In complex eukaryotes the accurate splicing of the precursors to messenger RNAs is essential for production of the correct protein components in all cells. Evidence for the importance of this process comes from the fact that splicing mutations account for 10-15% of human genetic disease. Both components of the spliceosome (the molecular machine for the removal of introns) and splicing mechanisms are highly conserved among eukaryotes (see, for example, Mount and Salz 2000), and splicing mutations account for comparable fraction of loss-of-function mutations in model organisms, such as Arabidopsis, with complex gene structures.

The boundaries between exons and introns are referred to as splice sites, and our research is directed towards understanding how these sites are selected during the splicing of precursors to mRNA. Although there are a number of distinct steps required for the formation of a functional spliceosome, the recognition of core splicing signals by a conserved set of factors occurs early in the process, and it is likely that the assembly of these early factors determines the outcome of splicing in most cases. An understanding of the rules governing splice site selection is very important for gene annotation, and accurate gene annotation in the absence of reliable experimental evidence remains difficult to achieve, in part because a number of aspects of pre-mRNA splicing limit our ability to predict how splicing will occur on the basis of genomic sequence data (Mount, 2000). An improved understanding of the rules that govern splice site selection in Arabidopsis will lead to improvements in our ability to find genes in all plants.

Conserved sequences at the splice sites make a major contribution to splice site selection, and the recognition of these signals is generally understood. The projects described here address the contributions of splicing signals other than those at the splice sites themselves. The first characterizes splicing enhancer sequences elements within exons. Best characterized in animal genes, exonic splicing enhancers activate nearby splice sites. The second project investigates the means by which adjacent splice sites facilitate each other's recognition. The requirement for productive interactions among splicing factors bound at either end of an exon often means that a splice site mutation will result in the skipping of the exon that bears the mutation, thereby affecting the splicing of the neighboring intron as well. This exon-skipping phenomenon has been described in Arabidopsis (Brown 1996). The third project will exploit the availability of a mutation in the lariat debranching enzyme to characterize branch sites in Arabidopsis.

This project seeks to identify and characterize exonic splicing enhancers (ESEs) in the Arabidopsis thaliana genome, and to use knowledge of these enhancers to improve gene annotation. Although splicing enhancers likely function in splice site selection in many plant genes, and contribute to the regulation of alternative splicing, plant ESEs have not yet been described. The role of computationally identified candidate splicing enhancer sequences, and sequences from genes that are known to be alternatively spliced, will be tested in transgenic Arabidopsis using a splicing reporter construct. The activity of these enhancers will be examined in transgenes that depend on exon inclusion for expression. This will include analysis of the tissue-specificity of splicing enhancer activity under standard growth conditions and different temperatures, and should provide an extensive database of information about the role of particular sequences in promoting splicing. This project will generate approximately 2,000 publicly available transgenic lines carrying splicing reporter genes with defined candidate splicing enhancer sequences and description of marker gene expression for each splicing enhancer candidate.

REFERENCES

Chapman, K. B., and Boeke, J. D. . (1991). Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65: 483—491.

Nam, K., Lee, G., Trambley, J., Devine, S.E., and Boeke, J. D. (1997). Severe Growth Defect in a Schizosaccharomyces pombe Mutant Defective in Intron Lariat Degradation. Mol. Cell. Biol. 17: 809-818.