this indicates that these fusion transcripts are not the result of chromosomal breakpoints that occur within exons, but rather use classical gene transcription mechanisms to generate alternative exon boundaries.
importantly,  <dig>  of the fusion transcripts maintain the original open reading frame of the parental genes , opening the possibility that fusion transcripts can be translated into distinct functional proteins with unique biological properties.
indeed,  <dig> fusion proteins have already been detected in various human tissues  <cit> .fig.
<dig>
a pie graph showing the proportion of fusion points that occur at the exon boundaries of one, both or neither genes that comprise the fusion transcript.
b bubble plot of the number of fusion transcripts that use the at-ac, ct-ac, ct-gc, gc-ag, and gt-ag donor-acceptor splice sites.
bubble size represents the average gene expression  for fusion transcripts within that donor-acceptor class.
c pie chart of the percentage of fusion transcripts that maintain the original reading frames of the genes that comprise the fusion transcripts .
the outer pie chart represents the nucleotide position  within the codon of the first  and second  genes at the fusion points of those respective genes
computational prediction indicates that fusion junctions and non-fusion splice sites have similar propensities to hybridize
it has been proposed that fusion transcripts might be the result of ‘chromosomal looping’ that brings distal loci together  <cit> .
thus, a computational analysis of the sequences flanking fusion junctions of the parental gene loci was performed to determine the capacity of these two loci to hybridize , thereby bringing distal regions together to mediate one continuous transcriptional event that produces a fusion transcript.
our hypothesis stems from the rna splicing process which similarly involves a series of steps comprising multiple nucleotide hybridizations between snrna/ribonucleoprotein complexes with the target pre-mrna  <cit> .
we found no obvious difference in both the regions of hybridization, and the total number of hybridized sequences between parental gene loci of fusion transcripts  compared to canonical exon-exon boundaries of genes from the ncbi refseq database .
meme analysis was also undertaken to assess for motifs that might promote genomic hybridization between parental gene loci.
the predicted hybridized nucleotides of two gene 1/gene  <dig> combinations have different motifs between fusion and non-fusion loci , while predicted hybridized nucleotides between gene  <dig> upstream and gene  <dig> upstream had g- and a-rich motifs .
notably, predicted hybridized nucleotides between gene  <dig> downstream and gene  <dig> upstream had similar g-rich motifs in both fusion and non-fusion loci .fig.
<dig>
a diagram showing 100 nt of genomic sequence upstream  and downstream  of the point of fusion at the two genes comprising the fusion transcript that were used for hybridisation analysis.
b the line graph represents the number of fusion transcripts that have complementary nucleotides  at the respective distance  from the point of fusion  between the up- and downstream sequences from gene  <dig> and gene  <dig>  the histogram represents the average number of complementary nucleotides between the up- and down-stream sequences from gene  <dig> and gene  <dig>  the meme result  represents motifs of complementary sequences between the up- and down-stream sequences from gene  <dig> and gene  <dig>  up- and down-stream sequences from random non fusion intron splice sites were used for comparison
fusion loci are depleted of repetitive sequences
we also assessed whether there was a selection of repetitive sequences at fusion loci as it has been found that alu repeats mediate aberrant gene transcription through exonization  <cit> .
sequence analyses of genomic sequences flanking the fusion junctions  at parental gene loci  indicate that they have a low abundance of repetitive sequences.
for example, only six repeat families were found within these regions  .
the highest prevalence of repeats corresponds to sines that were predominantly located further away from the point of fusion, but these account for only  <dig>  %  of all fusions .
apart from ltrs at the gene  <dig> parental loci, the number of repetitive sequences at non-fusion loci  was generally similar to parental gene loci of fusion transcripts .
given the likely use of similar transcriptional mechanisms between fusion and non-fusion loci from aforementioned observations, the lack of repetitive sequences at fusion loci is not surprising as exonic and splice regions are generally well conserved  <cit>  to ensure functionality of important genes.fig.
