as most of the human genes with multiple exons express more than one transcript , alternative splicing is considered to be a major mechanism for producing a complex proteome from a limited number of genes ( 1 ) .
the different transcripts of one gene can be translated into functionally different protein isoforms ( 2 ) or can be degraded by nonsense - mediated mrna decay ( 3 ) .
the regulation of alternative splicing plays a role in several important processes such as the formation and function of synapses ( 4 ) , axon guidance in drosophila ( 5,6 ) and t - cell activation ( 7 ) .
furthermore , defects in alternative splicing are causative for a number of human diseases ( 8,9 ) and thought to contribute to cancer development ( 10 ) .
while much research focused on larger alternative splice events such as exon skipping , it recently became clear that numerous alternative splice events result in only subtle changes of the mrna and of the protein ( 1214 ) .
the most widespread type is the alternative splicing at acceptor sites with the pattern nagnag ( n stands for a , c , g , or t , throughout the paper we write t instead of u also when referring to an rna sequence ) ( 12,15,16 ) .
in such a motif , both ags represent potential alternative acceptor sites which result in transcripts that differ by only 3 nt ( the nag ) .
( est)/mrna data 16% of all nagnags and noteworthy 39% of the tandem acceptors with a haghag pattern ( also denoted 
plausible nagnags , h stands for a , c , or t ) are currently known to be alternatively spliced .
furthermore , we recently found evidence for alternative splicing at donor splice sites with the motifs gtngtn , gcngtn and gtngcn ( denoted as gyngyn donors , y stands for c or t ) where both gt / gc donors are used ( 17 ) .
we denote a tandem splice site as confirmed if the usage of both splice sites is represented by at least one est / mrna and unconfirmed otherwise .
although the term  tandem splice site refers to any pair of neighboring splice sites , in our database we collected data about nagnag acceptors and gyngyn donors .
apart from their frequency , subtle alternative splice events are of interest since several cases are known to result in functionally different protein isoforms ( 16,1822 ) and alternative nagnag splicing in the untranslated region ( utr ) can affect the translational efficiency ( 23 ) . moreover ,
the effect for the protein might be drastic since a premature stop codon can be created ( 12,17 ) .
many nagnag acceptors are conserved between human and mouse and the ratio of the two splice forms can be highly controlled in a tissue - specific manner ( 12,16,24 ) .
furthermore , snps that affect a nagnag acceptor can be relevant for human disease as demonstrated for the abca4 gene ( 25 ) and suggested for many other genes ( 26 ) . while previous databases on alternative splicing do not store such subtle splice events ( 2729 ) , recent databases contain confirmed tandem splice sites ( 3032 ) .
however , they do not contain unconfirmed tandem splice sites and do not allow to search for tandem splice sites with specific features . to facilitate further experimental studies as well as large - scale bioinformatics analyses of tandem splice sites , we have developed a relational database , tassdb ( tandem splice site database ) , which provides large collections of gyngyn donors and nagnag acceptors in eight species .
since these subtle splice events can easily be overlooked in experimental systems ( a 3 nt difference between two bands is barely visible on an agarose gel ) and additional alternative splice events are likely to be missed in current est data , tassdb also stores unconfirmed tandem splice sites .
a user interface allows to search for genes of interest and to get all relevant information about the respective tandem splice sites .
it is also possible to search for genes harboring gyngyns / nagnags with specific features .
finally , tassdb annotates nagnags that are affected by a snp and also contains 51 tandem acceptors whose nagnag pattern can only be observed in another snp - allele and not in the human genome reference sequence .
as alternatively spliced nagnag acceptors and gyngyn donors occur in a large number of lineages including vertebrates , flies and nematodes , tassdb stores information about the tandem splice sites of eight species : homo sapiens , canis familiaris , mus musculus , rattus norvegicus , gallus gallus , danio rerio , drosophila melanogaster and caenorhabditis elegans .
