BACKGROUND
embryonic stem  cells are self-renewable cells able to differentiate into virtually any cell type, an ability called pluripotency .
besides obvious therapeutic potential, pluripotency provides an opportunity to understand how differentiation works in early embryonic development.
many groups aim to characterize the 'stemness' of es cells in terms of gene regulation and to identify genes responsible for maintaining pluripotency.
although the identification of the oct <dig> , sox <dig> and nanog regulatory network  <cit>  is a significant advance, an integrated understanding is still lacking.
some key approaches to understanding the molecular basis of pluripotency and early differentiation are the analysis of transcription factor binding site mapping  <cit> , epigenetics studies , as well as in-depth assessments of transcripts expressed in es cells.
transcriptome surveys of es cells by sage  <cit> , mpss  <cit> , gene trapping  <cit>  and est sequencing  <cit>  have been performed by several groups under the hypothesis that transcripts expressed specifically in es cells are instrumental for maintaining pluripotency.
another transcript profiling method which has been used to interogate es cell transcriptome and offers a marked improvement compared to those techniques is gene identification signature  analysis  <cit> .
gis analysis is a sage modification which isolates tags of  <dig> base pairs  from the 5'- and 3'-ends of a transcript and concatenates them to form paired-end ditag  structures.
whereas sage extracts a single tag per transcript, gis analysis presents paired sequence from transcript start and end sites, marking the boundaries of transcriptional units  on the genome.
gis analysis of mouse e <dig> es cells generated  <dig>  pet sequences.
among them were hundreds of novel, uncharacterized tus readily apparent on comparison of pet boundaries with known-gene boundaries mapped to the genome.
the novel tus were separated into four categories: new transcriptional start and end sites of known genes, intergenic splicing connecting two adjacent genes as a single transcript, trans-splicing events, and totally novel transcripts.
identification of novel transcripts from sage tags not matching known genes has been reported before  <cit> , and heretofore unrecognized novel es cell transcriptome components have been hypothesized to be involved in orchestrating the pluripotent state  <cit> .
in one major recent study, es-specific sage tags have been earmarked for 3' cdna cloning and subsequent evaluation of the novel transcripts as potential regulators of the stemness phenotype  <cit> .
we also hypothesized that some es-derived novel tus were expressed preferentially in undifferentiated es cells, as they had not been identified previously with other transcriptome characterisation techniques, and hence that they may potentially be involved in maintaining pluripotency.
another example of this approach is the identification of zfp <dig> as a transcription factor that controls pluripotency in es cells  <cit> .
building up from large-scale est and mpss data which had indicated that the previously uncharacterized zfp <dig> gene was differentially expressed between pluripotent and differentiated states, wang et al.
<cit>  found that zfp <dig> regulates pluripotency and is actively involved in the oct <dig> and nanog regulatory loop.
to similarly accomplish discovery of novel pluripotency-associated transcripts, we focused on two groups of novel tus inferred from gis pet data: the fully novel transcripts and the intergenic splicing events.
since gis analysis provided sequence from 5'- and 3'-ends of the transcripts, we physically validated and sequenced these tus to reveal their intron-exon structures.
we analysed their splice junctions, alternative isoforms, open reading frames , and homologies to predict protein-coding potential.
in an attempt to relate the tus to es-specific functions, we profiled their expression in tissue panels and differentiation timecourses, showing that the tus were either es-specific or downregulated upon differentiation.
we outline five novel tus to be further investigated for direct functional roles in es cells.
RESULTS
initial computational analysis of novel transcriptional units in mouse embryonic stem cells
the initial dataset of  <dig> candidate unconventional pet-supported tus consisted of  <dig> novel tus not corresponding to known genes and  <dig> representing potential intergenic splicing.
we eliminated tu candidates with multiple or ambiguous mappings, as described in materials and methods.
additionally, one novel and one intergenically spliced tu on the mm <dig> assembly matched known genes on mm <dig> and were not analysed further.
table  <dig> summarizes filtering results for novel and intergenically spliced candidate tus with e <dig> es pet support.
the numbers refer to pet counts in each named category corresponding to annotation-based filtering described in the text.
the 'mapping to multiple loci' and 'unmappable to the genome' columns are the combined results of both in-silico pcr and blastn.
<dig> tus were chosen for experimental validation:  <dig> novel tus with no known-gene support and  <dig> intergenically spliced tus.
their corresponding pets were visualized against the mm <dig> assembly to gauge the extent of mrna and est support  for these tus.
eight tus were supported at least partially by full-length cdnas and/or ests, but the other eight lacked support in public transcriptome databases and were therefore completely novel .
expression validation, sequence analysis, and genomic properties of novel tus in mouse embryonic stem cells
we assayed the  <dig> novel tus in mouse es cells by rtpcr.
we obtained pcr products for  <dig> of the tus.
they were sequenced and blat-aligned to the genome.
four tus  yielded non-specific rtpcr products which did not map to the expected locus after sequencing.
for all of those tus, there had been no cdna or est evidence of nearby transcription along the genome.
as a result, only the pet half-tag  sequences themselves were available for primer design.
the short length and low complexity of these sequences likely resulted in suboptimal primers which amplified non-specific products.
three other tus  gave specific products of sizes consistent with lack of splicing; two of these were also potentially problematic due to intronic same-strand localization relative to known genes.
these seven tus were therefore excluded.
of the other seven pcr-positive tus, five yielded full-length sequences and became the focus of remaining analyses .
tu <dig> : a novel pry and spry domain-containing stem cell gene
three isoforms of tu <dig> were recovered by full-length sequencing of rtpcr products.
they were  <dig>   <dig> and  <dig> bp long, named a , b  and c  respectively .
the longest isoform a had nine exons while isoforms b and c contained eight and seven, respectively.
all splice junctions were canonical .
no spliced ests were present at this locus.
of the nine unspliced ests overlapping exonic sequences of tu <dig>  one  was from mouse es cells and two  were from trophoblast stem cells, as was the single full-length cdna at the locus .
this cdna- and est-derived expression profile was consistent with expression of this tu in es cells.
the three ests supporting the 3'-halftag of the original pet  <dig> overlapped the last exon of tu <dig>  while the other ests and the cdna partially were internal to the tu.
this implies that the diversity of tu <dig> full-length isoforms may be considerably greater than demonstrated here.
the longest isoform had an orf of  <dig> amino acids , from nucleotide   <dig> to nt  <dig>  this orf contained the conserved domains pry and spry, an evolutionary innovation associated with immunity  <cit> .
it had homology to a predicted mouse protein similar to tripartite motif protein  <dig> and to a predicted rat protein similar to flj <dig>  which also contains pry and spry domains.
interestingly, tu <dig> shares a bidirectional promoter with bc <dig>  another uncharacterized protein-coding gene, through the est by <dig>  this est constituted an alternative 5' end of bc <dig> and was also of es-cell origin.
bc <dig> itself was supported by many other es-cell mrnas and ests, and contained a partial pry domain, though its orf was only  <dig> aa long.
however, its human putative ortholog, flj <dig>  had a 468-aa orf with ring, pry, and spry domains.