<dig>
a diagram showing 100 nt of genomic sequence upstream  and downstream  of the point of fusion at the two genes comprising the fusion transcript that were used to identify repetitive sequences.
b repeats from six families  were detected at fusion  and non-fusion  regions at both gene loci
fusion transcripts are regulated by androgen and/or therapeutic anti-androgens
we assessed whether fusion transcript expression correlates with the parental gene expression in order to better understand the coordination of fusion expression with their parental genes.
thus, an rt-qpcr analysis was undertaken on six fusion transcripts that were in frame, and from frameshift classes with the most fusion transcripts.
these include ctbs-gng <dig> , dhrs1-rabggta , sidt2-tagln , hars2-zmat <dig> , nos1ap-c1orf <dig> , and c17orf106-cdk <dig> .
three of these fusion transcripts comprise of genes that are adjacent to each other in the genome , with the other three derived from non-adjacent genes .
rt-qpcr was also carried out on the c1qtnf3-amacr  as a representative transcript with an interrupted reading frame.
our analysis indicates that all candidate fusion transcripts are either two-fold higher-  or lower-  expressed after androgen or anti-androgen treatment in lncap cells .
in many cases, the androgen and anti-androgen regulation of at least one of the parental genes is similar to the fusion transcript .
c1qtnf3-amacr was the notable exception .
the similarity in expression between fusion transcripts and their parental genes is not surprising considering that the splice site usage at fusion junctions is similar to those of non-fusion transcripts.
interestingly, all seven candidate fusion transcripts were regulated by androgen  and/or therapeutic anti-androgens , indicating that they might be important in disease progression and/or treatment resistance.table  <dig> summary of fusion expression by rt-qpcr
nos1ap-c1orf226
nos1ap
c1orf226
hars-zmat2
hars
zmat2
dhrs1-rabggta
dhrs1
rabggta
ctbs-gng5
ctbs
gng5
c17orf106-cdk3
c17orf106
cdk3
sidt2-tagln
sidt2
tagln
c1qtnf3-amacr
c1qtnf3
amacr
aat least two-fold higher- , lower- , or no change  in expression after -androgen treatment to mock treatments
bat least four of seven tumours with at least 2-fold over- , under- , or no change  in expression in tumors relative to non-tumor cells
dht dihydrotestosterone , bic bicalutamide , enz enzalutamide , ne not expressed
some fusion transcripts are differentially expressed in prostate tumors
of the seven candidate fusion transcripts, only four  could be readily detected in clinical prostate tumors and/or adjacent non-cancer cells .
of these, c1qtnf3-amacr has an expression profile that is distinct from both parental genes .
interestingly, the dhrs1-rabggta fusion transcript is less expressed in tumors compared to adjacent non-cancer cells .
furthermore, five of the fusion transcripts detected in this study  were found in both our clinical rnaseq dataset comprised of caucasian men, and in ren and colleagues dataset which comprised of han chinese men  <cit> .
thus, these fusion transcripts are great candidates for further studies as they are readily expressed in different ethnicities.
fusion loci undergo extensive alternative transcription
finally, we assessed for variant fusion transcripts given that most loci undergo variant transcription  <cit> .
a recent large-scale rnaseq study comprising  <dig> libraries from multiple cancers  <cit>  was interrogated, revealing that  <dig> transcripts harbored the same exon junctions as the transcripts detected in our prostate  data set .
these  <dig> transcripts accounted for only  <dig> of the  <dig> fusion transcripts detected in this study .
this indicates that multiple variant fusion transcripts use the same exon junctions.
in agreement, in addition to the  <dig> transcription-mediated fusion transcripts of our prostate-derived dataset, the iyer et al.
<cit>  dataset revealed that parental loci were extensively spliced, with 124/ <dig> presenting alternative fusion transcripts .
we thus propose that these loci are highly amendable to fusion and alternative transcription.
an example of extensive fusion transcription from the iyer et al.
dataset for the seven candidate fusion loci from this study is shown in fig.
<dig> fig.
<dig> diagram of other fusion transcripts expressed at the seven candidate fusion loci.
red ucsc bed tracks indicate fusion transcripts discovered by iyer et al.
