Raghavan, Madhura2018-10-232020-08-222018-08-30Raghavan_cornellgrad_0058F_10980http://dissertations.umi.com/cornellgrad:10980bibid: 10489811https://hdl.handle.net/1813/59715The molecular mechanisms by which the spliceosome achieves high fidelity during pre-mRNA splicing while simultaneously maintaining a high rate remain poorly understood. Here, I show a role for a core spliceosomal factor, Prp8, in balancing splicing speed with fidelity. An RNaseH-like domain (RH) within Prp8 contains a 17 amino acid insertion called the RH extension, which has been demonstrated to exist in two conformations: a β-hairpin, or an open loop structure. My work demonstrates that these two structures of the RH extension are associated with two distinct functional spliceosomal states. I demonstrate that mutations that increase the relative stability of the open loop conformation result in fast, but error-prone splicing. By contrast, mutations that increase the relative stability of the β-hairpin conformation result in hyper-accurate, but slow splicing. I propose a model where the RH extension toggles back and forth between the two conformations during splicing and helps balance speed with fidelity. Further, to better understand the functional role of RH extension in splice site usage, I have investigated its evolution across organisms which display varying levels of degeneracy in their splice site sequences. Remarkably, the RH extension residues are invariant among widely diverged species across almost all domains of eukaryotic life, the vast majority of which have degenerate splice site sequences. By contrast, in several organisms where the amino acid sequence of the RH extension has evolved, splice site sequences are seen to conform to a more rigid consensus, suggesting the fascinating possibility that the RH extension has driven the push towards constricting the splice site sequences present in these organisms.en-USGeneticsRNAMolecular biologyPrp8splicingdissertation or thesishttps://doi.org/10.7298/X4154F97