the promoter sharing potential may imply a coregulatory and conserved relationship between these two tandemly arranged pry-domain genes with es-specific expression.
tu <dig> : a putative novel es-specific transcription factor
tu <dig> had two isoforms, a  and b  , with five and four exons respectively.
the splice junctions were canonical.
exonic same-strand overlap with known ests , was observed.
the structurally closest est was ca <dig>  derived from a mouse es cell line.
the longest orf was  <dig> aa, coded by nt 107– <dig> of isoform b. the orf contained partial krab and fog zinc finger domains, homologous to a predicted protein similar to gonadotropin-inducible ovarian transcription factor  <dig>  this suggests that tu <dig> may encode an es-specific transcription factor.
tu <dig> : slc10a3-ubl <dig> intergenic splicing
tu <dig> connected the genes slc10a <dig> and ubl <dig>  taken separately, these two adjacent genes had numerous mrnas and ests in the public databases, but no public transcripts connect the genes by intergenic splicing.
we identified three isoforms of this tu from e <dig> es cells,  <dig>   <dig> and  <dig> bp long, labeled a , b , and c  respectively .
all isoforms spliced the 5'utr of slc10a <dig> gene to a novel splice acceptor in the intergenic region and continued into the remaining sequence of the downstream ubl <dig> gene.
the two longer isoforms consisted of seven exons while the shortest had six, skipping one exon of ubl <dig>  all splice junctions, including the novel splice acceptors, were canonical, suggesting that this tu was biologically transcribed and not a result of cdna library chimerism.
only the two longer isoforms are likely to encode a protein; the 98-aa longest orf of isoform c lacks any significant homology to slc10a <dig>  ubl <dig>  or any other proteins.
the longer isoforms a and b both potentially encoded a 157-aa orf.
this orf contained the conserved domain gdx_n in the first  <dig> aa and was homologous to ubl <dig>  blastp search of ncbi orf finder results did not detect any potential for chimeric protein formation, indicating that slc10a <dig> contributed exclusively the 5'utr to the slc10a3-ubl <dig> tu while the entire coding potential was contributed by ubl <dig>  therefore, despite intergenic splicing, the transcript would not encode a novel protein.
it is possible that regulatory sequences of slc10a <dig>  such as rna-binding protein cognate sites in the 5'utr, are functionally united with the ubl <dig> orf by this intergenic splicing and may result in slc10a <dig> sequence-mediated effects on ubl <dig> mrna or protein levels.
tu <dig> : clk2-scamp <dig> intergenic splicing
tu <dig> connected the genes clk <dig> and scamp <dig>  each of the component genes had good mrna and est support individually but none of the public transcripts bridged the two genes into a single tu.
in addition to the 2687-nt isoform of this tu  validated in  <cit>  , we identified two isoforms of  <dig> and  <dig> nt, labeled a  and b , respectively .
isoform a was similar to the 2687-nt published transcript  <cit>  but skipped exon  <dig> of clk <dig>  it consisted of  <dig> exons, with all splice junctions being gt-ag.
isoform b had  <dig> exons and, likewise, all splice junctions were canonical.
all three isoforms skipped the last exon of clk <dig> and spliced into different scamp <dig> splice acceptors, presumably as a consequence of transcriptional read-through.
isoform a and the published transcript spliced into the second exon of scamp <dig> while isoform b spliced into the penultimate exon of scamp <dig>
isoform a had a 407-aa orf  and, similarly to the published transcript, encoded a chimeric protein with components from both genes.
the predicted protein contained the stkc  domain from clk <dig> and the scamp domain from scamp <dig>
clk <dig> may be involved in regulation of alternative splicing  <cit>  and our result indicates that clk <dig> itself may be alternatively spliced.
human scamp <dig> is known to undergo a specific tyrosine phosphorylation event, leading potentially to downregulation of the human egf receptor  <cit> .
the presence of the clk <dig> kinase domain in the fused transcript might imply auto-regulation of the scamp <dig> fragment of the chimeric protein by self-phosphorylation, a regulatory context distinct from conventional post-translational processing of scamp <dig>
isoform b had a longer orf of  <dig> aa .
however, this 517-aa longest orf of the isoform b mrna only contained the stkc domain and was solely homologous to the clk <dig> protein, because the scamp <dig> protein sequence in this isoform was encoded in a different reading frame.
therefore, despite intergenic splicing, isoform b does not encode a chimeric protein.
tu <dig> : 1110034a24rik-rpl36al intergenic splicing
intergenically spliced tu <dig>  connected the 1110034a24rik and rpl36al genes.
the tu,  <dig> bp long, has four exons with canonical splice junctions .
there were numerous cdnas and ests supporting each gene separately, but none bridged the two.
this tu overlapped in the reverse  orientation the mgat <dig> gene between 1110034a24rik and rpl36al.
the longest orf of this tu was  <dig> aa .
it contained no conserved domains and was identical to the standalone 1110034a24rik orf.
after the stop codon at nt  <dig> there was another 100-aa orf identical to rpl36al.
therefore, this intergenically spliced tu was potentially bicistronic, containing the complete orfs of both component genes, but did not encode a fusion protein.
the cis-antisense overlap with mgat <dig> was  <dig> nt, comprising the entire third exon of this tu which overlaps part of the coding sequence of mgat <dig>  expression of the 1110034a24rik-rpl36al bicistronic tu may exert regulatory effect on mgat through the cis-antisense pairing, as genes in other cis-antisense gene pairs can affect each other's expression  <cit> .
we then investigated whether these new members of the es cell transcriptome are regulated by known es transcription factors.
to accomplish this, we searched two datasets of experimentally supported transcription factor binding events for oct <dig>  nanog, and sox <dig> binding sites in the vicinity of the tus: chromatin immunoprecipitation paired-end ditags   <cit>  and chip-sequencing by solexa technology .
in brief, all tus had evidence of oct <dig>  nanog, and/or sox <dig> binding events within  <dig> kb of the tu in one or both types of chip datasets .
most notably, tu <dig> had oct <dig> and nanog binding sites  <dig>  kb downstream of its 3' end, while tu <dig> had a sox <dig> binding site  <dig>  kb downstream of its 3' end supported by both sox <dig> chip datasets.
only one binding event – a chip-seq nanog binding site – was localized directly at the transcription start site, for tu <dig>
qualitative and quantitative expression profiling of novel tus
since the tus were newly recognized components of the mouse es cell transcriptome, the fact that they had never been detected before in any other cell type suggested that they might be preferentially expressed in es cells.
we thus hypothesized that they may be involved in the maintenance of pluripotency in these cells and accordingly, checked their expression in stem cells and a panel of embryonic and adult tissues, because higher expression in undifferentiated cells would be consistent with a function specific to such cells.
an example of this pattern of gene expression is lin <dig> which was found by sage to be expressed only in human es cells  <cit>  and showed downregulation upon es cell differentiation.
in fact, lin <dig> has only been recently implicated in human somatic cell reprogramming back to the pluripotent state  <cit> , indicating its importance in es cells.