<cit> .
parental genes that fusion transcripts were derived in our study are represented as green tracks, and other genes located at that locus are represented as blue tracks.
the fusion junctions discovered in this study is also shown
CONCLUSIONS
using a conservative filtering process, we have identified  <dig> high confidence fusion transcripts that are readily expressed in prostate  cells in order to accurately analyze the sequences around fusion junctions in order to better understand fusion transcription and/or splicing.
we reveal that fusion splices sites generally use the same nucleotide sequences as non-fusion transcripts, thereby indicating that fusion transcription likely co-opts the same transcriptional processes.
however, this hypothesis may only apply to “read-through” chimeras which comprise the majority  of fusions that were detected in this study.
regardless of the mechanism, the recombination of exons from biologically distinct genes provides an interesting source of genetic variation that warrants further study which will further our understanding of the transcriptional nuances of more evolutionary complex species such as humans.

 the latest estimates indicate that the human genome comprises only protein coding genes. this number seems surprisingly low, considering the phenotypic complexity of humans. adding another layer of complexity, it is now appreciated that fusion transcripts -- which are derived of exons from two or more distinct genes -- can exponentially increase the protein coding/functional capacity of the human genome. there is now a body of evidence to indicate that numerous genes within the human genome are transcribed as fusion transcripts. notably, some fusion transcripts are more tissue specific than non-fusion transcripts, and are translated into proteins. here, we use prostate cells as a model to study fusion transcription given extensive studies that indicate that the prostate readily expresses fusion transcripts. for example, the most studied fusion in prostate cancer is formed between the tmprss and erg genes, resulting in erg transcription being driven by the androgen-responsive tmprss promoter. this fusion is observed in ~50 % of primary prostate tumors, and ~41 % of lymph node metastatic tumors. hundreds of novel fusion genes that are formed by chromosomal rearrangements have since been discovered in prostate cancer genomes. interestingly, some of these chromosomal rearrangement fusion genes can produce fusion transcripts comprising exons from more than two genes. the chinnaiyan laboratory extended their seminal tmprss2-erg study by using rnaseq to identify other fusion transcripts that are not produced by chromosomal alterations. later studies using rnaseq estimated that there may be as many as transcription-mediated fusion transcripts that are expressed in the prostate. importantly, maher and colleagues revealed that some transcription-mediated fusion transcripts such as slc45a3-elk are more highly expressed in metastatic prostate cancers compared to benign cells. other studies have since correlated slc45a3-elk expression with an unfavorable prostate cancer prognosis, resulting in a growing interest in fusion transcription in the prostate cancer biomarker field. a recent study of diverse cancer cases has identified fusion transcripts, some of which comprise kinase genes that have great potential to be targeted therapeutically. additionally, a more recent extensive study of rnaseq libraries discovered transcription-mediated fusion transcripts, many of which are expressed in the prostate and/or associated with various types of cancer. interestingly, fusion transcripts have also been found to be formed between mitochondrial dna with nuclear dna, occurring at a similar frequency as fusion transcripts that comprise solely of nuclear dna. in this study we characterized the genomic sequences flanking fusion transcripts to better understand the mechanisms that mediate fusion transcription, using prostate as a model given the aforementioned extensive studies in this tissue. indeed, a study in prostate cells reveals that the ctcf transcription factor mediates changes in chromosomal conformation that results in the possible formation of at least fusion transcripts {qin, #33}. here, we reveal that the sequences flanking fusion loci are similar to non-fusion loci, indicating that the mechanisms adopted by fusion transcription are likely to be similar to non-fusion transcription and intron splicing.  identification of fusion transcripts in prostate cancer a recent study indicates that the number of protein coding genes in the human genome is similar to lower vertebrates. thus, there has been a growing interest in fusion transcription as a mechanism to account for some of the phenotypic complexities of humans. here, we used the fusionmap program to first identify fusion transcripts in prostate rna-seq data sets as this program offers one of the best compromises between sensitivity and false positives. predicted fusion transcripts were then searched against the genome using the blat function of the ucsc genome browser, and manually inspected to filter out predicted fusion transcripts that map to other regions of the genome. this resulted in the detection of a total of high-confidence fusion transcripts from ren and colleagues and our dataset of clinical prostate cancers and their adjacent non-cancer prostate cells, and from our dataset of cultured