BACKGROUND
eukaryotic mrna is frequently alternative spliced.
recent studies of human tissue transcriptomes by high-throughput sequencing have revealed that about 95% of multi-exon genes undergo alternative splicing   <cit> .
this greatly enhances previous estimate of human as events  <cit> , thus further adds complexity to transcripts and proteins.
alternative cleavage and polyadenylation  is also an important mechanism to produce diverse mrna isoforms.
in apa events, a key regulatory step in the formation of the mrna 3'-end, a nascent mrna is cleaved at its cleavage site and the poly tail is added to the mrna  <cit> .
polyadenylation is associated with important cis-elements, such as the upstream canonical aauaaa and its hexamer variants, the downstream u/gu-rich elements, the auxiliary upstream elements, and the downstream elements  <cit> .
these element combinations determine how mrna 3'-ends are processed.
in human, over half of genes have alternative polyadenylation products  <cit> .
these alternative transcripts are often expressed in a tissue-specific pattern, and contribute to some inherited disorders and tumor development  <cit> .
in addition to the 3' most exons, polyadenylation sites  sites) can also exist in introns and internal exons.
in human, at least 20% of the genes have intronic polyadenylation  <cit> .
alternative tandem or intronic poly sites can lead to alternative polyadenylation  <cit> .
bioinformatic analysis has revealed different polyadenylation configuration within gene structure  <cit> .
the mrna produced from an internal polyadenylation site often encodes truncated proteins or distinct protein isoforms.
these protein products often show different cellular localization and/or different functions compared to the protein produced from the 3'-most poly site  <cit> .
genome-wide searches for poly sites resulted in the polya_db and polya_db <dig>  databases  <cit> .
to date,  <dig>  poly sites have been identified  <cit> .
however, these poly sites are mainly limited to coding regions, and the frequency of poly sites in large introns and intergenic regions remains largely unstudied.
in addition, the sequence selection for these databases was biased towards sequences in the unigene database  <cit> .
because intron did not overlap with known exons or cdna sequences, most intronic expressed sequence tag  sequences were excluded.
for example, ests located in large introns were removed in lee's research  <cit> , because these sequences usually did not overlap with other sequences for the same gene.
no doubt, this bias leaves a large pool of undiscovered transcript variants with intronic polyadenylation sites.
to identify these underrepresented poly sites, we preferentially selected intronic single-block ests considering that ests that span multiple exons often have been included in known unigene clusters and have been used for study of alternative splicing.
however, single-block ests, which span just one exon on chromosome, were not well considered  <cit> .
we focused on the intronic 3'-end exon sites associated with poly-tailed ests derived from single-block ests.
an intronic 3'-end exon site is defined as a terminal exon site located in introns upstream of the 3'-most exon of the gene.
herein we use the term "3'-end exon site", but not "3'-end exon" to describe intronic poly-tailed single-block ests because these 3'-end exons are usually incomplete at their 5'-ends and the closest exon junction is ambiguous.
as a result,  <dig>  intronic 3'-end exon sites from  <dig>  human genes were identified.
45% of all these sites represent novel transcript variants that are absent from other alternative splicing related databases.
to confirm that these sites are transcribed, we collected expression data from non-poly-tailed ests, full-length cdnas, end-pair sequencing of cdna clones, and affymetrix genomic tiling arrays.
these data confirm that about 84% of the predicted sites represent true transcripts.
we also successfully verified some predicted transcripts by rt-pcr experiments.
RESULTS
mapping and clustering intronic poly sites in the human genome
to identify novel transcript variants resulting from previously unidentified intronic poly sites, an annotated est alignment file from ucsc genome browser http://genome.ucsc.edu was analyzed .
we focused on single-block ests that did not overlap known mrna sequences.
initially,  <dig> , <dig> aligned est entries were analyzed for poly sites.
among these,  <dig> , <dig> single-block est entries were obtained, containing  <dig> , <dig> non-redundant ests.
these ests could be further divided into two type: poly-tailed or non-poly-tailed, with the number of  <dig>  and  <dig> , <dig>  respectively .
for the poly-tailed ests, by blasting these sequences against the refseq mrna database, known poly sites were identified and removed, leaving  <dig>  sequences for further analysis.
these ests were finally clustered into  <dig>  groups  clusters) from  <dig>  human genes  according to their position overlapping in the chromosome alignment.
these clusters represented 3'-end exon sites.
thus, the involved gene increased previous reported number that at least  <dig>  human genes contained intronic poly sites  <cit> .
the various poly-tailed ests in the same cluster may represent heterogeneous cleavage or different polyadenylation pattern if they contain different poly sites  <cit> .
most 3'-end exon sites were flanked by exons containing coding sequences .
see results for details.
among the single-block ests,  <dig> , <dig> ests without poly-tail were grouped in  <dig>  clusters.
non-poly clusters that overlapped with the poly-tailed 3'-end exon clusters would support the expression of the novel transcripts.
of the intronic 3'-end exon sites,  <dig>   from  <dig>  genes had at least one supporting non-poly-tailed est , and  <dig>   of the poly clusters contained at least two poly-tailed ests .
totally, 75%  of the identified 3'-end sites were supported by at least two ests.
among the resting 25% supported by only a single est, 37%  got further supported by transcriptional data from affymetrix genomic tiling array .
there were  <dig> independent poly clusters that could overlap with their adjacent clusters via the bridge of non-poly clusters.
some poly clusters bridged several non-poly clusters into a single large cluster .
these large clusters could just manifest the heterogeneity of the polyadenylation pattern at the 3'-end exons in the local genomic context  <cit> .