conventional rtpcr and quantitative real-time pcr  were used to profile expression of the novel tus.
the results, discussed further, showed that all five tus were either es-specific or were expressed most highly in es cells despite being also transcribed elsewhere.
to further confirm the pluripotency association of the tus, we also measured the changes in expression levels of the tus in es cells upon chemical induction of differentiation.
es cells were differentiated using retinoic acid , dimethyl sulfoxide , and hexamethylene bisacetamide  treatments, in separate experiments.
the three treatments served also to eliminate the possibility of chemical-specific expression responses.
to validate that the three chemical treatments reduced pluripotency marker levels and led the cells to differentiate, we tested expression levels of four principal pluripotency markers  by taqman qrtpcr.
the results clearly indicated that all pluripotency marker mrna levels decreased over time in all three treatment timecourses, proving that the treatments led to the expected differentiation outcomes .
qrtpcr was performed to determine the effect of differentiation on the rna level of the novel tus.
we demonstrated that most of the tus were consistently downregulated upon differentiation, regardless of the differentiation agents used.
tu <dig> : a novel stem cell gene with pry and spry domains
qualitative rtpcr on the multitissue panel suggested that tu <dig> had an es-cell-specific expression pattern .
we did not detect expression of this tu outside of es cells by rtpcr .
qrtpcr demonstrated that expression of this tu was low though not absent in non-es samples.
the second-highest expression level was in adult skeletal muscle, at 30% of the es-cell level; in the few other tissues where the tu was expressed, its rna level was less than 20% of the es-cell level .
consistent downregulation of this tu upon es cell differentiation was evident in all three differentiation timecourses, although it was most pronounced with dmso treatment .
tu <dig> : a putative novel es-specific transcription factor
this novel tu showed a similar expression pattern to tu <dig>  qualitative rtpcr indicated that besides expression in es cells this tu had barely-detectable expression in the embryo at days seven and eleven .
qrtpcr confirmed the es specificity of tu <dig> .
the expression level of tu <dig>  similarly to tu <dig>  showed a decreasing trend upon es cell differentiation .
this gradual effect was observed in all three treatments but the decrease was not as rapid as that of tu <dig>  a significant reduction in tu <dig> expression was observed after four days of ra treatment whereas for tu <dig> the effect was observed as early as day two.
in the other differentiation treatments, the effect was similar between the two genes, with dmso treatment yielding the strongest response.
this expression profile argues in favor of potential relevance of tu <dig> to es cell pluripotency.
tu <dig> : slc10a3-ubl <dig> intergenic splicing
qualitative rtpcr suggested that the intergenically spliced slc10a3-ubl <dig> tu had a close-to-ubiquitous expression profile , although it nevertheless was preferentially expressed in embryonic rather than adult tissues.
one adult sample with higher expression of slc10a3-ubl <dig> than es cells was testes .
this is intriguing because adult testes reportedly contain multipotent cells capable of forming derivatives of the three embryonic layers  <cit> .
qrtpcr showed that this bicistronic tu was downregulated upon differentiation .
dmso and hmba differentiation timecourses resulted in a more rapid downregulation of slc10a3-ubl <dig>  giving it an expression profile recapitulating that of tu <dig>
to verify that this intergenically spliced tu was involved in pluripotency but its component genes were not, we individually measured expression levels of the component genes, slc10a <dig> and ubl <dig>  a correlation between the expression level of the bicistronic tu and expression levels of its component genes would suggest that slc10a3-ubl <dig> was not an independently regulated tu.
however, the expression level of this tu was markedly different from those of the component genes, both in the multitissue panel and in all three differentiation timecourses .
in the multitissue panel slc10a <dig> was highly upregulated in adult tissues, especially in heart, kidney, and liver, while ubl <dig> expression was high in the heart and skeletal muscle relative to e <dig> es cells.
in the differentiation timecourses, the component genes did not show any down regulation effect upon induction of differentiation.
in fact, they were upregulated, especially upon dmso treatment.
this result showed that although this bicistronic tu contained the 5' utr of slc10a <dig>  its regulation did not simply reflect the expression profile of that gene.
we conclude that slc10a3-ubl <dig> is a genuine novel transcript regulated independently of its component genes.
furthermore, we propose that this locus may undergo expression regulation by a combination of alternative splicing and preferential transcription initiation at the downstream ubl <dig> promoter, such that intergenic splicing is favored in es cells while independent transcription of the component genes occurs preferentially in differentiated tissues.
tu <dig> : clk2-scamp <dig> intergenic splicing
based on qualitative rtpcr, tu <dig> appeared largely es-specific, with some embryonic expression .
the only adult tissue samples that showed a visible band were skeletal muscle and testes.
qrtpcr showed that expression level in testes was approximately fivefold greater than the es-cell level .
upon es cell differentiation, the clk2-scamp <dig> tu showed significant downregulation .
in ra- and hmba-treated cells the gene showed a similar expression profile with a maximum decrease to 20% of the es-cell level after four days of treatment.
in the dmso timecourse this effect was less pronounced.
tu <dig> : 1110034a24rik-rpl36al intergenic splicing
qualitative rtpcr of the intergenically spliced 1110034a24rik-rpl36al tu showed that it was not es cell-specific, as multiple embryonic and adult samples showed detectable expression .
however, qrtpcr indicated that the highest expression level was still in es cells .
accordingly, ra, dmso and hmba treatments all showed that expression of 1110034a24rik-rpl36al decreased as the cells differentiated .
this decrease was significant in all of the timecourses tested with up to 80% reduction in mrna expression in ra-treated cells at day six and up to 90% decrease in both dmso- and hmba-treated cells.
rnai-based functional analysis of novel tus
we designed 4-sirna combinations targeting tu <dig> and tu <dig> and individual sirnas uniquely targeting the intergenically spliced tu <dig>  tu <dig>  and tu <dig> transcripts .
we were unable to achieve satisfactory tu <dig> knockdown .
we accomplished knockdowns of the other four tus to 25% – 55% of control  mrna levels .
we hypothesized that, if the novel tus were relevant to maintaining es cell pluripotency instead of merely associated with pluripotency, then depletion of rna levels of the tus could result in depletion of mrna levels of some, or all, of the four principal pluripotency-driving transcription factors .
although both nanog and klf <dig> expression levels were reduced to 70% of reference upon knockdown of tu <dig>  no downregulation of the oct <dig> or sox <dig> was observed.
there was no downregulation of oct <dig>  sox <dig>  klf <dig>  or nanog upon knock down of the other novel tus .
we also reasoned that, if the tus were indeed required for maintaining the es state, then rnai suppression of the tu rna levels could lead to upregulation of well-known ectodermal, endodermal, and mesodermal lineage markers.
we therefore tested, by qrtpcr with commercially available primer sets and fluorescent probes , the mrna levels of ectodermal , endodermal , and mesodermal  differentiation markers after independent tu <dig>  tu <dig>  tu <dig>  and tu <dig> rnai transfections.
differentiation marker levels were unaffected by tu <dig>  tu <dig>  and tu <dig> rnai.