lncap cells treated with androgens and anti-androgens. the majority of these fusion transcripts are derived from genes that are located next to each other in the genome, otherwise referred to as “read-through transcripts”, or transcription induced chimeras. this observation is supported by a recent study in prostate cancer cells that indicates that a high percentage of fusion transcripts involve neighbouring genes {qin, #33}. of the other fusion transcripts, are derived from genes that are located on different chromosomes, and are derived from non-adjacent genes that are on the same chromosome. notably, a majority of fusion transcripts were solely detected in either ren and colleagues or the lncap datasets, and some fusion transcripts were exclusively detected in lncap cells that were treated with either bicalutamide, enzalutamide, or dht. this highlights the importance of using many different biological data sets to identify fusion transcripts.fig. a circos plot from rnaseq data of fusion transcripts from the ren et. al. dataset, from our clinical prostate cancers and adjacent non-cancer prostate cells, and from lncap prostate cancer cells that were treated with either nm androgen or 10 μm anti-androgen. b venn diagram detailing how many unique fusion transcripts were detected between the different rnaseq datasets. c venn diagram detailing how many unique fusion transcripts were detected between androgen or anti-androgen treated lncap cells most fusion transcripts are formed at annotated exon junctions, use classical gt-ag intron donor-acceptor motifs, and preserve the open reading frame previous studies have characterized fusion transcript splice sites at the exon usage level or at the rna sequence level, but to our knowledge, there has been no attempt to characterize the genomic sequence surrounding fusion junctions. thus, we have characterized the nucleotide sequences flanking fusion junctions at the genes that fusion transcripts are derived to better understand the mechanisms that mediate fusion expression. an analysis of fusion junctions revealed that most fusion transcripts detected in our fusionmap analysis are formed at the exon borders of either one or both parental genes, and that most use the canonical gt-ag intron donor-acceptor splice sites. interestingly, this observation also applied to fusion transcripts that are the result of chromosomal rearrangements such as tmprss2-erg. thus, fusion transcripts that are located within genomic regions that undergo genomic rearrangements still use the same transcriptional machinery as non-fusion loci, unless chromosomal breakpoints occur within exons. notably, 50 % and 70 % of fusion transcripts that use the classical gt-ag intron donor-acceptor sites correspond to fusion junctions that were located at neither or only one exon boundary of the parental genes, respectively. this indicates that these fusion transcripts are not the result of chromosomal breakpoints that occur within exons, but rather use classical gene transcription mechanisms to generate alternative exon boundaries. importantly, of the fusion transcripts maintain the original open reading frame of the parental genes, opening the possibility that fusion transcripts can be translated into distinct functional proteins with unique biological properties. indeed, fusion proteins have already been detected in various human tissues.fig. a pie graph showing the proportion of fusion points that occur at the exon boundaries of one, both or neither genes that comprise the fusion transcript. b bubble plot of the number of fusion transcripts that use the at-ac, ct-ac, ct-gc, gc-ag, and gt-ag donor-acceptor splice sites. bubble size represents the average gene expression for fusion transcripts within that donor-acceptor class. c pie chart of the percentage of fusion transcripts that maintain the original reading frames of the genes that comprise the fusion transcripts. the outer pie chart represents the nucleotide position within the codon of the first and second genes at the fusion points of those respective genes computational prediction indicates that fusion junctions and non-fusion splice sites have similar propensities to hybridize it has been proposed that fusion transcripts might be the result of ‘chromosomal looping’ that brings distal loci together. thus, a computational analysis of the sequences flanking fusion junctions of the parental gene loci was performed to determine the capacity of these two loci to hybridize, thereby bringing distal regions together to mediate one continuous transcriptional event that produces a fusion transcript. our hypothesis stems from the rna splicing process which similarly involves a series of steps comprising multiple nucleotide hybridizations between snrna/ribonucleoprotein complexes with the target pre-mrna. we found no obvious difference in both the regions of hybridization, and the total number of hybridized sequences between parental gene loci of fusion transcripts compared to canonical exon-exon boundaries of genes from the ncbi refseq database. meme analysis was also undertaken to assess for motifs that might promote genomic hybridization between parental gene loci. the predicted hybridized nucleotides of two gene 1/gene combinations have different motifs between fusion and non-fusion loci, while predicted hybridized nucleotides between gene upstream and gene upstream had g- and a-rich motifs. notably, predicted hybridized nucleotides