our annotation pipeline is based on transcript - to - genome mappings taken from the ucsc genome browser ( 33 ) .
apart from the refseq annotation that was used for all species , we additionally used ensembl transcripts for human , rat , chicken , zebrafish , the ucsc 
knowngene set for human , mouse , rat ( 34 ) , flybase transcripts for drosophila and wormbase transcripts for c.elegans .
intron structure as well as the annotation of the open reading frame was taken from the ucsc annotation .
to identify alternatively spliced nagnags and gyngyns , we used blast against all ests and mrnas from the respective species as described in refs ( 12,17 ) .
the primary aim of tassdb is to provide information that is specific for the tandem splice site and the putative alternative splice event .
thus , we collected the following data : the splice site motif , its genomic locus , its location in the transcript ( 5/3-utr or cds with intron phase 0/1/2 ) , the impact of the splice event on the protein , the sequences and length of the up-/downstream exon and the intron , and information about the ests / mrnas that indicate usage of one of the two splice sites . as the degree of similarity of a splice site to the overall consensus is an important criterion to distinguish alternatively from non - alternatively spliced nagnag acceptors ( 15 ) , we also computed the maximum entropy scores for both splice sites in a tandem ( 35 ) .
the basic design of tassdb was driven by the idea to separate splice site specific data from transcript specific data .
for example , the gyngyn / nagnag motif , the genomic locus and the splice site scores are independent of transcript annotation .
however , features such as intron phase , protein impact and est confirmation depend on the annotation and the exon 
for example , the intron 13 of the phf1 gene that contains a cagcag acceptor is in intron phase 2 according to the annotation of nm_024165 . due to skipping of the upstream 95 nt exon ,
thus , the protein impact of the cagcag is the insertion / deletion ( indel ) of a ser in nm_024165 but indel ala in nm_002636 ( figure 1a ) .
another example is the cagcag acceptor of intron 1 of the cbx1 gene having two alternative first exons ( represented by the transcripts enst00000225603 and nm_006807 ) .
we found est evidences for alternative cagcag splicing if the upstream first exon is used ( enst00000225603 ) but not if the downstream first exon is used ( nm_006807 ) ( figure 1b ) . whether this is a biological phenomenon or simply the consequence of a lack of sufficient est data , deserves further research .
( a ) example of a nagnag acceptor whose protein impact differs in the annotation of two transcripts .
( b ) example of a nagnag acceptor that is only confirmed according the exon - intron structure of one but not a second transcript .
the most frequent use of tassdb might be a search for tandem splice sites of a given gene .
to this end , tassdb provides a quick search interface where a user only specifies a gene symbol or a transcript accession and gets the entire information of both confirmed and unconfirmed gyngyns and nagnags for this gene .
a more advanced task is to select tandem splice sites with specific features for further experimental or computational analysis . to this end
, tassdb provides an advanced search interface where the user can restrict the search to gyngyns or nagnags with the following features : ( i ) pattern of the splice site , ( ii ) number of ests / mrnas that match both splice forms , ( iii ) location in the utr or in the coding sequence ( cds ) ( as well as specific intron phases ) , and ( iv ) protein impact ( figure 2 ) .
thus , it is easy to formulate queries such as : ( i ) show all confirmed nagnags that result in single amino acid events .
( ii ) show all tandem splice sites where both splice forms are represented by at least three ests / mrnas and that are located in the 5-utr .
the result of the search consists of two parts : ( i ) summary table of affected genes and their number of tandem splice sites and ( ii ) detailed tables with information about the tandem splice sites .
the detailed result tables also provide links to the ests / mrnas for both splice forms as well as links to the ucsc genome browser .
if the transcript specific data differ between transcripts , tassdb will show detailed result tables with more than two columns ( figure 1 ) .
features that differ between transcripts are shown in black while those that are equal in all transcripts are shown in grey colour .