3'-end novel transcript variants are expressed
to confirm that these poly sites represent novel alternative transcript variants and not genomic dna contamination, our analysis pipeline had four steps.
first, we did blast searches against all known mrnas excluding sequences from refseq.
second, for ests with clone id, we traced their partner sequences of the same clones and checked for splicing signals within the sequences.
third, we compared the 3'-end exon sites with the data of affymetrix tiling array.
finally, we selected some novel transcript variants and verified them via rt-pcr experiments.
in our analysis, poly-tailed ests that had hits in the refseq mrna database by blast searching were eliminated as known transcripts.
however, there are many mrnas are not included in the refseq database.
most of the sequences are produced by full-length cdna sequencing projects.
if our 3'-end ests could be aligned well to such cdnas, the ests were thought to be potential novel transcripts.
among the  <dig>  3'-end exon sites,  <dig>   from  <dig>  genes had hits from at least one mrna .
this indicates the transcript variants have been cloned by others.
as the full-length cdna sequencing projects have been conducted with state-of-art quality control as well as manual verification, it is appropriate that most of these supported ests stand for bona fide mrnas.
the remaining  <dig>  sites, involving  <dig>  human genes, may represent unidentified 3'-end exon sites for novel transcript variants.
ests often have clone ids, which identify the plasmid clones of source cdna fragments.
est sequences are produced from single-pass sequencing of 5'- and/or 3'-end of the clones.
as we have got the 3'-end single-block ests, we could trace their corresponding 5'-end ests with the same clone ids.
if the 5'-end est could be split into multiple blocks, with adjacent gt/ag splicing signal on the human genome, which could be taken as the exons in mature mrnas, it was concluded that the pair of 5'-end and 3'-end ests comprised a bona fide mrna.
in our data, 3'-end exon sites contained two types of ests: poly-tailed and non-poly-tailed but overlapped with the former.
if either type of the ests had multi-block 5'-end ests, the 3'-end exon site was thought to be supported.
first, traced sequences were obtained for the poly ests, and  <dig> novel transcript variants were obtained .
none of the intronic poly-tailed ests for the  <dig> clones were in polya_db <dig> database.
second, traced sequences were also obtained for the non-poly ests.
from  <dig>  non-poly-tailed sequences,  <dig>  clone ids were extracted and  <dig>  of these clones had multiple traced sequences.
of these traces sequences,  <dig>  had multiple blocks aligned with the human genome with at least one block overlapped with refseq mrnas.
these sequences offered supporting evidence for novel 3'-end exons.
overall, est clones provided evidence that  <dig>  poly sites from  <dig>  genes represented expressed transcript variants .
transcriptional fragments from the affymetrix genomic tiling array  <cit> , which could support the existence of transcripts through the specified chromosome region, were integrated in our analysis.
affymetrix fragments overlapped with  <dig>  3'-end exon sites  from  <dig>  genes .
finally, we selected novel isoforms of a couple of genes which have roles in signal transduction and did nested-pcr verification.
our interest was to explore the function of novel protein products encoded by the transcripts.
it was expected that the full coding sequence should be included in pcr products.
the primer strategy was that the upstream primer  was located nearby the translational start site  of the refseq mrna, while the downstream primer  should be located in the poly-tailed ests.
primer sets were listed in additional file  <dig>  the electrophoresis bands of the second pcr products were shown in figure  <dig>  sequences of the pcr products were made blast search, and were revealed to be novel .
sequence analysis was also made with the blat program.
as a result, rt-pcrs confirmed transcription of at least 38%  of selected intronic poly sites.
acquired novel sequences and their accession numbers in genbank database were listed in additional file  <dig>  in case of mapk <dig> , two novel alternative splicing variants were obtained, fj <dig> and fj <dig>  the latter had an extra  <dig> nt resulting from alternative receptor site in exon  <dig>  just like caspase- <dig> gamma  <cit> , and a in-frame pre-stop codon is therefore introduced.
the 3'-end exons that defined novel transcript variants could either be "hidden exons", not overlapping with any known exons, or "composite exons", extending known exons  <cit> .
one "composite exon" and one "hidden exon" examples were shown in figure 3a and 3b, respectively.
the 3'-end exon for dll <dig>  was "composite exon" , whereas the pattern for stambp  was "hidden exon" .
the submitted sequences were indicated as "yourseq" in each panel .
a prolonged  or an additional block  relative to the reference sequences was shown.
these blocks represented prolonged or novel exons previously unidentified, that is, the "composite exons" and "hidden exons".
further analysis of these transcript variants suggested that these variants complied with gt/ag rules and were not incompletely processed mrnas.
for the resting  <dig> candidates that were not successfully cloned, it might be relevant to limited tissue cdna sources, unsuitable primers, pcr condition, low expression level of target transcripts, and non-specific amplification, and so on.
in summary, the majority  of our poly sites were supported by at least one of validation steps, as well as non-poly-tailed ests .
among these validation tests, the new isoforms supported by the rt-pcr, est clone id and blast hit against full cdnas are more trustable than those only from affymetrix validation because the single-block ests in the 3'-end exon sites can be joined to the upstream part of the annotated genes, whereas sites supported by affymetrix tiling array may belong to independent genes hidden in the introns.
totally,  <dig>   3'-end exon sites from  <dig>  genes were taken as high-reliability .
the resting sites, whether they were supported by affymetrix transcriptional fragments or not, were relatively less reliable and more dependent of experimental validation to exclude independently expressed transcripts.
among our  <dig> validated novel isoforms from rt-pcr experiments,  <dig> were still successfully cloned although their 3'-end exons were not supported by above mentioned est clone id and blast hit against full cdnas.
the average length of the 3'-end exon sites in our result was  <dig> nucleotides, whereas the average length of the 3'-end exons of all the human refseqs was about  <dig> nucleotides.
this could be explained by that ests just represent segments of complete transcriptional sequences, and therefore, a 3'-end exon site just represents partial sequence of a full 3'-end exon.
no doubt, the actual 3'-end exon could extend toward upstream , and the precise nearest exon-exon boundary could be revealed.
theoretically, a 3'-end exon site corresponds to one full-length transcript which needs pcr validation to reveal the complete 3'-end exon.
this is the reason that we would like to use the term "3'-end exon site", but not "3'-end exon" to describe these incomplete 3'-end exons in our study.
comparison with other alternative splicing related databases
to make a comparison with the polya_db <dig> database, the accession numbers of poly-tailed ests as well as the positions of chromosome alignment of each cluster were used.
as a result, total  <dig>   3'-end exon sites from  <dig>  genes were covered by polya_db <dig> database .
among some of these overlapping poly sites, we found more supporting ests.
for example, the poly site in polya_db <dig>  hs.