between gene downstream and gene upstream had similar g-rich motifs in both fusion and non-fusion loci.fig. a diagram showing 100 nt of genomic sequence upstream and downstream of the point of fusion at the two genes comprising the fusion transcript that were used for hybridisation analysis. b the line graph represents the number of fusion transcripts that have complementary nucleotides at the respective distance from the point of fusion between the up- and downstream sequences from gene and gene the histogram represents the average number of complementary nucleotides between the up- and down-stream sequences from gene and gene the meme result represents motifs of complementary sequences between the up- and down-stream sequences from gene and gene up- and down-stream sequences from random non fusion intron splice sites were used for comparison fusion loci are depleted of repetitive sequences we also assessed whether there was a selection of repetitive sequences at fusion loci as it has been found that alu repeats mediate aberrant gene transcription through exonization. sequence analyses of genomic sequences flanking the fusion junctions at parental gene loci indicate that they have a low abundance of repetitive sequences. for example, only six repeat families were found within these regions. the highest prevalence of repeats corresponds to sines that were predominantly located further away from the point of fusion, but these account for only  % of all fusions. apart from ltrs at the gene parental loci, the number of repetitive sequences at non-fusion loci was generally similar to parental gene loci of fusion transcripts. given the likely use of similar transcriptional mechanisms between fusion and non-fusion loci from aforementioned observations, the lack of repetitive sequences at fusion loci is not surprising as exonic and splice regions are generally well conserved to ensure functionality of important genes.fig. a diagram showing 100 nt of genomic sequence upstream and downstream of the point of fusion at the two genes comprising the fusion transcript that were used to identify repetitive sequences. b repeats from six families were detected at fusion and non-fusion regions at both gene loci fusion transcripts are regulated by androgen and/or therapeutic anti-androgens we assessed whether fusion transcript expression correlates with the parental gene expression in order to better understand the coordination of fusion expression with their parental genes. thus, an rt-qpcr analysis was undertaken on six fusion transcripts that were in frame, and from frameshift classes with the most fusion transcripts. these include ctbs-gng, dhrs1-rabggta, sidt2-tagln, hars2-zmat, nos1ap-c1orf, and c17orf106-cdk. three of these fusion transcripts comprise of genes that are adjacent to each other in the genome, with the other three derived from non-adjacent genes. rt-qpcr was also carried out on the c1qtnf3-amacr as a representative transcript with an interrupted reading frame. our analysis indicates that all candidate fusion transcripts are either two-fold higher- or lower- expressed after androgen or anti-androgen treatment in lncap cells. in many cases, the androgen and anti-androgen regulation of at least one of the parental genes is similar to the fusion transcript. c1qtnf3-amacr was the notable exception. the similarity in expression between fusion transcripts and their parental genes is not surprising considering that the splice site usage at fusion junctions is similar to those of non-fusion transcripts. interestingly, all seven candidate fusion transcripts were regulated by androgen and/or therapeutic anti-androgens, indicating that they might be important in disease progression and/or treatment resistance.table summary of fusion expression by rt-qpcr nos1ap-c1orf226 nos1ap c1orf226 hars-zmat2 hars zmat2 dhrs1-rabggta dhrs1 rabggta ctbs-gng5 ctbs gng5 c17orf106-cdk3 c17orf106 cdk3 sidt2-tagln sidt2 tagln c1qtnf3-amacr c1qtnf3 amacr aat least two-fold higher-, lower-, or no change in expression after -androgen treatment to mock treatments bat least four of seven tumours with at least 2-fold over-, under-, or no change in expression in tumors relative to non-tumor cells dht dihydrotestosterone, bic bicalutamide, enz enzalutamide, ne not expressed some fusion transcripts are differentially expressed in prostate tumors of the seven candidate fusion transcripts, only four could be readily detected in clinical prostate tumors and/or adjacent non-cancer cells. of these, c1qtnf3-amacr has an expression profile that is distinct from both parental genes. interestingly, the dhrs1-rabggta fusion transcript is less expressed in tumors compared to adjacent non-cancer cells. furthermore, five of the fusion transcripts detected in this study were found in both our clinical rnaseq dataset comprised of caucasian men, and in ren and colleagues dataset which comprised of han chinese men. thus, these fusion transcripts are great candidates for further studies as they are readily expressed in different ethnicities. fusion loci undergo extensive alternative transcription finally, we assessed for variant fusion transcripts given that most loci undergo variant transcription. a recent large-scale rnaseq study comprising libraries from multiple cancers was interrogated, revealing that transcripts harbored the same exon junctions as the transcripts detected in our prostate data set. these transcripts accounted