Characterization of U2AF26, a paralog of the splicing factor U2AF35

by Shepard, Jeremiah Brian.

The essential splicing factor U2 auxiliary factor (U2AF) mediates 3’ splice site recognition during spliceosome assembly. The mammalian U2AF is composed of a large subunit, U2AF65, and a small subunit, U2AF 35. U2AF65 recognizes the pyrimidine tract and U2AF35 binds to the AG dinucleotide, both of which are specific 3’ splice site sequence motifs. In the present work U2AF26, a paralog of the conventional U2AF35, has been studied. U2AF26 shares 84% primary amino acid identity with U2AF35, suggesting vi functional homology. However, U2AF26 has two amino acid substitutions in ribonulceoprotein consensus sequence-2 (RNP-2) and significant differences within the RS domain, two regions thought to be important for the function of U2AF35. The goal of this study was to characterize the functional differences between the two small subunits. Western blot analysis revealed that U2AF26 protein expression varies relative to U2AF35 in different mouse tissues. Site-specific crosslinking analysis of sixteen permutations of the nucleotide composition upstream and downstream of the AG indicates that U2AF26 and U2AF35 bind to the UAGG motif with the highest affinity. Interestingly, U2AF26 binds the UAGU motif better than U2AF35. This observation suggests that U2AF26 and U2AF35 have overlapping binding affinities, but that U2AF26 might be capable of recognizing a specific 3’ splice site motif better than U2AF 35. Initial evidence suggested that U2AF26 is regulated by circadian rhythm. Analysis of U2AF26 over a 24-hour period in the mouse forebrain indicates that expression of the full length transcript does not change significantly, but the alternative splicing of the U2AF26 transcript fluctuates during the day:night cycle. Examination of U2AF26 alternative splicing in other tissues revealed that this splicing event is temporallyregulated in the liver, but with a two-peaked pattern of splicing. Further analysis of other alternative splicing events in the liver indicates that the polypyrimidine tract binding (PTB) transcript is regulated in a similar manner. The two-peaked pattern of splicing in the liver suggests that the alternative splicing of U2AF26 and PTB is not regulated by circadian rhythm. However, this is the first time it has been observed that pre-mRNA splicing changes as a function of the day:night cycle. vii