<dig> .
<dig>  included only one poly-tailed sequence , but in our dataset, the corresponding 3'-end exon site  was supported by two poly-tailed ests , and seven non-poly-tailed ests .
it suggests from above analysis that our data can well complement previous studies.
up to date, many alternative splicing databases have been developed  <cit> , the main purpose is to collect all the alternative splicing candidates.
it seems that one important common aspect for these databases is that multiple-block exons are used for analysis, and precise exon-intron boundary is required, whereas single-block ests are not well considered.
we made a comparison between our data and two reputed alternative splicing databases, asap ii database   <cit>  and astd   <cit> , which superseded the asd   <cit>  and atd   <cit>  databases.
as shown in table  <dig> and additional file  <dig>  among  <dig>  3'-end exon sites, only 6%  sites from  <dig> genes were covered by asap ii, whereas 11%  sites from  <dig>  genes were covered by astd database .
this suggests that most of our data are novel.
during our process, muro et al recently identified 3'-ends of human and murine genes by automated est cluster analysis  <cit> , we compared their data and ours, and found that about 37%  sites from  <dig>  genes were same .
excluding all the above crossing 3'-end exon sites and the sites having blast hit against full cdnas, a total 45%  from  <dig>  genes are novel and unique in our data.
novel transcript variants are derived from processed mature mrnas
from the sequence analysis shown in additional file  <dig>  the canonical splice boundaries  were implicated.
these novel isoforms showed that they were processed with introns deletion.
the gene structures  of two examples further confirmed that the rt-pcr products were derived from processed mature mrnas, but not unspliced precursor mrnas.
on the other hand, the clone id tracing analysis  also revealed that the novel transcripts were derived from processed mature mrnas.
polyadenylation usually requires a hexamer motif as a primary 3'-end processing element, which is usually called the polyadenylation signal .
a  <dig> nt nucleotide region preceding the potential cleavage sites of all  <dig>  ests was searched for the motifs to match at least one of the thirteen known pas hexamers   <cit> .
as a result, about 65%  of all the 3'-end exons had at least one of these pas hexamers .
among  <dig>   3'-end sites having mrna hits , also about 63%  had at least one of thirteen above mentioned pas.
it suggests from above analysis that the novel transcript variants be derived from processed mature mrnas, but not unspliced precursor mrnas or degradation products of pre-mrna.
novel transcript variants are truncated and missing functional domains
intronic poly sites often lead to truncated isoforms that lose important functional domain or localization signals.
to evaluate if domains are lost in the novel transcript variants from intronic poly sites, all protein products containing the intronic poly  sites had been annotated.
domains were deleted or truncated in transcript variants from  <dig>  poly sites from  <dig>  genes .
the detailed information of involved domains in additional file  <dig> was shown in additional file  <dig>  among all poly sites,  <dig>  could lead to deletion of trans-membrane domain.
as an example, the novel isoform for tnfrsf1a , herein designated as tnfrsf1aβ as it represents the second isoform of tnfrsf1a, was analyzed.
tnfrsf1a is a death receptor with two known ligands, tumor necrosis factor and lymphotoxin-α.
through interactions with these ligands, tnfrsf1a initiates cellular signals and regulates many cellular functions including inflammation, immune response, proliferation, and apoptosis  <cit> .
the length of pcr product is  <dig>  bp which contains an open reading frame of  <dig> bp .
tnfrsf1aβ consists of  <dig> amino acids , and is generated from an intronic "hidden exon" between exon  <dig> and exon  <dig> .
tnfrsf1aβ lacks the trans-membrane helix and the full cytoplasmic region including the death domain compared to the full-length protein , while retaining the signal peptide and the conserved binding domain, that is tnfr  domain.
soluble tnfrsf1a, which functions as natural inhibitors for tumor necrosis factor, was observed and widely investigated  <cit> .
soluble tnfrsf1a is likely produced when tace , a metalloprotease that cleaves transmembrane proteins, cleaves the tnfrsf1a ecodomain  <cit> .
however, tnfrsf1aβ we found is a natural transcript and likely encodes a secretory protein product, and may play a regulatory role preferentially by competitively binding tnfr ligand .
as alternate poly sites may be regulated in a tissue- or disease-specific pattern  <cit> , in addition to domain annotation, expression profiles for novel 3'-end exon sites were provided .
we compared the est distribution in normal and cancerous tissues for each cluster, it revealed that some transcript variants may be cancer-specifically expressed.
moreover, an additional supplemental file  provided all the candidate poly sites of each human genes, integrating polya_db <dig>  muro et al's  <cit>  and our results.
totally  <dig>  sites of  <dig>  genes were included.
discussion
in this genome-wide analysis, we showed that alternative polyadenylation in intronic sites can generate lots of novel transcript variants.
we preferentially selected intronic single-block ests for analysis in that these ests were not well considered in previous studies  <cit> , including lee's research  <cit> .
so, our work is a good complement for previous study  <cit> .
single-block ests within the intergenic region were not included in our analysis though some of them represent gene extensions  <cit> .
single-block ests are often suspected as contamination of genomic dna.
however, in our analysis, we showed that about 84% of the est clusters were supported by at least one evidence: hit from full-length cdna, multiple-block 5'-end ests, overlapping with transcribing sites from affymetrix tiling array, or having multiple supporting ests.
so by carefully screening, the single-block ests could be used as valuable resources for discovering novel transcripts.
besides focusing on single-block ests, the pipeline in our analysis was designed to improve poly site detection, all these contribute to the discovery of novel intronic 3'-end exons.
during our analysis, we found that more than 90% of the est entries in our results were created before the polya_db <dig> was released.
