ProSplicer: a database of putative alternative splicing information derived from protein, mRNA and expressed sequence tag sequence data
© Huang et al.; licensee BioMed Central Ltd. 2003
Received: 4 September 2002
Accepted: 24 January 2003
Published: 14 March 2003
ProSplicer is a database of putative alternative splicing information derived from the alignment of proteins, mRNA sequences and expressed sequence tags (ESTs) against human genomic DNA sequences. Proteins, mRNA and ESTs provide valuable evidence that can reveal splice variants of genes. The alternative splicing information in the database can help users investigate the alternative splicing and tissue-specific expression of genes.
Alternative splicing is a widely occurring and important mechanism for controlling the expression of cellular and viral genes. It changes the effects of a gene in different tissues and developmental states by generating distinct mRNA isoforms composed of different selections of exons, which produce variant proteins. This phenomenon is widespread in the human genome and it was commonly believed that alternative splicing existed in only about 30 to 40% of all genes [1, 2].
Because the number of sequences - that is, proteins, mRNAs, and ESTs - in the databases is increasing exponentially, it is possible to decipher alternative splicing forms by computational alignment methods such as BLAST  and SIM4 . mRNA and EST sequences, as well as protein sequences that are theoretically translated into nucleotides in six reading frames, provide gene-expression evidence revealing alternative splicing events when aligned against genomic sequences. The alignment tools BLAST and SIM4 can both align mRNA and EST sequences against the reference genomic sequence. However, only SIM4 provides boundary information that can be used to postulate the splicing sites (donor sites and acceptor sites) when considering alternative splicing issues.
In ProSplicer, complete genomic sequences of known and novel human genes are used to investigate alternative splicing variants. ProSplicer provides alternative splicing information for known and novel genes by aligning three major types of expressed gene evidence, that is protein, mRNA and EST sequences. The alternative splicing forms predicted by considering protein, mRNA, and EST sequences together are more complete than just the exons predicted by EST sequences. The tissue-specific expression information provided by mRNA and EST sequences is also helpful in revealing alternative splicing forms favored in different tissues, for example by exon skipping. The database provides keyword search for retrieving and searching the contents and a graphical user interface displays the alternative splice forms. The alternative splice sites predicted by protein, mRNA or EST sequences individually are also given.
Numbers of sequences and exon candidates in ProSplicer
Number of sequences
Number of exon candidates
In ProSplicer, all the related evidence sequences, that is, mRNA, EST and protein sequences that are maintained in the database, are pre-aligned to the genomic gene sequences. All alternative splicing variants revealed by the alignment after the filtering phase are also stored. Both text and graphical information are provided in ProSplicer, as well as the query interfaces provided via the web.
ProSplicer provides several keyword search criteria, such as Ensembl gene identification numbers, gene symbols or names, protein id, and UniGene id. Users can submit a gene symbol as keyword and the database returns the query result containing the keyword. All the gene-related information, including supporting evidence, that is mRNA, EST and protein sequences, are also provided in the interface. Protein id and UniGene id can also be submitted by the user and the query result returns the genes that are supported by the query protein sequences or UniGene clusters.
Graphical splicing view
Jumping to specific region. You can jump to a user-specified region of the genomic sequence (see A in Figure 4) where all the related sequences and alignment result are also shown in the detailed view.
Scaling the view. You can scale the view to 1/8,1/4,1/2, 2, 4 or 8 times its current window size (see B in Figure 4).
Moving the view. You can move to the left or right of the current view (see C in Figure 4).
Also shown in Figure 4 is the main graphical view of the alternative splicing view. This comprises basic gene information: gene id (D), gene symbol (E), and gene description (F). The items provided in the splicing view are: the quality of alignment (G), with the degree of similarity between matching blocks, that is, exon candidates, represented by different colors; the length of the selected gene region (H); and sequence identification (I) - each 'Sequence ID' of a nucleotide or protein sequence is hyperlinked to SWISS-PROT, GenBank and dbEST. When you click on an exon candidate (J), a new browsing window opens showing the alignment flat file. The different color fills of the exon blocks refer to the alignment quality as set out in G. When you click on an intron block, a new browsing window showing the alignment flat file opens. The display also includes tissue information, with different tissues represented in different colors, and species information on the source organism of the protein or mRNA sequences.