upregulation of fgf <dig>  and t-brachyury , to approximately 150% of reference levels each, was observed at day  <dig> and day  <dig> post-tu <dig> rnai .
nanog, klf <dig>  fgf <dig>  and t-brachyury were reproducibly affected by tu <dig> perturbation, making a direct function of tu <dig> in pluripotency maintenance possible in principle.
however, the lack of generalized effects on pluripotency transcription factors and differentiation marker classes suggests that even tu <dig> may not be an essential pluripotency regulator.
discussion
validation rate of novel tus from a pet-transcriptome library
the five novel tus whose transcription and full-length sequence we validated represent only a small subset  of candidate novel tus revealed by pet-based mes transcriptome sampling.
we ascribe this small fraction to our selection criteria implemented in selecting tus to be validated.
the criteria were designed to maximize the chance of experimental validation but do not imply that all remaining tus are artefactual.
to sample a larger number of tus for future physical validation, selection criteria can be relaxed.
for example, the  <dig> kb cutoff value for maximum genomic span can be increased since some genes are an order of magnitude longer .
two putative bicistronic tus which were excluded from the original dataset would have been detected if the cutoff had been increased to  <dig> mb.
among tus selected for physical validation, only five yielded specific products.
one reason for the low efficiency is likely the limited pet sequence available for primer selection, the crucial point of the validation process.
it is possible that the primers designed from the pet sequence were suboptimal and unable to bind to the correct transcript resulting in both false negative and false positive pcr results.
the presence of ests and flcdnas in the locus clearly aids validations as it increases the amount of known exonic sequence space putatively belonging to the pet-supported tu; utilizing this space benefits primer design.
ng et al.
verified a large proportion of pet-supported mes transcripts .
however, they used their own gis flcdna library as the template source.
thus, some of their confirmed tus were likely due either to experimental artefacts or to extremely rare transcripts which were clonally propagated in that library, but might not be present at detectable levels in normal e <dig> cells.
we used rna from e <dig> cells for our analysis.
furthermore, the high success rate obtained by ng et al.
was for a combination of novel and known genes; the validation rate for novel tus alone was not explicitly specified.
these factors might account for our lower validation rate.
intergenic splicing
three of our five novel tus join two adjacent genes by forming a single transcript containing exonic sequences of both genes.
despite sporadic reports of intergenic splicing , its biological significance is largely unknown.
several models speculate on how such transcriptional gene fusion may have a physiological role.
first, the fusion may be a byproduct of transcriptional read-through, whereby the transcriptional machinery does not stop at the transcription stop site of the first gene but "reads-through" to the 3' end of the next gene on the same strand instead.
these can be rare unregulated stochastic events with no functional significance.
however, we showed that our transcripts were not present just in es cells; they were frequently detectable in several, though not all, cell and tissue types, but never ubiquitous, and the expression levels differed reproducibly between samples.
consequently, the intergenic splicing events we observed are unlikely to be random.
second, chimeric tus – those with fusion orfs in addition to intergenic splicing – can form novel and truly functional proteins.
the fusion can create a new bifunctional protein, as is the case in the tweak-april fusion  <cit> , or change the properties of one of the participating proteins.
an example of the latter case is the kua-ube2v <dig> fusion  <cit>  where the fused protein is cytoplasmic while the original ube2v <dig> is nuclear.
only one of our three intergenically spliced tus has a putatively chimeric fusion orf, however.
only 25% of intergenically spliced genes detected in a recent study encode chimeric proteins  <cit> .
a less clear functional impact of intergenic splicing centers on expression regulation.
a theoretical example is the bicistronic tu <dig> which contained the 5' utr of the upstream slc10a <dig> gene and the entire coding sequence of the downstream ubl <dig> gene.
since tu <dig> contained the 5'utr of slc10a <dig> it could be hypothesized that the bicistronic transcript would be co-regulated together with the upstream gene.
however, analysis of their expression level showed that tu <dig> was regulated independently from its two component genes.
there might be other consequences of the hybrid "5'utr of gene  <dig> – orf of gene 2" composition of this tu, for example potential for regulation of ubl <dig> by rna-binding proteins which only recognize specific sites in the 5'utr of slc10a <dig>
despite the scale of the intergenic splicing datasets reported in  <cit>  and  <cit> , neither study identified intergenic splicing at the three loci corresponding to our tus.
in fact, even a recent high-throughput effort to catalog novel components of the mouse es transcriptome  <cit>  did not yield any evidence of intergenic splicing at our three loci, although gene trap tags corresponding individually to ubl <dig>  clk <dig>  scamp <dig>  and rpl36al were isolated in that project.
this shows that diversity and genomewide incidence of this phenomenon exceed current estimates, and emphasizes the need for targeted validation of candidates from specific libraries, such as our mes transcriptome library, to gain insight into the diversity of intergenic splicing in transcriptomes previously not subjected to deep sampling.
relevance to pluripotency of es cells
in addition to reporting novel tus in mouse es cells, we provide preliminary evidence for their functional significance.
since the novel tus were initially found in undifferentiated es cells, we hypothesized that they were involved in es cell pluripotency.
our findings initially supported this hypothesis since almost all of the tus we reported here were preferentially expressed in es cells compared to adult tissues, and all tus had nearby oct <dig>  sox <dig>  and nanog binding sites confirmed by two independent lines of chip evidence.
this suggests an importance of these tus in es, rather than differentiated, cells, although it does not preclude functional roles in other contexts.
more direct support for our hypothesis is provided by the downregulation of these tus upon es cell differentiation.
the effect was common to all three differentiation treatments tested, indicating that it was not a chemical-specific response, but rather a reproducible outcome of differentiation.
combined with the est-derived expression profiles and multi tissue expression analysis, this result further implicates the five tus in maintaining es cell pluripotency.
loss-of-function experiments for four of these novel tus did not lead to cell differentiation as can be seen from the levels of pluripotency markers oct <dig>  sox <dig>  nanog and klf <dig> upon sirna transfections.
however, tu <dig> was intriguing because its knockdown led to approximately 30% concurrent reduction in klf <dig> and nanog levels, as well as to  <dig> -fold upregulation of fgf <dig> and t-brachyury.
it has been known that t-brachyury is able to upregulate nanog, which in turn suppresses t-brachyury's own expression in an indirect, smad-facilitated negative feedback loop in early mesodermal progenitors  <cit> .
if an identical t-brachyury/nanog relationship exists in undifferentiated es cells or in an early mesodermal progenitor subpopulation thereof, then it is possible that tu <dig> may be a contributor in maintaining nanog levels in undifferentiated es cells so that when its expression is knocked down nanog level is no longer maintained and the suppression of t-brachyury is lost.
summarily however, these five tus alone, without unknown cofactors, are not necessary or sufficient for maintaining pluripotency.
overexpression of the tus, in transient or stable transfectants, could be attempted as an additional functional test in order to determine whether higher levels of the tu rnas enable es cells to resist chemical differentiation drivers.
while an in-silico or computational subtractive-hybridization comparison with a non-es transcriptome would be appropriate, there was no non-es pet transcriptome dataset generated by ng et al.