it implied that most of the novel transcript variants were derived by the improvement of our detection methods and the consideration of single-block ests, but not merely by the growth of the transcript databases.
although different methods have been used for poly site prediction  <cit> , current methods achieve only moderate sensitivity and specificity.
for example, about 47% of known poly sequences in the polya_db database were not predicted the support vector machine   <cit> .
among our predicted 3'-end exon sites, less than thirty can be predicted by polya_svm  cluster region ±  <dig> nucleotides was used for predictions).
however, 68% of the  <dig>  ests, which correspond to about 63% of the  <dig>  3'-end exons , have at least one of thirteen known pas hexamers.
this low detection rate of prediction by polya_svm likely results from heterogeneity of the intronic poly sites compared to the conventional 3'-most poly sites.
it is worthy of note, a method different to ours for identification of 3'-ends of genes was made according to est frequency histogram along the genome by muro et al  <cit> .
they show that 22-52% of sequences in commonly used human and murine "full-length" transcript databases may not currently end at bona fide polyadenylation sites.
since the average length of the 3'-end exons of all the current human refseqs is about  <dig> nucleotides, they will get longer according to muro et al 's results.
as the comparison in the text has shown, muro et al's method and ours have respective advantages, and complement each other.
both methods will contribute to identification of full-length transcripts.
novel 3'-end exons we detected could be defined as "hidden exons" and "composite exons" described previously  <cit> .
however, some apparent "hidden exons" could be actually "composite", because ests only represent partial cdna sequences and may be extended to overlap with known exons.
not all intronic poly sites correspond to actual novel transcript variants.
for example, internal priming, due to a consecutive string of 'a's in the mrnas, results in false positives.
for cdna library construction, oligo-dt is often used as the primer for first strand cdna synthesis.
this primer can anneal to the internal priming site, producing truncated sequences.
internal priming accounts for about 12% for the total 3' ests in the database  <cit> .
in previous study like tian's  <cit> , the genomic dna sequence around the predicted poly site was checked.
if there were more than  <dig> consecutive 'a's or at least  <dig> 'a's in  <dig> nt window, it was suspected to be an internal priming site.
however, when applied the criterion to study the adjacent dna sequence of 3'-end of human refseq mrnas, it was found that  <dig> %  mrnas had such a trait at their 3'-ends.
so if using the above criterion, many true positive sites might be missed.
in our analysis, we tried to reduce internal priming sites by eliminating all ests that could be aligned well with known refseq mrnas .
in order to find novel transcript variants as many as possible, we did not request an accurate signature of exon junction and cleavage site.
this is different to those previous reported  <cit> .
the 3'-end exon site provides the approximate locus of the "composite exons" or the "hidden exons" for novel isoforms.
the supporting ests of a 3'-end exon site further provide proper sites for downstream primer designing to amplify the full coding region of corresponding novel isoforms.
we performed rt-pcr to validate some interested candidates with success rate of about 38% .
sequence analysis revealed they were derived from processed mature mrna, but not unspliced precursor.
in our analysis, although most of the sites are supported by at least two types of evidences, there are still  <dig>  sites containing only one est sequence without supporting in other way.
some of these sites may truly represent novel transcript variants associated with low expression levels.
for example, the sites db <dig> , db <dig> , db <dig> , and db <dig> , and db <dig> , they contain only one est sequence, but the est is from a full-length cdna clone .
one type of rna polyadenylation controls rna degradation in the nucleus  <cit> .
the exosome plays a key role in the surveillance of nuclear mrna synthesis and maturation.
poly tails guiding rna to be degraded by the exosome are usually shorter than those increasing mrna stability, and these poly tails are not made strictly of 'a's.
these sites were not actively eliminated in our analysis, but they are unlikely to greatly affect the results because they would not be detected under our stringent criteria.
on the other hand, sequence analysis of the poly-tailed ests revealed that pas did exist in most of our ests.
this result combined with other evidences, suggest our predicted poly sites should represent bona fide mrnas, but not unspliced precursor mrnas, neither the degradation products.
another type of rna quality control is nonsense-mediated mrna decay , which selectively degrades mrnas that contain a premature translation termination codon   <cit> .
although nmd mainly acts as quality control to eliminate faulty transcripts in gene expression, it is also involved in physiological and pathological functions  <cit> .
usually, nmd occurs when translation terminates more than 50- <dig> nucleotides upstream of the exon-exon junction, in which case components of the termination complex are thought to interact with the exon-junction complex  to elicit nmd  <cit> .
although 45% of alternatively spliced mrnas are predicted to be an nmd target  <cit> , an mrna is immune to nmd if translation terminates less than 50- <dig> nucleotides upstream of the 3'-most exon-exon junction or downstream of the junction.
this means if a natural stop codon of an mrna exists in the 3'-end exon, it is not subject to nmd.
the transcripts predicted in our study use an alternative 3'-utrs, assuming that upstream exons do not change.
because we have not got the full-length form for each transcripts, we can not estimate the proportion of our results that would be affected by nmd.
however, it has been reported that alternative polyadenylation may be an nmd-rescue regulatory mechanism in ptc-containing mrnas  <cit> .
our data seem to be consistent with the view.
actually all the novel transcripts proved by rt-pcr experiments in our study comprise the natural stop codon in the last exon.
a further analysis revealed that in nearly all the 3'-end ests except some very short ones, stop codons exist in all three reading frames .
so if there were no correct stop codons in the 5'-exons, the stop codon in the 3'-end exons of our result would be used.
this is different to middle exons that may not contain in-frame stop codons and could not help conveniently clone transcripts with complete coding regions.
it should be noted that a large number of non-coding rnas  are expressed from the mammalian genome  <cit> .
these ncrnas include mirnas, snornas, snrnas, and pirnas, and so on, which are involved in controlling various levels of gene expression in physiology and development.
non-coding rnas can be derived from antisense or sense transcripts with overlapping or interlacing exons, or retained introns.
to investigate that whether the internal intronic transcripts in our data actually represent known ncrnas, we compared the chromosome alignment position between the 3'-end exon sites in our study and those of human ncrnas from noncode v <dig>   <cit> .
in  <dig>  human ncrna entries collected in noncode v <dig> , less than one hundred 3'-end exon sites were overlapped .
so it seems that most of our 3'-end exons do not represent known ncrnas.
whereas, we found many poly sites were located in the introns before the coding exons.
if they were real, the potential novel transcripts would be composed of the 5'-utr of the original mrna.
whether the transcripts encode small orfs or regulatory small rnas needs to study in the future.