A comparison of existing alternative splicing databases and tools
Several alternative splicing databases, such as AsMamDB , ASDB  and SpliceDB , are constructed on the basis of genes annotated containing the keywords 'alternative splicing'. AsMamDB contains information about alternative splicing in several mammals. SpliceNest , SpliceDB, AsMmDB, and HASDB  map clustered ESTs onto human genomic DNA to compute gene structures and splice variants. PALS db  takes the longest mRNA sequence in each UniGene  cluster as the reference sequence, which is aligned with ESTs and mRNA sequences in the same cluster to predict alternative splicing sites. The BLAT server  is a BLAST-like alignment tool that aligns an input nucleotide sequence to human genomic sequences, mRNA, EST and protein sequences. BLAT builds an index of the database and then scans linearly through the query sequence for local alignments. It then stitches them together into a larger alignment. Finally, BLAT revisits small internal exons possibly missed at the first stage and, where feasible, adjusts large gap boundaries that have canonical splice sites. BLAT is more accurate and 500 times faster than popular existing tools for mRNA/DNA alignments. BLAT is very effective for doing alignments between mRNA and genomic DNA from the same species, and can reveal splicing variants from the alignment result. ProSplicer pre-aligns known and novel gene sequences to the available mRNA, EST and protein sequences. ProSplicer is useful when the user wants to find alternative splicing variants by inputing a gene. We briefly summarize the difference between ProSplicer and BLAT as follows.
First, researchers can input gene names in ProSplicer as opposed to nucleotide sequences in the query stage. Second, the methods of alignment and filtering of sequences are most likely to be very different. We describe our method more fully in the Materials and method section. Third, in ProSplicer, links to various databases and functional information on particular genes (OMIM, RefSeq, GO, HUGO, and so on) are provided.
A comparison of different alternative splicing databases and tools
Types of sequence supported
90,000 EST clusters
Human and Arabidopsis
PALS db (Release 2)
Human and mouse
43,337 splice site pairs
AsMmDB (version 1.0)
1,563 alternative splicing genes
Human, mouse and rat
ASDB (version 2.1)
6,201 UniGene clusters
Human and mouse
BLAST (protein) SIM4 (mRNA and ESTs)
The future of ProSplicer
Novel genes can also be analyzed and integrated into the ProSplicer database. The ProSplicer database does not search by input raw nucleotide sequence because of the limitations of computational power. To handle the database search successfully, the query nucleotide sequences need to be aligned to a BLAST database of nucleotide, EST and protein sequences. We would like to support the query feature in the future by improving the computational power.
Materials and methods
The genomic sequences and gene annotation information are obtained from ENSEMBL  (Release 28 May, 2002) and contains 21,786 genes, including known and novel genes. The related mRNA sequences and EST sequences of the genes are retrieved from UniGene  (Release 147), and contain 96,105 human gene clusters. The EST sequences are from dbEST (Release 22 February, 2002), and there are about 3,991,208 human EST. The protein sequences are obtained from the SWISS-PROT and TrEMBL (Release 20 March, 2002) . SWISS-PROT (Release 40) contains 101,602 entries in total and 29,751 human entries. From TrEMBL 25,972 rodent entries are taken into account.
The preprocessing phase
In this phase, all the sequences and gene information are first collected and maintained. The data files are obtained from dbEST , UniGene , SWISS-PROT  and TrEMBL  databases. These files are then parsed and imported into a MySQL database. Ensembl  provides database files of MySQL, which we import directly into the database under construction. In the human genome data in Ensembl, each chromosome is represented as assembled fragments of nucleotide sequences. For each known gene and novel gene, Ensembl provides its location and direction in one or multiple chromosome. The genomic sequence of a gene is extracted from the assembled chromosome sequences.
The alignment phase
In ProSplicer, alternative splicing events are revealed by aligning the EST, mRNA and protein sequences against the gene genomic sequences. When aligning the nucleotide sequences, that is, mRNA and EST sequences, both types of nucleotide sequence are viewed as query sequences which are aligned to the gene genomic sequences using the alignment tool SIM4 . As the EST sequences are partial sequences of gene transcripts, the alignment result may show partial matching regions when they are compared to the genomic coding sequences. The mRNA sequences, which in general are cloned and sequenced from mature mRNA, provide more complete alignment results than do EST sequences to reveal the alternative splice forms.
The filtering phase
Aligning the mRNA, EST and protein sequences to the genomic sequences gives matching blocks which can be considered as exon candidates. However, there is some noise within the alignment result. The major sources of noise are small match blocks, repeat sequences and matching blocks on the complementary strand. The exon candidates generated by aligning the mRNA and EST sequences are eliminated if they are less than 15 base-pairs (bp) long, and those generated by protein comparisons if their length is less than 10 bp. Exon candidates are eliminated if the sequences are matched on the complementary strands of the gene genomic sequences. The remaining exon candidates generated from a single mRNA, EST or protein sequence are assembled into a complete splicing form by considering the sequential order of the matching blocks on the corresponding genomic sequence. To generate the splicing variants for each evidential sequence, we connect those exon candidates to reconstruct a splicing form by reference to the genomic sequence. This is done by comparing the position of the start and end sites of the matching blocks generated by aligning the EST, mRNA and protein sequences. The matching blocks in each evidential sequence are connected as a splicing form.
ProSplicer is now available at .
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