for only of the fusion transcripts detected in this study. this indicates that multiple variant fusion transcripts use the same exon junctions. in agreement, in addition to the transcription-mediated fusion transcripts of our prostate-derived dataset, the iyer et al. dataset revealed that parental loci were extensively spliced, with 124/ presenting alternative fusion transcripts. we thus propose that these loci are highly amendable to fusion and alternative transcription. an example of extensive fusion transcription from the iyer et al. dataset for the seven candidate fusion loci from this study is shown in fig.  fig. diagram of other fusion transcripts expressed at the seven candidate fusion loci. red ucsc bed tracks indicate fusion transcripts discovered by iyer et al.. parental genes that fusion transcripts were derived in our study are represented as green tracks, and other genes located at that locus are represented as blue tracks. the fusion junctions discovered in this study is also shown  using a conservative filtering process, we have identified high confidence fusion transcripts that are readily expressed in prostate cells in order to accurately analyze the sequences around fusion junctions in order to better understand fusion transcription and/or splicing. we reveal that fusion splices sites generally use the same nucleotide sequences as non-fusion transcripts, thereby indicating that fusion transcription likely co-opts the same transcriptional processes. however, this hypothesis may only apply to “read-through” chimeras which comprise the majority of fusions that were detected in this study. regardless of the mechanism, the recombination of exons from biologically distinct genes provides an interesting source of genetic variation that warrants further study which will further our understanding of the transcriptional nuances of more evolutionary complex species such as humans. 
our computational analysis indicated that most fusion junctions use the consensus gt-ag intron donor-acceptor splice site, and most fusion transcripts maintained the open reading frame. our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. the rnaseq datasets were from our and other clinical prostate tumors with adjacent non-cancer cells, and from the lncap prostate cancer cell line that were mock-, androgen-, and anti-androgen- treated. finally, rt-qpcr was performed on rna from both clinical prostate tumors and adjacent non-cancer cells, and lncap cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. we assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. we reveal that fusion transcript expression is similar to the expression of parental genes. fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions. the abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that sine repeats are involved in aberrant gene transcription. fusion transcripts are found in many tissues and have the potential to create novel functional products. we used the fusionmap program to identify high-confidence fusion transcripts from rnaseq data. in total, fusion transcripts were identified from all rnaseq datasets. the majority of these fusion transcripts were ‘read-through chimeras’ derived from adjacent genes in the genome. characterization of sequences at fusion loci were carried out using a combination of the fusionmap program, custom perl scripts, and the rnafold program. we analyzed data from prostate cells as previous studies have shown extensively that these cells readily undergo fusion transcription. we found few instances of repetitive sequences at both fusion and non-fusion junctions. here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing.
 fusion transcripts are found in many tissues and have the potential to create novel functional products. here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. we analyzed data from prostate cells as previous studies have shown extensively that these cells readily undergo fusion transcription.  we used the fusionmap program to identify high-confidence fusion transcripts from rnaseq data. the rnaseq datasets were from our and other clinical prostate tumors with adjacent non-cancer cells, and from the lncap prostate cancer cell line that were mock-, androgen-, and anti-androgen- treated. in total, fusion transcripts were identified from all rnaseq datasets. the majority of these fusion transcripts were ‘read-through chimeras’ derived from adjacent genes in the genome. characterization of sequences at fusion loci were carried out using a combination of the fusionmap program, custom perl scripts, and the rnafold program. our computational analysis indicated that most fusion junctions use the consensus gt-ag intron donor-acceptor splice site, and most fusion transcripts maintained the open reading frame. we assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. the abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that sine repeats are involved in aberrant gene transcription. we found few instances of repetitive sequences at both fusion and non-fusion junctions. finally, rt-qpcr was performed on rna from both clinical prostate tumors and adjacent non-cancer cells, and lncap cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. we reveal that fusion transcript expression is similar to the expression of parental genes.  fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fusionrnarnaseqprostate cancerissue-copyright-statement© the author 2015 