Characterization of the specificity and affinity of the splicing factor BBP/SF1

The yeast branchpoint binding protein (BBP) is an RNA binding protein that is well conserved with its human orthologue, splicing factor 1 (SF1). BBP/SF1, referring to both the yeast and human protein, has been implicated in playing a role in the early stages of RNA splicing, however, its precise role remains unclear. Evidence that it is involved in splicing includes the observation that it binds directly to the branchpoint sequence (BPS) during the early stages of spliceosome assembly in both yeast and human.;Yeast introns have a highly conserved BPS consisting of a nearly invariant UACUAAC. Yeast BBP interacts with this sequence with high specificity and affinity. Human introns, however, do not contain a conserved BPS. The BPS sequence is degenerate in human introns with most containing a consensus sequence of CURAY (R purine). In order to accommodate the degenerate BPS found in most human introns, it is necessary that SF1 recognize a wider range of RNA targets. This means SF1 is significantly less specific than BBP and binds with more than a 100 fold weaker affinity compared to yeast BBP protein.;To understand the role of BBP in splicing and how it achieves its specificity and high affinity, SELEX experiments were carried out to identify the highest affinity RNA targets for yeast BBP. It was found that yeast BBP binds the BPS UACUAAC with highest affinity and a stem-loop upstream of the BPS enhances binding. This enhanced binding is facilitated by BBP's zinc knuckle domains.;To elucidate how BBP and SF1 differ in their RNA binding characteristics, a comparative biochemical approach was taken. This was done by exchanging protein regions within SF1 for regions of BBP and vice versa. The results from these studies led to the discovery of the amino acids within BBP and SF1 that determine their respective RNA binding characteristics. This dissertation includes my previously published co-authored material and my previously unpublished co-authored material.