screenshot of the advanced search interface : one can search for genes harboring tandem splice sites with a specific pattern [ such as gtngcn for tandem donors or haghag ( plausible ) for tandem acceptors ] , a certain number of ests / mrnas matching both splice forms , a location in the utr or cds and a specific protein impact .
as snps that affect nagnag acceptors are predictive for variation in alternative splicing , tassdb also annotates the snp data found in our previous study ( 26 ) including 51 polymorphic tandem acceptors whose nagnag pattern is not visible in the genome reference sequence .
such an example is the appbp1 gene where acceptor pattern of intron 6 is aaacag in the human reference genome sequence .
the snp rs363209 leads to a second allele with an aagcag pattern , thus this g - allele creates a novel tandem acceptor .
tassdb always contains the sequence with the nagnag acceptor ( in this case the g - allele ) .
finally , tassdb provides an interface where the user can send an arbitrary sql select query to the database . the relational database schema with table and
thus , tassdb can be used to retrieve large datasets for further computational analysis of tandem splice sites .
it is evident that tandem splice sites are widespread in all eight species and that with the exception of c.elegans for nagnags numerous of them are already known to be alternatively spliced .
summary of the current content of tassdb total number of transcripts used for detecting tandem splice sites .
as alternatively spliced nagnag acceptors and gyngyn donors occur in a large number of lineages including vertebrates , flies and nematodes , tassdb stores information about the tandem splice sites of eight species : homo sapiens , canis familiaris , mus musculus , rattus norvegicus , gallus gallus , danio rerio , drosophila melanogaster and caenorhabditis elegans .
our annotation pipeline is based on transcript - to - genome mappings taken from the ucsc genome browser ( 33 ) .
apart from the refseq annotation that was used for all species , we additionally used ensembl transcripts for human , rat , chicken , zebrafish , the ucsc 
knowngene set for human , mouse , rat ( 34 ) , flybase transcripts for drosophila and wormbase transcripts for c.elegans .
intron structure as well as the annotation of the open reading frame was taken from the ucsc annotation .
to identify alternatively spliced nagnags and gyngyns , we used blast against all ests and mrnas from the respective species as described in refs ( 12,17 ) .
the primary aim of tassdb is to provide information that is specific for the tandem splice site and the putative alternative splice event .
thus , we collected the following data : the splice site motif , its genomic locus , its location in the transcript ( 5/3-utr or cds with intron phase 0/1/2 ) , the impact of the splice event on the protein , the sequences and length of the up-/downstream exon and the intron , and information about the ests / mrnas that indicate usage of one of the two splice sites . as the degree of similarity of a splice site to the overall consensus is an important criterion to distinguish alternatively from non - alternatively spliced nagnag acceptors ( 15 ) , we also computed the maximum entropy scores for both splice sites in a tandem ( 35 ) .
the basic design of tassdb was driven by the idea to separate splice site specific data from transcript specific data .
for example , the gyngyn / nagnag motif , the genomic locus and the splice site scores are independent of transcript annotation .
however , features such as intron phase , protein impact and est confirmation depend on the annotation and the exon 
for example , the intron 13 of the phf1 gene that contains a cagcag acceptor is in intron phase 2 according to the annotation of nm_024165 . due to skipping of the upstream 95 nt exon ,
thus , the protein impact of the cagcag is the insertion / deletion ( indel ) of a ser in nm_024165 but indel ala in nm_002636 ( figure 1a ) .
another example is the cagcag acceptor of intron 1 of the cbx1 gene having two alternative first exons ( represented by the transcripts enst00000225603 and nm_006807 ) .
we found est evidences for alternative cagcag splicing if the upstream first exon is used ( enst00000225603 ) but not if the downstream first exon is used ( nm_006807 ) ( figure 1b ) .
whether this is a biological phenomenon or simply the consequence of a lack of sufficient est data , deserves further research .
( a ) example of a nagnag acceptor whose protein impact differs in the annotation of two transcripts .