<cit> , precluding direct quantitative comparison of these novel tus' expression in es vs. non-es states.
CONCLUSIONS
our observations summarily demonstrate that all five tus we analyzed represent genuine novel transcripts potentially important in maintaining pluripotency.
to elucidate the biological importance of these novel tus, rnai knock-downs were performed, which indicated that rna level ablation of tu <dig> leads to a modicum of reproducible expression profile changes associated with the three principal cell differentiation fates while such ablation of the other four tus failed to change expression phenotypes.
nevertheless, our results establish a foundation for functional analysis of a small number of novel tus filtered from a large set of candidates, and show that the gis mes pet-transcriptome library is a good resource for identification of novel tus highly expressed in, and specific to, es cells.

 embryonic stem cells are self-renewable cells able to differentiate into virtually any cell type, an ability called pluripotency. besides obvious therapeutic potential, pluripotency provides an opportunity to understand how differentiation works in early embryonic development. many groups aim to characterize the'stemness' of es cells in terms of gene regulation and to identify genes responsible for maintaining pluripotency. although the identification of the oct, sox and nanog regulatory network is a significant advance, an integrated understanding is still lacking. some key approaches to understanding the molecular basis of pluripotency and early differentiation are the analysis of transcription factor binding site mapping, epigenetics studies, as well as in-depth assessments of transcripts expressed in es cells. transcriptome surveys of es cells by sage, mpss, gene trapping and est sequencing have been performed by several groups under the hypothesis that transcripts expressed specifically in es cells are instrumental for maintaining pluripotency. another transcript profiling method which has been used to interogate es cell transcriptome and offers a marked improvement compared to those techniques is gene identification signature analysis. gis analysis is a sage modification which isolates tags of base pairs from the 5'- and 3'-ends of a transcript and concatenates them to form paired-end ditag structures. whereas sage extracts a single tag per transcript, gis analysis presents paired sequence from transcript start and end sites, marking the boundaries of transcriptional units on the genome. gis analysis of mouse e es cells generated pet sequences. among them were hundreds of novel, uncharacterized tus readily apparent on comparison of pet boundaries with known-gene boundaries mapped to the genome. the novel tus were separated into four categories: new transcriptional start and end sites of known genes, intergenic splicing connecting two adjacent genes as a single transcript, trans-splicing events, and totally novel transcripts. identification of novel transcripts from sage tags not matching known genes has been reported before, and heretofore unrecognized novel es cell transcriptome components have been hypothesized to be involved in orchestrating the pluripotent state. in one major recent study, es-specific sage tags have been earmarked for 3' cdna cloning and subsequent evaluation of the novel transcripts as potential regulators of the stemness phenotype. we also hypothesized that some es-derived novel tus were expressed preferentially in undifferentiated es cells, as they had not been identified previously with other transcriptome characterisation techniques, and hence that they may potentially be involved in maintaining pluripotency. another example of this approach is the identification of zfp as a transcription factor that controls pluripotency in es cells. building up from large-scale est and mpss data which had indicated that the previously uncharacterized zfp gene was differentially expressed between pluripotent and differentiated states, wang et al. found that zfp regulates pluripotency and is actively involved in the oct and nanog regulatory loop. to similarly accomplish discovery of novel pluripotency-associated transcripts, we focused on two groups of novel tus inferred from gis pet data: the fully novel transcripts and the intergenic splicing events. since gis analysis provided sequence from 5'- and 3'-ends of the transcripts, we physically validated and sequenced these tus to reveal their intron-exon structures. we analysed their splice junctions, alternative isoforms, open reading frames, and homologies to predict protein-coding potential. in an attempt to relate the tus to es-specific functions, we profiled their expression in tissue panels and differentiation timecourses, showing that the tus were either es-specific or downregulated upon differentiation. we outline five novel tus to be further investigated for direct functional roles in es cells.  initial computational analysis of novel transcriptional units in mouse embryonic stem cells the initial dataset of candidate unconventional pet-supported tus consisted of novel tus not corresponding to known genes and representing potential intergenic splicing. we eliminated tu candidates with multiple or ambiguous mappings, as described in materials and methods. additionally, one novel and one intergenically spliced tu on the mm assembly matched known genes on mm and were not analysed further. table summarizes filtering results for novel and intergenically spliced candidate tus with e es pet support. the numbers refer to pet counts in each named category corresponding to annotation-based filtering described in the text. the'mapping to multiple loci' and'unmappable to the genome' columns are the combined results of both in-silico pcr and blastn. tus were chosen for experimental validation: novel tus with no known-gene support and intergenically spliced tus. their corresponding pets were visualized against the mm assembly to gauge the extent of mrna and est support for these tus. eight tus were supported at least partially by full-length cdnas and/or ests, but the other eight lacked support in public transcriptome databases and were therefore completely novel. expression validation, sequence analysis, and genomic properties of novel tus in mouse embryonic stem cells we assayed the novel tus in mouse es cells by rtpcr. we obtained pcr products for of the tus. they were sequenced and blat-aligned to the genome. four tus yielded non-specific rtpcr products which did not map to the expected locus after sequencing. for all of those tus, there had been no cdna or est evidence of nearby transcription along the genome. as a result, only the pet half-tag sequences themselves were available for primer design. the short length and low complexity of these sequences likely resulted in suboptimal primers which amplified non-specific products. three other tus gave specific products of sizes consistent with lack of splicing; two of these were also potentially problematic due to intronic same-strand localization relative to known genes. these seven tus were therefore excluded. of the other seven pcr-positive tus, five yielded full-length sequences and became the focus of remaining analyses. tu: a novel pry and spry domain-containing stem cell gene three isoforms of tu were recovered by full-length sequencing of rtpcr products. they were and bp long, named a, b and c respectively. the longest isoform a had nine exons while isoforms b and c contained eight and seven, respectively. all splice junctions were canonical. no spliced ests were present at this locus. of the nine unspliced ests overlapping exonic sequences of tu one was from mouse es cells and two were from trophoblast stem cells, as was the single full-length cdna at the locus. this cdna- and est-derived expression profile was consistent with expression of this tu in es cells. the three ests supporting the 3'-halftag of the original pet overlapped the last exon of tu while the other ests and the cdna partially were internal to the tu. this implies that the diversity of tu full-length isoforms may be considerably greater than demonstrated here. the longest isoform had an orf of amino acids, from nucleotide to nt this orf contained the conserved domains pry and spry, an evolutionary innovation associated with immunity. it had homology to a predicted mouse protein similar to tripartite motif protein and to a predicted rat protein similar to flj which also contains pry and spry domains. interestingly, tu shares a bidirectional promoter with bc another uncharacterized protein-coding gene, through the est by this est constituted an alternative 5' end of bc and was also of es-cell origin. bc itself was supported by many other es-cell mrnas and ests, and contained a partial pry domain, though its orf was only aa long. however, its human putative ortholog, flj had a 468-aa orf with ring, pry, and spry domains. the promoter sharing potential may imply a coregulatory and conserved relationship between these two