Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology.

With a painting, although the close view may be indistinct, the view of the whole can resolve itself into a discernable picture. Similarly, the global view of the genome will provide a unique vantage for understanding splicing. Since only a few systems of alternative splicing are likely to be analyzed in biochemical detail at least in the near term, bioinformatics approaches will be important in predicting alternative splicing patterns from genomic sequence. Simple sequence comparisons are a first step. Microexons and intronic splicing regulatory regions are often much more highly conserved between species than other intron sequence (see for example Thackeray and Ganetzky 1995xThackeray, J.R and Ganetzky, B. Genetics. 1995; 141: 203–214PubMedSee all ReferencesThackeray and Ganetzky 1995). Comparison of the mouse and human or the D. melanogaster and D. virilis genomes will yield a great deal of information on exon location and on splicing regulatory sequences.Work is also underway to align the EST and cDNA databases with genome sequences (9xKent, W.J and Zahler, A.M. Nucleic Acids Res. 2000; 28: 91–93Crossref | PubMedSee all References, 19xWolfsberg, T.G and Landsman, D. Nucleic Acids Res. 1997; 25: 1626–1632Crossref | PubMed | Scopus (102)See all References). Although this alignment approach again has difficulties in identifying short exons and exon termini correctly (Florea et al. 1998xFlorea, L, Hartzell, G, Zhang, Z, Rubin, G.M, and Miller, W. Genome Res. 1998; 8: 967–974PubMedSee all ReferencesFlorea et al. 1998), it can in principle identify all the splicing occurring within the sequenced portion of existing cDNAs. At the moment, this is only a small portion of the splicing events in the genome, but projects to generate large databases of full-length cDNA sequences will greatly improve its coverage (17xStrausberg, R.L, Feingold, E.A, Klausner, R.D, and Collins, F.S. Science. 1999; 286: 455–457Crossref | PubMed | Scopus (212)See all References, 13xRubin, G.M, Hong, L, Brokstein, P, Evans-Holm, M, Frise, E, Stapleton, M, and Harvey, D.A. Science. 2000; 287: 2222–2224Crossref | PubMed | Scopus (277)See all References). These efforts are unlikely to identify many low abundance mRNAs and will miss potentially important products. However, they will provide an initial reference splicing pattern for many genes. Such a large-scale identification of constitutive and regulated exons should also give us a great deal of information on the features and sequences that distinguish actual exons from the cryptic splice sites not normally recognized by the splicing apparatus. In addition to improving algorithms for predicting spliced segments within genomic sequence, this information will give insight into the mechanisms of alternative splicing and help experimentalists make sense of their complex biochemical systems.Much more difficult than identifying exons correctly will be predicting splicing regulatory patterns from genomic sequence. This issue can be seen in the DSCAM gene sequence where the alternative exons were relatively easy to identify through protein homology (Schmucker et al. 2000xSchmucker, D, Clemens, J, Shu, J, Worby, C, Xiao, J, Muda, M, Dixon, J, and Zipursky, L. Cell. 2000; 101: 671–684Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesSchmucker et al. 2000). However, the same sequence gives no clues about how these exons are regulated or which exons might be used in particular cells. Alternative exons have binding sites for multiple regulatory proteins that often show only subtle variation between tissues, and the sequence elements that control exons of very different tissue specificity can look similar (Smith and Valcarcel 2000xSmith, C.W and Valcarcel, J. Trends Biochem. Sci. 2000; 25: 349–404Abstract | Full Text | Full Text PDF | PubMed | Scopus (143)See all ReferencesSmith and Valcarcel 2000). Thus, even where we identify important regulatory sequences and proteins for an exon, it will be difficult to predict the precise tissues or conditions that lead to splicing activation or repression.The combinatorial nature of splicing regulation is similar to the control of transcription through promoter and enhancer elements and poses similar problems (Smith and Valcarcel 2000xSmith, C.W and Valcarcel, J. Trends Biochem. Sci. 2000; 25: 349–404Abstract | Full Text | Full Text PDF | PubMed | Scopus (143)See all ReferencesSmith and Valcarcel 2000). Many of the whole genome experimental approaches already being applied to transcriptional regulation can be informative for splicing as well (Young 2000xYoung, R. Cell. 2000; 102: 9–15Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesYoung 2000). Microarray technologies that allow the simultaneous assessment of the splicing of many exons within an RNA sample should prove particularly helpful. Unlike the detection of RNAs to measure whole transcript levels, these alternative splicing detector arrays will monitor the inclusion of particular exons in different populations of mRNAs. In one strategy, a position on the array would contain an oligonucleotide complementary to either a differentially included exon or to the exon/exon junction generated when this exon is skipped. The relative hybridization of a sample to these two sequences will give a measure of the relative inclusion of the exon. One limitation of this approach is that it does not give information correlating the exons within a single mRNA. For example, in the DSCAM gene, one would identify the alternatives for exons 4 and 6 that were present in a total mRNA sample, but which particular exons 4 and 6 were used together in the same mRNA molecule would not be discernable. Most importantly, however, such a technology permits one to examine the coordinate regulation of large groups of exons depending on development, cell type, or extracellular stimulus. This system-wide data about exon use may lead to the identification of sequence features that determine particular patterns of expression.The presence or absence of particular exons in an mRNA can also be correlated with data on the expression patterns of potential splicing regulators, such as SR proteins or hnRNPs. Combined with conditional knockouts of these regulators, we can ask precise questions about what combinations of regulatory proteins are needed for particular exons. Although it is recognized as a significant problem in the understanding transcriptional regulation, the question of combinatorial control of splicing is only beginning to be addressed. Nevertheless, this understanding of how exons are regulated on a system-wide scale will ultimately be essential in interpreting genome sequence and predicting how and when certain proteins are produced from it.Only a global view of splicing regulation combined with a detailed understanding of its mechanisms will allow us to paint a picture of an organism's total complement of proteins and of how this complement changes with development and the environment. Working toward this goal should keep bioinformatics researchers and molecular biologists busy for some time into the post-genome era.*E-mail: dougb@microbio.lifesci.ucla.edu