Alternative splicing is one of the principal means by which organisms create cell and tissue diversity. Currently no technique is available for conducting large-scale alternative splicing analyses. We propose to develop two novel technologies to measure alternative splicing on a genomic scale using Arabidopsis thaliana as a model system. We selected Arabidopsis as an assay development platform because of three competitive advantages: 1) the project is cost effective because three types of custom DNA microarrays uniquely suited for this project have already been designed, fabricated and tested, including the first whole genome tiling array. 2) More than 1000 examples of alternatively spliced Arabidopsis genes have been described, providing a stringent training set for our analyses and 3) obtaining tissue specific and developmentally distinct samples is straightforward. During this project we will develop the necessary protocols and software for extracting alternative splicing data from DNA microarrays. We will critically analyze the three different microarray designs and assess each one as a potential model for subsequent technology transfer to human splicing arrays. In parallel, we will design and test a highly multiplexed sequence-tagged Molecular Inversion Probe (MIP) assay to measure RNA splicing. The MIP assay is highly adaptable and can be utilized to monitor alternative splicing in Arabidopsis by redesigning variable regions on the probes to interrogate exon-exon splice junctions. Developing a MIP-based assay to measure splicing in Arabidopsis is tantamount to producing an assay for human splicing because translating the technology is a simple matter of redesigning the probes. Once completed, this project will provide a rigorous comparison of the two technologies. We are capitalizing on an extraordinary opportunity to use state-of-the-art biotechnological tools to build two genome-wide alternative splicing assays. Information derived from this project will give us insight into the tissue variation and overall scale of alternative splicing in Arabidopsis as well as, allow for more efficient and economical development of analogous splicing assays for humans.
| Juneau, Kara; Palm, Curtis; Miranda, Molly et al. (2007) High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing. Proc Natl Acad Sci U S A 104:1522-7 |
| Juneau, Kara; Miranda, Molly; Hillenmeyer, Maureen E et al. (2006) Introns regulate RNA and protein abundance in yeast. Genetics 174:511-8 |