tandemly arranged pry-domain genes with es-specific expression. tu: a putative novel es-specific transcription factor tu had two isoforms, a and b, with five and four exons respectively. the splice junctions were canonical. exonic same-strand overlap with known ests, was observed. the structurally closest est was ca derived from a mouse es cell line. the longest orf was aa, coded by nt 107– of isoform b. the orf contained partial krab and fog zinc finger domains, homologous to a predicted protein similar to gonadotropin-inducible ovarian transcription factor this suggests that tu may encode an es-specific transcription factor. tu: slc10a3-ubl intergenic splicing tu connected the genes slc10a and ubl taken separately, these two adjacent genes had numerous mrnas and ests in the public databases, but no public transcripts connect the genes by intergenic splicing. we identified three isoforms of this tu from e es cells, and bp long, labeled a, b, and c respectively. all isoforms spliced the 5'utr of slc10a gene to a novel splice acceptor in the intergenic region and continued into the remaining sequence of the downstream ubl gene. the two longer isoforms consisted of seven exons while the shortest had six, skipping one exon of ubl all splice junctions, including the novel splice acceptors, were canonical, suggesting that this tu was biologically transcribed and not a result of cdna library chimerism. only the two longer isoforms are likely to encode a protein; the 98-aa longest orf of isoform c lacks any significant homology to slc10a ubl or any other proteins. the longer isoforms a and b both potentially encoded a 157-aa orf. this orf contained the conserved domain gdx_n in the first aa and was homologous to ubl blastp search of ncbi orf finder results did not detect any potential for chimeric protein formation, indicating that slc10a contributed exclusively the 5'utr to the slc10a3-ubl tu while the entire coding potential was contributed by ubl therefore, despite intergenic splicing, the transcript would not encode a novel protein. it is possible that regulatory sequences of slc10a such as rna-binding protein cognate sites in the 5'utr, are functionally united with the ubl orf by this intergenic splicing and may result in slc10a sequence-mediated effects on ubl mrna or protein levels. tu: clk2-scamp intergenic splicing tu connected the genes clk and scamp each of the component genes had good mrna and est support individually but none of the public transcripts bridged the two genes into a single tu. in addition to the 2687-nt isoform of this tu validated in, we identified two isoforms of and nt, labeled a and b, respectively. isoform a was similar to the 2687-nt published transcript but skipped exon of clk it consisted of exons, with all splice junctions being gt-ag. isoform b had exons and, likewise, all splice junctions were canonical. all three isoforms skipped the last exon of clk and spliced into different scamp splice acceptors, presumably as a consequence of transcriptional read-through. isoform a and the published transcript spliced into the second exon of scamp while isoform b spliced into the penultimate exon of scamp isoform a had a 407-aa orf and, similarly to the published transcript, encoded a chimeric protein with components from both genes. the predicted protein contained the stkc domain from clk and the scamp domain from scamp clk may be involved in regulation of alternative splicing and our result indicates that clk itself may be alternatively spliced. human scamp is known to undergo a specific tyrosine phosphorylation event, leading potentially to downregulation of the human egf receptor. the presence of the clk kinase domain in the fused transcript might imply auto-regulation of the scamp fragment of the chimeric protein by self-phosphorylation, a regulatory context distinct from conventional post-translational processing of scamp isoform b had a longer orf of aa. however, this 517-aa longest orf of the isoform b mrna only contained the stkc domain and was solely homologous to the clk protein, because the scamp protein sequence in this isoform was encoded in a different reading frame. therefore, despite intergenic splicing, isoform b does not encode a chimeric protein. tu: 1110034a24rik-rpl36al intergenic splicing intergenically spliced tu connected the 1110034a24rik and rpl36al genes. the tu, bp long, has four exons with canonical splice junctions. there were numerous cdnas and ests supporting each gene separately, but none bridged the two. this tu overlapped in the reverse orientation the mgat gene between 1110034a24rik and rpl36al. the longest orf of this tu was aa. it contained no conserved domains and was identical to the standalone 1110034a24rik orf. after the stop codon at nt there was another 100-aa orf identical to rpl36al. therefore, this intergenically spliced tu was potentially bicistronic, containing the complete orfs of both component genes, but did not encode a fusion protein. the cis-antisense overlap with mgat was nt, comprising the entire third exon of this tu which overlaps part of the coding sequence of mgat expression of the 1110034a24rik-rpl36al bicistronic tu may exert regulatory effect on mgat through the cis-antisense pairing, as genes in other cis-antisense gene pairs can affect each other's expression. we then investigated whether these new members of the es cell transcriptome are regulated by known es transcription factors. to accomplish this, we searched two datasets of experimentally supported transcription factor binding events for oct nanog, and sox binding sites in the vicinity of the tus: chromatin immunoprecipitation paired-end ditags and chip-sequencing by solexa technology. in brief, all tus had evidence of oct nanog, and/or sox binding events within kb of the tu in one or both types of chip datasets. most notably, tu had oct and nanog binding sites kb downstream of its 3' end, while tu had a sox binding site kb downstream of its 3' end supported by both sox chip datasets. only one binding event – a chip-seq nanog binding site – was localized directly at the transcription start site, for tu qualitative and quantitative expression profiling of novel tus since the tus were newly recognized components of the mouse es cell transcriptome, the fact that they had never been detected before in any other cell type suggested that they might be preferentially expressed in es cells. we thus hypothesized that they may be involved in the maintenance of pluripotency in these cells and accordingly, checked their expression in stem cells and a panel of embryonic and adult tissues, because higher expression in undifferentiated cells would be consistent with a function specific to such cells. an example of this pattern of gene expression is lin which was found by sage to be expressed only in human es cells and showed downregulation upon es cell differentiation. in fact, lin has only been recently implicated in human somatic cell reprogramming back to the pluripotent state, indicating its importance in es cells. conventional rtpcr and quantitative real-time pcr were used to profile expression of the novel tus. the results, discussed further, showed that all five tus were either es-specific or were expressed most highly in es cells despite being also transcribed elsewhere. to further confirm the pluripotency association of the tus, we also measured the changes in expression levels of the tus in es cells upon chemical induction of differentiation. es cells were differentiated using retinoic acid, dimethyl sulfoxide, and hexamethylene bisacetamide treatments, in separate experiments. the three treatments served also to eliminate the possibility of chemical-specific expression responses. to validate that the three chemical treatments reduced pluripotency marker levels and led the cells to differentiate, we tested expression levels of four principal pluripotency markers by taqman qrtpcr. the results clearly indicated that all pluripotency marker mrna levels decreased over time in all three treatment timecourses, proving that the treatments led to the expected differentiation outcomes. qrtpcr was performed to determine the effect of differentiation on the rna level of the novel tus. we demonstrated that most of the tus were consistently downregulated upon differentiation, regardless of the differentiation agents used. tu: a novel stem cell gene with pry and spry domains qualitative rtpcr on the multitissue panel suggested that tu had an es-cell-specific expression pattern. we did not detect expression of this tu outside of es cells by rtpcr. qrtpcr demonstrated that expression of this tu was low though not absent in non-es samples. the second-highest expression level was in adult skeletal muscle, at 30% of the es-cell level; in the few other tissues where the tu was expressed, its rna level was less than 20% of the es-cell level. consistent downregulation of this tu upon es cell differentiation was evident in all three differentiation timecourses, although it was most pronounced with dmso