CONCLUSIONS
in conclusion, our results identify novel 3'-end alternative splicing isoforms.
the expression of these novel variants was confirmed with computational and experimental tools.
these data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome.

 eukaryotic mrna is frequently alternative spliced. recent studies of human tissue transcriptomes by high-throughput sequencing have revealed that about 95% of multi-exon genes undergo alternative splicing. this greatly enhances previous estimate of human as events, thus further adds complexity to transcripts and proteins. alternative cleavage and polyadenylation is also an important mechanism to produce diverse mrna isoforms. in apa events, a key regulatory step in the formation of the mrna 3'-end, a nascent mrna is cleaved at its cleavage site and the poly tail is added to the mrna. polyadenylation is associated with important cis-elements, such as the upstream canonical aauaaa and its hexamer variants, the downstream u/gu-rich elements, the auxiliary upstream elements, and the downstream elements. these element combinations determine how mrna 3'-ends are processed. in human, over half of genes have alternative polyadenylation products. these alternative transcripts are often expressed in a tissue-specific pattern, and contribute to some inherited disorders and tumor development. in addition to the 3' most exons, polyadenylation sites sites) can also exist in introns and internal exons. in human, at least 20% of the genes have intronic polyadenylation. alternative tandem or intronic poly sites can lead to alternative polyadenylation. bioinformatic analysis has revealed different polyadenylation configuration within gene structure. the mrna produced from an internal polyadenylation site often encodes truncated proteins or distinct protein isoforms. these protein products often show different cellular localization and/or different functions compared to the protein produced from the 3'-most poly site. genome-wide searches for poly sites resulted in the polya_db and polya_db databases. to date, poly sites have been identified. however, these poly sites are mainly limited to coding regions, and the frequency of poly sites in large introns and intergenic regions remains largely unstudied. in addition, the sequence selection for these databases was biased towards sequences in the unigene database. because intron did not overlap with known exons or cdna sequences, most intronic expressed sequence tag sequences were excluded. for example, ests located in large introns were removed in lee's research, because these sequences usually did not overlap with other sequences for the same gene. no doubt, this bias leaves a large pool of undiscovered transcript variants with intronic polyadenylation sites. to identify these underrepresented poly sites, we preferentially selected intronic single-block ests considering that ests that span multiple exons often have been included in known unigene clusters and have been used for study of alternative splicing. however, single-block ests, which span just one exon on chromosome, were not well considered. we focused on the intronic 3'-end exon sites associated with poly-tailed ests derived from single-block ests. an intronic 3'-end exon site is defined as a terminal exon site located in introns upstream of the 3'-most exon of the gene. herein we use the term "3'-end exon site", but not "3'-end exon" to describe intronic poly-tailed single-block ests because these 3'-end exons are usually incomplete at their 5'-ends and the closest exon junction is ambiguous. as a result, intronic 3'-end exon sites from human genes were identified. 45% of all these sites represent novel transcript variants that are absent from other alternative splicing related databases. to confirm that these sites are transcribed, we collected expression data from non-poly-tailed ests, full-length cdnas, end-pair sequencing of cdna clones, and affymetrix genomic tiling arrays. these data confirm that about 84% of the predicted sites represent true transcripts. we also successfully verified some predicted transcripts by rt-pcr experiments.  mapping and clustering intronic poly sites in the human genome to identify novel transcript variants resulting from previously unidentified intronic poly sites, an annotated est alignment file from ucsc genome browser http://genome.ucsc.edu was analyzed. we focused on single-block ests that did not overlap known mrna sequences. initially,, aligned est entries were analyzed for poly sites. among these,, single-block est entries were obtained, containing, non-redundant ests. these ests could be further divided into two type: poly-tailed or non-poly-tailed, with the number of and, respectively. for the poly-tailed ests, by blasting these sequences against the refseq mrna database, known poly sites were identified and removed, leaving sequences for further analysis. these ests were finally clustered into groups clusters) from human genes according to their position overlapping in the chromosome alignment. these clusters represented 3'-end exon sites. thus, the involved gene increased previous reported number that at least human genes contained intronic poly sites. the various poly-tailed ests in the same cluster may represent heterogeneous cleavage or different polyadenylation pattern if they contain different poly sites. most 3'-end exon sites were flanked by exons containing coding sequences. see results for details. among the single-block ests,, ests without poly-tail were grouped in clusters. non-poly clusters that overlapped with the poly-tailed 3'-end exon clusters would support the expression of the novel transcripts. of the intronic 3'-end exon sites, from genes had at least one supporting non-poly-tailed est, and of the poly clusters contained at least two poly-tailed ests. totally, 75% of the identified 3'-end sites were supported by at least two ests. among the resting 25% supported by only a single est, 37% got further supported by transcriptional data from affymetrix genomic tiling array. there were independent poly clusters that could overlap with their adjacent clusters via the bridge of non-poly clusters. some poly clusters bridged several non-poly clusters into a single large cluster. these large clusters could just manifest the heterogeneity of the polyadenylation pattern at the 3'-end exons in the local genomic context. 3'-end novel transcript variants are expressed to confirm that these poly sites represent novel alternative transcript variants and not genomic dna contamination, our analysis pipeline had four steps. first, we did blast searches against all known mrnas excluding sequences from refseq. second, for ests with clone id, we traced their partner sequences of the same clones and checked for splicing signals within the sequences. third, we compared the 3'-end exon sites with the data of affymetrix tiling array. finally, we selected some novel transcript variants and verified them via rt-pcr experiments. in our analysis, poly-tailed ests that had hits in the refseq mrna database by blast searching were eliminated as known transcripts. however, there are many mrnas are not included in the refseq database. most of the sequences are produced by full-length cdna sequencing projects. if our 3'-end ests could be aligned well to such cdnas, the ests were thought to be potential novel transcripts. among the 3'-end exon sites, from genes had hits from at least one mrna. this indicates the transcript variants have been cloned by others. as the full-length cdna sequencing projects have