Genome-wide detection of alternative splicing in expressed sequences of human genes

Abstract

We have identified 6201 alternative splice relationships in human genes, through a genome-wide analysis of expressed sequence tags (ESTs). Starting with approximately 2.1 million human mRNA and EST sequences, we mapped expressed sequences onto the draft human genome sequence and only accepted splices that obeyed the standard splice site consensus. A large fraction (47%) of these were observed multiple times, indicating that they comprise a substantial fraction of the mRNA species. The vast majority of the detected alternative forms appear to be novel, and produce highly specific, biologically meaningful control of function in both known and novel human genes, e.g. specific removal of the lysosomal targeting signal from HLA-DM beta chain, replacement of the C-terminal transmembrane domain and cytoplasmic tail in an FC receptor beta chain homolog with a different transmembrane domain and cytoplasmic tail, likely modulating its signal transduction activity. Our data indicate that a large proportion of human genes, probably 42% or more, are alternatively spliced, but that this appears to be observed mainly in certain types of molecules (e.g. cell surface receptors) and systemic functions, particularly the immune system and nervous system. These results provide a comprehensive dataset for understanding the role of alternative splicing in the human genome, accessible at http://www.bioinformatics.ucla.edu/HASDB.

Figure 1

Detection and validation of alternative splicing. (A) Types of evidence for alternative splicing (see text). (B) Types of alternative splicing detected in this study include exon skipping, alternative 5′ splice donor sites and alternative 3′ splice acceptor sites.

Figure 2

Alternative splicing of DMPK. (A) Gene structure for exons XII–XV of the dystrophia myotonica protein kinase gene (DMPK), in contig NT000991 of chromosome 19. Two splice forms are shown, one observed in an mRNA (mRNA1) and one in an EST (EST1). (B) Example sequence evidence for the two splice forms. Sequence EST1 skips directly from exon XII to exon XV. We detected three alternative splice forms in DMPK; all are confirmed by the experimental literature (23).

Figure 3

Alternative splicing of HLA-DMB. (A) Genomic structure of the HLA-DM β gene, in contig NT001520 of chromosome 6. Exons are shown as filled boxes, and the observed splices are shown on top of the genomic sequence. (B) The four alternative forms of HLA-DM β mRNA inferred from the expressed sequence data, colored to show the exons. The protein reading frame is indicated by an arrow beneath each form, showing the transmembrane domain (TM) and lysosomal targeting signal (LT). (C) The splice donor and acceptor sites for the eight putative splices observed in HLA-DM β. The primary consensus site sequences are highlighted in black and secondary consensus sequences (5) are marked in magenta.

Figure 4

Alternative splicing of Hs.11090, a putative FCɛ receptor β chain homolog. (A) Genomic structure of exons and splices, as in Figure 3. Potential polyadenylation sites important for the alternative gene forms are indicated. (B) Three alternative forms inferred from the expressed sequences. Predicted transmembrane domains (TM) are indicated (see text). (C) The corresponding protein forms, indicating topology across the membrane.

Figure 5

Analysis of a random sample of alternative spliced genes. (A) Fraction of the observed alternative splice in protein coding region, 5′ and 3′ UTR. (B) Fraction replacing the protein N-terminus, C-terminus or internal regions. (C) Fraction causing truncation of protein product due to frame-shift; extension of the protein product due to frame-shift; switch to a new initiator codon while preserving protein reading frame; switch to a new terminator codon from an alternative exon; in-frame deletion of codons, preserving reading frame; in-frame insertion of codons, preserving frame. (D) Origin of alternative splicing evidence: detected in mRNA (presumed not novel); detected in EST (by comparison with an mRNA); detected in EST (by comparison with other ESTs). (E) Type of alternative splice. (F) Categorization of alternatively spliced genes by systemic function (see text). (G) Categorization by gene product (see text).