treatment. tu: a putative novel es-specific transcription factor this novel tu showed a similar expression pattern to tu qualitative rtpcr indicated that besides expression in es cells this tu had barely-detectable expression in the embryo at days seven and eleven. qrtpcr confirmed the es specificity of tu. the expression level of tu similarly to tu showed a decreasing trend upon es cell differentiation. this gradual effect was observed in all three treatments but the decrease was not as rapid as that of tu a significant reduction in tu expression was observed after four days of ra treatment whereas for tu the effect was observed as early as day two. in the other differentiation treatments, the effect was similar between the two genes, with dmso treatment yielding the strongest response. this expression profile argues in favor of potential relevance of tu to es cell pluripotency. tu: slc10a3-ubl intergenic splicing qualitative rtpcr suggested that the intergenically spliced slc10a3-ubl tu had a close-to-ubiquitous expression profile, although it nevertheless was preferentially expressed in embryonic rather than adult tissues. one adult sample with higher expression of slc10a3-ubl than es cells was testes. this is intriguing because adult testes reportedly contain multipotent cells capable of forming derivatives of the three embryonic layers. qrtpcr showed that this bicistronic tu was downregulated upon differentiation. dmso and hmba differentiation timecourses resulted in a more rapid downregulation of slc10a3-ubl giving it an expression profile recapitulating that of tu to verify that this intergenically spliced tu was involved in pluripotency but its component genes were not, we individually measured expression levels of the component genes, slc10a and ubl a correlation between the expression level of the bicistronic tu and expression levels of its component genes would suggest that slc10a3-ubl was not an independently regulated tu. however, the expression level of this tu was markedly different from those of the component genes, both in the multitissue panel and in all three differentiation timecourses. in the multitissue panel slc10a was highly upregulated in adult tissues, especially in heart, kidney, and liver, while ubl expression was high in the heart and skeletal muscle relative to e es cells. in the differentiation timecourses, the component genes did not show any down regulation effect upon induction of differentiation. in fact, they were upregulated, especially upon dmso treatment. this result showed that although this bicistronic tu contained the 5' utr of slc10a its regulation did not simply reflect the expression profile of that gene. we conclude that slc10a3-ubl is a genuine novel transcript regulated independently of its component genes. furthermore, we propose that this locus may undergo expression regulation by a combination of alternative splicing and preferential transcription initiation at the downstream ubl promoter, such that intergenic splicing is favored in es cells while independent transcription of the component genes occurs preferentially in differentiated tissues. tu: clk2-scamp intergenic splicing based on qualitative rtpcr, tu appeared largely es-specific, with some embryonic expression. the only adult tissue samples that showed a visible band were skeletal muscle and testes. qrtpcr showed that expression level in testes was approximately fivefold greater than the es-cell level. upon es cell differentiation, the clk2-scamp tu showed significant downregulation. in ra- and hmba-treated cells the gene showed a similar expression profile with a maximum decrease to 20% of the es-cell level after four days of treatment. in the dmso timecourse this effect was less pronounced. tu: 1110034a24rik-rpl36al intergenic splicing qualitative rtpcr of the intergenically spliced 1110034a24rik-rpl36al tu showed that it was not es cell-specific, as multiple embryonic and adult samples showed detectable expression. however, qrtpcr indicated that the highest expression level was still in es cells. accordingly, ra, dmso and hmba treatments all showed that expression of 1110034a24rik-rpl36al decreased as the cells differentiated. this decrease was significant in all of the timecourses tested with up to 80% reduction in mrna expression in ra-treated cells at day six and up to 90% decrease in both dmso- and hmba-treated cells. rnai-based functional analysis of novel tus we designed 4-sirna combinations targeting tu and tu and individual sirnas uniquely targeting the intergenically spliced tu tu and tu transcripts. we were unable to achieve satisfactory tu knockdown. we accomplished knockdowns of the other four tus to 25% – 55% of control mrna levels. we hypothesized that, if the novel tus were relevant to maintaining es cell pluripotency instead of merely associated with pluripotency, then depletion of rna levels of the tus could result in depletion of mrna levels of some, or all, of the four principal pluripotency-driving transcription factors. although both nanog and klf expression levels were reduced to 70% of reference upon knockdown of tu no downregulation of the oct or sox was observed. there was no downregulation of oct sox klf or nanog upon knock down of the other novel tus. we also reasoned that, if the tus were indeed required for maintaining the es state, then rnai suppression of the tu rna levels could lead to upregulation of well-known ectodermal, endodermal, and mesodermal lineage markers. we therefore tested, by qrtpcr with commercially available primer sets and fluorescent probes, the mrna levels of ectodermal, endodermal, and mesodermal differentiation markers after independent tu tu tu and tu rnai transfections. differentiation marker levels were unaffected by tu tu and tu rnai. upregulation of fgf and t-brachyury, to approximately 150% of reference levels each, was observed at day and day post-tu rnai. nanog, klf fgf and t-brachyury were reproducibly affected by tu perturbation, making a direct function of tu in pluripotency maintenance possible in principle. however, the lack of generalized effects on pluripotency transcription factors and differentiation marker classes suggests that even tu may not be an essential pluripotency regulator. discussion validation rate of novel tus from a pet-transcriptome library the five novel tus whose transcription and full-length sequence we validated represent only a small subset of candidate novel tus revealed by pet-based mes transcriptome sampling. we ascribe this small fraction to our selection criteria implemented in selecting tus to be validated. the criteria were designed to maximize the chance of experimental validation but do not imply that all remaining tus are artefactual. to sample a larger number of tus for future physical validation, selection criteria can be relaxed. for example, the kb cutoff value for maximum genomic span can be increased since some genes are an order of magnitude longer. two putative bicistronic tus which were excluded from the original dataset would have been detected if the cutoff had been increased to mb. among tus selected for physical validation, only five yielded specific products. one reason for the low efficiency is likely the limited pet sequence available for primer selection, the crucial point of the validation process. it is possible that the primers designed from the pet sequence were suboptimal and unable to bind to the correct transcript resulting in both false negative and false positive pcr results. the presence of ests and flcdnas in the locus clearly aids validations as it increases the amount of known exonic sequence space putatively belonging to the pet-supported tu; utilizing this space benefits primer design. ng et al. verified a large proportion of pet-supported mes transcripts. however, they used their own gis flcdna library as the template source. thus, some of their confirmed tus were likely due either to experimental artefacts or to extremely rare transcripts which were clonally propagated in that library, but might not be present at detectable levels in normal e cells. we used rna from e cells for our analysis. furthermore, the high success rate obtained by ng et al. was for a combination of novel and known genes; the validation rate for novel tus alone was not explicitly specified. these factors might account for our lower validation rate. intergenic splicing three of our five novel tus join two adjacent genes by forming a single transcript containing exonic sequences of both genes. despite sporadic reports of intergenic splicing, its biological significance is largely unknown. several models speculate on how such transcriptional gene fusion may have a physiological role. first, the fusion may be a byproduct of transcriptional read-through, whereby the transcriptional machinery does not stop at the transcription stop site of the first gene but "reads-through" to the 3' end of the next gene on the same strand instead. these can be rare unregulated stochastic events with no functional