been conducted with state-of-art quality control as well as manual verification, it is appropriate that most of these supported ests stand for bona fide mrnas. the remaining sites, involving human genes, may represent unidentified 3'-end exon sites for novel transcript variants. ests often have clone ids, which identify the plasmid clones of source cdna fragments. est sequences are produced from single-pass sequencing of 5'- and/or 3'-end of the clones. as we have got the 3'-end single-block ests, we could trace their corresponding 5'-end ests with the same clone ids. if the 5'-end est could be split into multiple blocks, with adjacent gt/ag splicing signal on the human genome, which could be taken as the exons in mature mrnas, it was concluded that the pair of 5'-end and 3'-end ests comprised a bona fide mrna. in our data, 3'-end exon sites contained two types of ests: poly-tailed and non-poly-tailed but overlapped with the former. if either type of the ests had multi-block 5'-end ests, the 3'-end exon site was thought to be supported. first, traced sequences were obtained for the poly ests, and novel transcript variants were obtained. none of the intronic poly-tailed ests for the clones were in polya_db database. second, traced sequences were also obtained for the non-poly ests. from non-poly-tailed sequences, clone ids were extracted and of these clones had multiple traced sequences. of these traces sequences, had multiple blocks aligned with the human genome with at least one block overlapped with refseq mrnas. these sequences offered supporting evidence for novel 3'-end exons. overall, est clones provided evidence that poly sites from genes represented expressed transcript variants. transcriptional fragments from the affymetrix genomic tiling array, which could support the existence of transcripts through the specified chromosome region, were integrated in our analysis. affymetrix fragments overlapped with 3'-end exon sites from genes. finally, we selected novel isoforms of a couple of genes which have roles in signal transduction and did nested-pcr verification. our interest was to explore the function of novel protein products encoded by the transcripts. it was expected that the full coding sequence should be included in pcr products. the primer strategy was that the upstream primer was located nearby the translational start site of the refseq mrna, while the downstream primer should be located in the poly-tailed ests. primer sets were listed in additional file the electrophoresis bands of the second pcr products were shown in figure sequences of the pcr products were made blast search, and were revealed to be novel. sequence analysis was also made with the blat program. as a result, rt-pcrs confirmed transcription of at least 38% of selected intronic poly sites. acquired novel sequences and their accession numbers in genbank database were listed in additional file in case of mapk, two novel alternative splicing variants were obtained, fj and fj the latter had an extra nt resulting from alternative receptor site in exon just like caspase- gamma, and a in-frame pre-stop codon is therefore introduced. the 3'-end exons that defined novel transcript variants could either be "hidden exons", not overlapping with any known exons, or "composite exons", extending known exons. one "composite exon" and one "hidden exon" examples were shown in figure 3a and 3b, respectively. the 3'-end exon for dll was "composite exon", whereas the pattern for stambp was "hidden exon". the submitted sequences were indicated as "yourseq" in each panel. a prolonged or an additional block relative to the reference sequences was shown. these blocks represented prolonged or novel exons previously unidentified, that is, the "composite exons" and "hidden exons". further analysis of these transcript variants suggested that these variants complied with gt/ag rules and were not incompletely processed mrnas. for the resting candidates that were not successfully cloned, it might be relevant to limited tissue cdna sources, unsuitable primers, pcr condition, low expression level of target transcripts, and non-specific amplification, and so on. in summary, the majority of our poly sites were supported by at least one of validation steps, as well as non-poly-tailed ests. among these validation tests, the new isoforms supported by the rt-pcr, est clone id and blast hit against full cdnas are more trustable than those only from affymetrix validation because the single-block ests in the 3'-end exon sites can be joined to the upstream part of the annotated genes, whereas sites supported by affymetrix tiling array may belong to independent genes hidden in the introns. totally, 3'-end exon sites from genes were taken as high-reliability. the resting sites, whether they were supported by affymetrix transcriptional fragments or not, were relatively less reliable and more dependent of experimental validation to exclude independently expressed transcripts. among our validated novel isoforms from rt-pcr experiments, were still successfully cloned although their 3'-end exons were not supported by above mentioned est clone id and blast hit against full cdnas. the average length of the 3'-end exon sites in our result was nucleotides, whereas the average length of the 3'-end exons of all the human refseqs was about nucleotides. this could be explained by that ests just represent segments of complete transcriptional sequences, and therefore, a 3'-end exon site just represents partial sequence of a full 3'-end exon. no doubt, the actual 3'-end exon could extend toward upstream, and the precise nearest exon-exon boundary could be revealed. theoretically, a 3'-end exon site corresponds to one full-length transcript which needs pcr validation to reveal the complete 3'-end exon. this is the reason that we would like to use the term "3'-end exon site", but not "3'-end exon" to describe these incomplete 3'-end exons in our study. comparison with other alternative splicing related databases to make a comparison with the polya_db database, the accession numbers of poly-tailed ests as well as the positions of chromosome alignment of each cluster were used. as a result, total 3'-end exon sites from genes were covered by polya_db database. among some of these overlapping poly sites, we found more supporting ests. for example, the poly site in polya_db hs.. included only one poly-tailed sequence, but in our dataset, the corresponding 3'-end exon site was supported by two poly-tailed ests, and seven non-poly-tailed ests. it suggests from above analysis that our data can well complement previous studies. up to date, many alternative splicing databases have been developed, the main purpose is to collect all the alternative splicing candidates. it seems that one important common aspect for these databases is that multiple-block exons are used for analysis, and precise exon-intron boundary is required, whereas single-block ests are not well considered. we made a comparison between our data and two reputed alternative splicing databases, asap ii database and astd, which superseded the asd and atd databases. as shown in table and additional file among 3'-end exon sites, only 6% sites from genes were covered by asap ii, whereas 11% sites from genes were covered by astd database. this suggests that most of our data are novel. during our process, muro et al recently identified 3'-ends of human and murine genes by automated est cluster analysis, we compared their data and ours, and found that about 37% sites from genes were same. excluding all the