PRE-MRNA SPLICE SITE RECOGNITION IN HUMAN DISEASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of our research is to understand the three-dimensional shapes that guide interaction of pre-mRNA splicing factors with the 3¿¿" splice site, thereby providing a foundation to develop treatments for inherited diseases associated with errors in splice site recognition. The essential human splicing factor, U2AF binds to the pre-mRNA 3¿¿" splice site consensus sequence during the critical early stages of splice site choice, and recruits the core splicing machinery. We recently determined the structure of U2AF in complex with poly-uridine with the aid of synchrotron radiation [Sickmier et al. (2006) Mol. Cell). This structure demonstrates the mechanism of uridine recognition by U2AF. However, the in vivo RNA targets of U2AF contain cytosine. The goal of the proposed work is to understand how U2AF changes conformation to recognize a cytosine instead of uracil. To this end we have crystallized U2AF with three different uridine-rich oligonucleotides with single-positions changed to cytosine. Specific nucleotides are brominated to unambiguously identify the nucleic acid register. Cocrystals with one of the sequences diffract to 2.9¿ resolution, and two other cocrystals diffract to better than 3.5¿ resolution in-house. Synchrotron radiation is required to obtain the high resolution data sets to reveal subtle conformational changes of this essential splicing factor bound to target RNA sequences.