This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. These structures would contribute to understanding the basis for many inherited diseases that are associated with errors in splice site recognition, including cancers, metabolic disorders, muscular dystrophies, and cystic fibrosis among others. The essential splicing factor, U2AF binds to the pre-mRNA 3'splice site consensus sequence during the critical early stages of splice site choice, and recruits further components of the splicing machinery. The pre-mRNA sequence recognized by U2AF is a polypyrimidine tract composed primarily of uridines and cytidines. A few years ago, we determined the structure of U2AF bound to an optimal polyuridine splice site with the aid of synchrotron radiation [Sickmier et al. (2006) Mol. Cell]. This structure demonstrates that U2AF recognizes uridines through hydrogen bonds with the edges of the bases. However, the in vivo RNA targets of human U2AF are frequently interrupted by cytidines. To investigate how the U2AF structure adapts to accommodate cytidines, we determined a series of U2AF bound to cytidine-containing RNAs using the microfocus F1 beamline at CHESS. From these structures, we concluded that U2AF (1) rearranges side-chain and water-mediated hydrogen bonds and (2) adjusts its binding register to adapt to cytidine-containing sites. We complemented these crystallographic results by measuring the affinities of U2AF for the cytidine-containing site, and found that U2AF can tolerate up to four consecutive cytidines in the polypyrimidine tract with little difference among affinities, but beyond this limit can no longer adapt. The polypyrimidine tract-binding domain of U2AF contains two modular RNA recognition motifs (RRMs) separated by a poorly conserved interdomain linker. By using the ensemble optimization method (EOM) to analyze small angle X-ray scattering data collected at the SIBYLS beamline at ALS, we found that the relative arrangement of the U2AF RRMs appears to be flexible. Based on these data, we hypothesize that in solution, the flexible RRM arrangement contributes to the ability of U2AF to adapt to variable splice sites. Here, we propose to compare the low resolution shapes of U2AF complexes with (1) a natural polypyrimidine tract sequence (CCCUUUUUUUUCC), (2) the polyuridine site (UUUUUUUUUUUUU), and (3) a sequence that represents the limit of cytidine-substitutions that U2AF binds without detectable decrease in affinity (CCCUUUCUCCUCC) using small angle X-ray scattering at CHESS beamline G1. Sequence-dependent changes in the molecular dimensions of the complexes would support the hypothesis that adjustable inter-RRM register contributes to U2AF ability to adapt to cytidine-containing sequences. All samples are monodisperse. SAXS data was previously collected for an analogous complex of U2AF with a 12-nucleotide polyuridine RNA at the SIBYLS beamline at ALS. This data produced consistent Guinier plots over a concentration range (2-8 mg/mL) and resulted in Chi values <2 when modeled using Dammin. This work is funded by the National Institutes of Health, R01-GM 070503.
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