above crossing 3'-end exon sites and the sites having blast hit against full cdnas, a total 45% from genes are novel and unique in our data. novel transcript variants are derived from processed mature mrnas from the sequence analysis shown in additional file the canonical splice boundaries were implicated. these novel isoforms showed that they were processed with introns deletion. the gene structures of two examples further confirmed that the rt-pcr products were derived from processed mature mrnas, but not unspliced precursor mrnas. on the other hand, the clone id tracing analysis also revealed that the novel transcripts were derived from processed mature mrnas. polyadenylation usually requires a hexamer motif as a primary 3'-end processing element, which is usually called the polyadenylation signal. a nt nucleotide region preceding the potential cleavage sites of all ests was searched for the motifs to match at least one of the thirteen known pas hexamers. as a result, about 65% of all the 3'-end exons had at least one of these pas hexamers. among 3'-end sites having mrna hits, also about 63% had at least one of thirteen above mentioned pas. it suggests from above analysis that the novel transcript variants be derived from processed mature mrnas, but not unspliced precursor mrnas or degradation products of pre-mrna. novel transcript variants are truncated and missing functional domains intronic poly sites often lead to truncated isoforms that lose important functional domain or localization signals. to evaluate if domains are lost in the novel transcript variants from intronic poly sites, all protein products containing the intronic poly sites had been annotated. domains were deleted or truncated in transcript variants from poly sites from genes. the detailed information of involved domains in additional file was shown in additional file among all poly sites, could lead to deletion of trans-membrane domain. as an example, the novel isoform for tnfrsf1a, herein designated as tnfrsf1aβ as it represents the second isoform of tnfrsf1a, was analyzed. tnfrsf1a is a death receptor with two known ligands, tumor necrosis factor and lymphotoxin-α. through interactions with these ligands, tnfrsf1a initiates cellular signals and regulates many cellular functions including inflammation, immune response, proliferation, and apoptosis. the length of pcr product is bp which contains an open reading frame of bp. tnfrsf1aβ consists of amino acids, and is generated from an intronic "hidden exon" between exon and exon. tnfrsf1aβ lacks the trans-membrane helix and the full cytoplasmic region including the death domain compared to the full-length protein, while retaining the signal peptide and the conserved binding domain, that is tnfr domain. soluble tnfrsf1a, which functions as natural inhibitors for tumor necrosis factor, was observed and widely investigated. soluble tnfrsf1a is likely produced when tace, a metalloprotease that cleaves transmembrane proteins, cleaves the tnfrsf1a ecodomain. however, tnfrsf1aβ we found is a natural transcript and likely encodes a secretory protein product, and may play a regulatory role preferentially by competitively binding tnfr ligand. as alternate poly sites may be regulated in a tissue- or disease-specific pattern, in addition to domain annotation, expression profiles for novel 3'-end exon sites were provided. we compared the est distribution in normal and cancerous tissues for each cluster, it revealed that some transcript variants may be cancer-specifically expressed. moreover, an additional supplemental file provided all the candidate poly sites of each human genes, integrating polya_db muro et al's and our results. totally sites of genes were included. discussion in this genome-wide analysis, we showed that alternative polyadenylation in intronic sites can generate lots of novel transcript variants. we preferentially selected intronic single-block ests for analysis in that these ests were not well considered in previous studies, including lee's research. so, our work is a good complement for previous study. single-block ests within the intergenic region were not included in our analysis though some of them represent gene extensions. single-block ests are often suspected as contamination of genomic dna. however, in our analysis, we showed that about 84% of the est clusters were supported by at least one evidence: hit from full-length cdna, multiple-block 5'-end ests, overlapping with transcribing sites from affymetrix tiling array, or having multiple supporting ests. so by carefully screening, the single-block ests could be used as valuable resources for discovering novel transcripts. besides focusing on single-block ests, the pipeline in our analysis was designed to improve poly site detection, all these contribute to the discovery of novel intronic 3'-end exons. during our analysis, we found that more than 90% of the est entries in our results were created before the polya_db was released. it implied that most of the novel transcript variants were derived by the improvement of our detection methods and the consideration of single-block ests, but not merely by the growth of the transcript databases. although different methods have been used for poly site prediction, current methods achieve only moderate sensitivity and specificity. for example, about 47% of known poly sequences in the polya_db database were not predicted the support vector machine. among our predicted 3'-end exon sites, less than thirty can be predicted by polya_svm cluster region ± nucleotides was used for predictions). however, 68% of the ests, which correspond to about 63% of the 3'-end exons, have at least one of thirteen known pas hexamers. this low detection rate of prediction by polya_svm likely results from heterogeneity of the intronic poly sites compared to the conventional 3'-most poly sites. it is worthy of note, a method different to ours for identification of 3'-ends of genes was made according to est frequency histogram along the genome by muro et al. they show that 22-52% of sequences in commonly used human and murine "full-length" transcript databases may not currently end at bona fide polyadenylation sites. since the average length of the 3'-end exons of all the current human refseqs is about nucleotides, they will get longer according to muro et al's results. as the comparison in the text has shown, muro et al's method and ours have respective advantages, and complement each other. both methods will contribute to identification of full-length transcripts. novel 3'-end exons we detected could be defined as "hidden exons" and "composite exons" described previously. however, some apparent "hidden exons" could be actually "composite", because ests only represent partial cdna sequences and may be extended to overlap with known exons. not all intronic poly sites correspond to actual novel transcript variants. for example, internal priming, due to a consecutive string of'a's in the mrnas, results in false positives. for cdna library construction, oligo-dt is often used as the primer for first strand cdna synthesis. this primer can anneal to the internal priming site, producing truncated sequences. internal priming accounts for about 12% for the total 3' ests in the database. in previous study like tian's, the genomic dna sequence around the predicted poly site was checked. if there were more than consecutive'a's or at least'a's in nt window, it was suspected to be an internal priming site. however, when applied the criterion to study the adjacent dna sequence of 3'-end of