significance. however, we showed that our transcripts were not present just in es cells; they were frequently detectable in several, though not all, cell and tissue types, but never ubiquitous, and the expression levels differed reproducibly between samples. consequently, the intergenic splicing events we observed are unlikely to be random. second, chimeric tus – those with fusion orfs in addition to intergenic splicing – can form novel and truly functional proteins. the fusion can create a new bifunctional protein, as is the case in the tweak-april fusion, or change the properties of one of the participating proteins. an example of the latter case is the kua-ube2v fusion where the fused protein is cytoplasmic while the original ube2v is nuclear. only one of our three intergenically spliced tus has a putatively chimeric fusion orf, however. only 25% of intergenically spliced genes detected in a recent study encode chimeric proteins. a less clear functional impact of intergenic splicing centers on expression regulation. a theoretical example is the bicistronic tu which contained the 5' utr of the upstream slc10a gene and the entire coding sequence of the downstream ubl gene. since tu contained the 5'utr of slc10a it could be hypothesized that the bicistronic transcript would be co-regulated together with the upstream gene. however, analysis of their expression level showed that tu was regulated independently from its two component genes. there might be other consequences of the hybrid "5'utr of gene – orf of gene 2" composition of this tu, for example potential for regulation of ubl by rna-binding proteins which only recognize specific sites in the 5'utr of slc10a despite the scale of the intergenic splicing datasets reported in and, neither study identified intergenic splicing at the three loci corresponding to our tus. in fact, even a recent high-throughput effort to catalog novel components of the mouse es transcriptome did not yield any evidence of intergenic splicing at our three loci, although gene trap tags corresponding individually to ubl clk scamp and rpl36al were isolated in that project. this shows that diversity and genomewide incidence of this phenomenon exceed current estimates, and emphasizes the need for targeted validation of candidates from specific libraries, such as our mes transcriptome library, to gain insight into the diversity of intergenic splicing in transcriptomes previously not subjected to deep sampling. relevance to pluripotency of es cells in addition to reporting novel tus in mouse es cells, we provide preliminary evidence for their functional significance. since the novel tus were initially found in undifferentiated es cells, we hypothesized that they were involved in es cell pluripotency. our findings initially supported this hypothesis since almost all of the tus we reported here were preferentially expressed in es cells compared to adult tissues, and all tus had nearby oct sox and nanog binding sites confirmed by two independent lines of chip evidence. this suggests an importance of these tus in es, rather than differentiated, cells, although it does not preclude functional roles in other contexts. more direct support for our hypothesis is provided by the downregulation of these tus upon es cell differentiation. the effect was common to all three differentiation treatments tested, indicating that it was not a chemical-specific response, but rather a reproducible outcome of differentiation. combined with the est-derived expression profiles and multi tissue expression analysis, this result further implicates the five tus in maintaining es cell pluripotency. loss-of-function experiments for four of these novel tus did not lead to cell differentiation as can be seen from the levels of pluripotency markers oct sox nanog and klf upon sirna transfections. however, tu was intriguing because its knockdown led to approximately 30% concurrent reduction in klf and nanog levels, as well as to -fold upregulation of fgf and t-brachyury. it has been known that t-brachyury is able to upregulate nanog, which in turn suppresses t-brachyury's own expression in an indirect, smad-facilitated negative feedback loop in early mesodermal progenitors. if an identical t-brachyury/nanog relationship exists in undifferentiated es cells or in an early mesodermal progenitor subpopulation thereof, then it is possible that tu may be a contributor in maintaining nanog levels in undifferentiated es cells so that when its expression is knocked down nanog level is no longer maintained and the suppression of t-brachyury is lost. summarily however, these five tus alone, without unknown cofactors, are not necessary or sufficient for maintaining pluripotency. overexpression of the tus, in transient or stable transfectants, could be attempted as an additional functional test in order to determine whether higher levels of the tu rnas enable es cells to resist chemical differentiation drivers. while an in-silico or computational subtractive-hybridization comparison with a non-es transcriptome would be appropriate, there was no non-es pet transcriptome dataset generated by ng et al., precluding direct quantitative comparison of these novel tus' expression in es vs. non-es states.  our observations summarily demonstrate that all five tus we analyzed represent genuine novel transcripts potentially important in maintaining pluripotency. to elucidate the biological importance of these novel tus, rnai knock-downs were performed, which indicated that rna level ablation of tu leads to a modicum of reproducible expression profile changes associated with the three principal cell differentiation fates while such ablation of the other four tus failed to change expression phenotypes. nevertheless, our results establish a foundation for functional analysis of a small number of novel tus filtered from a large set of candidates, and show that the gis mes pet-transcriptome library is a good resource for identification of novel tus highly expressed in, and specific to, es cells. 
expression levels of all five tus dropped dramatically during three distinct chemically induced differentiation treatments of es cells in culture. expression profiling using embryonic samples and adult tissue panels confirmed that three of the tus were unique to or most highly expressed in es cells. transcriptome libraries retain considerable potential for novel gene discovery despite massive recent cdna and est sequencing efforts; cdna and est evidence for these es cell tus had been limited or absent. rnai results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it. however, the es cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, mpss, cdna/est sequencing, and sage technologies. rtpcr and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. however, sirna knockdowns of the tus did not alter mrna levels of pluripotency or differentiation markers, and did not affect cell morphology. the other three tus represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes. to identify novel genes associated with pluripotency, we globally searched for es transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed rnai to test their function.
 transcriptional control of embryonic stem cell pluripotency has been a subject of intense study. transcriptional regulators including oct, sox and nanog are fundamental for maintaining the undifferentiated state. however, the es cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, mpss, cdna/est sequencing, and sage technologies. to identify novel genes associated with pluripotency, we globally searched for es transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed rnai to test their function.  gene identification signature analysis, a sage derivative distinguished by paired 5' and 3' transcript end tags, identified candidate novel transcriptional units distinct from known genes in a mouse e es mrna library. we focused on tus free of artefacts and mapping discrepancies, five of which were validated by rtpcr product sequencing. two of the tus were revealed by annotation to represent novel protein-coding genes: a pry-domain cluster member and a krab-domain zinc finger. the other three tus represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes. expression profiling using embryonic samples and adult tissue panels confirmed that three of the tus were unique to or most highly expressed in es cells. expression levels of all five tus dropped dramatically during three distinct chemically induced differentiation treatments of es cells in culture. however, sirna knockdowns of the tus did not alter mrna levels of pluripotency or differentiation markers, and did not affect cell morphology.  transcriptome libraries retain considerable potential for novel gene discovery despite massive recent cdna and est sequencing efforts; cdna and est evidence for these es cell tus had been limited or absent. rtpcr and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. rnai results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it. 