( b ) example of a nagnag acceptor that is only confirmed according the exon - intron structure of one but not a second transcript .
the most frequent use of tassdb might be a search for tandem splice sites of a given gene . to this end , tassdb provides a quick search interface where a user only specifies a gene symbol or a transcript accession and gets the entire information of both confirmed and unconfirmed gyngyns and nagnags for this gene .
a more advanced task is to select tandem splice sites with specific features for further experimental or computational analysis . to this end
, tassdb provides an advanced search interface where the user can restrict the search to gyngyns or nagnags with the following features : ( i ) pattern of the splice site , ( ii ) number of ests / mrnas that match both splice forms , ( iii ) location in the utr or in the coding sequence ( cds ) ( as well as specific intron phases ) , and ( iv ) protein impact ( figure 2 ) .
thus , it is easy to formulate queries such as : ( i ) show all confirmed nagnags that result in single amino acid events .
( ii ) show all tandem splice sites where both splice forms are represented by at least three ests / mrnas and that are located in the 5-utr .
the result of the search consists of two parts : ( i ) summary table of affected genes and their number of tandem splice sites and ( ii ) detailed tables with information about the tandem splice sites .
the detailed result tables also provide links to the ests / mrnas for both splice forms as well as links to the ucsc genome browser .
if the transcript specific data differ between transcripts , tassdb will show detailed result tables with more than two columns ( figure 1 ) .
features that differ between transcripts are shown in black while those that are equal in all transcripts are shown in grey colour .
screenshot of the advanced search interface : one can search for genes harboring tandem splice sites with a specific pattern [ such as gtngcn for tandem donors or haghag ( plausible ) for tandem acceptors ] , a certain number of ests / mrnas matching both splice forms , a location in the utr or cds and a specific protein impact .
as snps that affect nagnag acceptors are predictive for variation in alternative splicing , tassdb also annotates the snp data found in our previous study ( 26 ) including 51 polymorphic tandem acceptors whose nagnag pattern is not visible in the genome reference sequence .
such an example is the appbp1 gene where acceptor pattern of intron 6 is aaacag in the human reference genome sequence .
the snp rs363209 leads to a second allele with an aagcag pattern , thus this g - allele creates a novel tandem acceptor .
tassdb always contains the sequence with the nagnag acceptor ( in this case the g - allele ) .
finally , tassdb provides an interface where the user can send an arbitrary sql select query to the database . the relational database schema with table and
thus , tassdb can be used to retrieve large datasets for further computational analysis of tandem splice sites .
it is evident that tandem splice sites are widespread in all eight species and that with the exception of c.elegans for nagnags numerous of them are already known to be alternatively spliced .
summary of the current content of tassdb total number of transcripts used for detecting tandem splice sites .
as tandem splice sites seem to occur in all species allowing alternative splicing , it would be desirable to include information for other species . in the future
, we plan to include bos taurus , xenopus tropicalis , takifugu rubripes and anopheles gambiae as more ests and annotated genes become available .
furthermore , information about conservation of these splice events in other species as well as more data about the protein impact may be included .
since the database was designed to store information about any tandem splice site , an extension to tandem splice sites that are more than 3 nt apart is conceivable .
 subtle alternative splice events at tandem splice sites are frequent in eukaryotes and substantially increase the complexity of transcriptomes and proteomes . we have developed a relational database , tassdb ( tandem splice site database ) , which stores extensive data about alternative splice events at gyngyn donors and nagnag acceptors . 
 these splice events are of subtle nature since they mostly result in the insertion / deletion of a single amino acid or the substitution of one amino acid by two others . 
 currently , tassdb contains 114  554 tandem splice sites of eight species , 5209 of which have est / mrna evidence for alternative splicing . 
 in addition , human snps that affect nagnag acceptors are annotated . 
 the database provides a user - friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download large datasets . 
 this database should facilitate further experimental studies and large - scale bioinformatics analyses of tandem splice sites . 
 the database is available at . 