human refseq mrnas, it was found that % mrnas had such a trait at their 3'-ends. so if using the above criterion, many true positive sites might be missed. in our analysis, we tried to reduce internal priming sites by eliminating all ests that could be aligned well with known refseq mrnas. in order to find novel transcript variants as many as possible, we did not request an accurate signature of exon junction and cleavage site. this is different to those previous reported. the 3'-end exon site provides the approximate locus of the "composite exons" or the "hidden exons" for novel isoforms. the supporting ests of a 3'-end exon site further provide proper sites for downstream primer designing to amplify the full coding region of corresponding novel isoforms. we performed rt-pcr to validate some interested candidates with success rate of about 38%. sequence analysis revealed they were derived from processed mature mrna, but not unspliced precursor. in our analysis, although most of the sites are supported by at least two types of evidences, there are still sites containing only one est sequence without supporting in other way. some of these sites may truly represent novel transcript variants associated with low expression levels. for example, the sites db, db, db, and db, and db, they contain only one est sequence, but the est is from a full-length cdna clone. one type of rna polyadenylation controls rna degradation in the nucleus. the exosome plays a key role in the surveillance of nuclear mrna synthesis and maturation. poly tails guiding rna to be degraded by the exosome are usually shorter than those increasing mrna stability, and these poly tails are not made strictly of'a's. these sites were not actively eliminated in our analysis, but they are unlikely to greatly affect the results because they would not be detected under our stringent criteria. on the other hand, sequence analysis of the poly-tailed ests revealed that pas did exist in most of our ests. this result combined with other evidences, suggest our predicted poly sites should represent bona fide mrnas, but not unspliced precursor mrnas, neither the degradation products. another type of rna quality control is nonsense-mediated mrna decay, which selectively degrades mrnas that contain a premature translation termination codon. although nmd mainly acts as quality control to eliminate faulty transcripts in gene expression, it is also involved in physiological and pathological functions. usually, nmd occurs when translation terminates more than 50- nucleotides upstream of the exon-exon junction, in which case components of the termination complex are thought to interact with the exon-junction complex to elicit nmd. although 45% of alternatively spliced mrnas are predicted to be an nmd target, an mrna is immune to nmd if translation terminates less than 50- nucleotides upstream of the 3'-most exon-exon junction or downstream of the junction. this means if a natural stop codon of an mrna exists in the 3'-end exon, it is not subject to nmd. the transcripts predicted in our study use an alternative 3'-utrs, assuming that upstream exons do not change. because we have not got the full-length form for each transcripts, we can not estimate the proportion of our results that would be affected by nmd. however, it has been reported that alternative polyadenylation may be an nmd-rescue regulatory mechanism in ptc-containing mrnas. our data seem to be consistent with the view. actually all the novel transcripts proved by rt-pcr experiments in our study comprise the natural stop codon in the last exon. a further analysis revealed that in nearly all the 3'-end ests except some very short ones, stop codons exist in all three reading frames. so if there were no correct stop codons in the 5'-exons, the stop codon in the 3'-end exons of our result would be used. this is different to middle exons that may not contain in-frame stop codons and could not help conveniently clone transcripts with complete coding regions. it should be noted that a large number of non-coding rnas are expressed from the mammalian genome. these ncrnas include mirnas, snornas, snrnas, and pirnas, and so on, which are involved in controlling various levels of gene expression in physiology and development. non-coding rnas can be derived from antisense or sense transcripts with overlapping or interlacing exons, or retained introns. to investigate that whether the internal intronic transcripts in our data actually represent known ncrnas, we compared the chromosome alignment position between the 3'-end exon sites in our study and those of human ncrnas from noncode v. in human ncrna entries collected in noncode v, less than one hundred 3'-end exon sites were overlapped. so it seems that most of our 3'-end exons do not represent known ncrnas. whereas, we found many poly sites were located in the introns before the coding exons. if they were real, the potential novel transcripts would be composed of the 5'-utr of the original mrna. whether the transcripts encode small orfs or regulatory small rnas needs to study in the future.  in conclusion, our results identify novel 3'-end alternative splicing isoforms. the expression of these novel variants was confirmed with computational and experimental tools. these data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome. 
although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences in the public databases, one special type, single-block est is much less emphasized. in the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. to verify that these sites are from expressed transcripts, we analyzed the supporting est number of each site, blasted representative ests against known mrna sequences, traced terminal sequences from cdna clones, and compared with the data of affymetrix tiling array. our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome. the expression of these novel variants was confirmed with computational and experimental tools. our results provide evidence for novel transcript variants with intronic poly sites. this bias leaves a large space to discover novel transcript variants. poly-tailed ests were obtained from single-block ests and clustered into groups standing for genes. additionally, rt-pcr confirmed 38% of predicted novel transcript variants.
 alternative polyadenylation sites within a gene can lead to alternative transcript variants. although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences in the public databases, one special type, single-block est is much less emphasized. this bias leaves a large space to discover novel transcript variants.  in the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. poly-tailed ests were obtained from single-block ests and clustered into groups standing for genes. most sites were not found in other alternative splicing databases. to verify that these sites are from expressed transcripts, we analyzed the supporting est number of each site, blasted representative ests against known mrna sequences, traced terminal sequences from cdna clones, and compared with the data of affymetrix tiling array. these analyses confirmed about 84% of the novel alternative transcripts, especially, 33% of the transcripts from genes were taken as high-reliability. additionally, rt-pcr confirmed 38% of predicted novel transcript variants.  our results provide evidence for novel transcript variants with intronic poly sites. the expression of these novel variants was confirmed with computational and experimental tools. our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome. 