Binding Interactions Between Splicing Factor Cus2 And U2 snRNA

Author(s): Sanchez, Santiago F | Advisor(s): Ares, Manuel | Abstract: Assembly of the U2 small nuclear ribonucleoprotein (snRNP) to form the pre-spliceosome (PSP) is the first ATP dependent step of splicing (Kim a Lin 1993). This assembly requires the rearrangement of the U2 stem II region from a stem IIc conformation to its competing stem IIa conformation (Zavanelli a Ares 1991, Perriman a Ares 2000). Both smFRET and genetic studies have shown splicing factor Cus2 is able to influence refolding of U2 into its IIa conformation. (Yan 1998, Perriman a Ares 2007, Rodgers et al. 2016). The Saccharomyces cerevisiae splicing factor CUS2 (Cold sensitive U2 snRNA Suppressor 2), contains an RNA recognition motif (RRM) which binds to U2 snRNA, as well as an acidic C-terminus which contains amino acid residues involved in Cus2’s RNA refolding and its cold sensitivity suppression. A Cus2-Y48D mutant (hereafter referred to as Y48D) reduces the protein’s ability to bind U2 RNA in vitro, and the proteins ability to suppress cold sensitivity seen in stem IIa folding mutants (Yan et al. 1998, Rodgers et al. 2016). Additionally, the mutations in Cus2’s C-terminus enhance its RNA refolding and cold sensitivity suppression, implying that the C-terminus is involved in both processes. Lastly, this enhanced suppression and enhanced refolding is only possible in the absence of the Y48D mutation, suggesting that the C-terminus interacts with CUS2’s RRM, though the nature of this interaction remains unclear.Surprisingly, although the Y48D mutation weakens Cus2’s ability to bind wild type U2, base pairing mutations that stabilize U2 in the IIc form allow this same mutant to bind the snRNA through a novel interaction (Rodgers et al. 2016). This interaction, along with knowledge that Cus2 preferentially binds to IIc-U2 over IIa (Perriman a Ares 2007), presents a model in which a novel RNA binding site outside Cus2’s RRM binds specifically to stem IIc U2, and then hands off the refolded stem IIa to the canonical RRM. Furthermore, it seems possible that the C-terminus may be interacting with this putative binding site or the canonical RRM to possibly inhibit RNA binding.Here I have tested parts of this model using genetic and biochemical approaches. To observe the effects of a hypothetical second binding site, I tested for predicted growth defects in yeast expressing only a stem IIc stable U2 and the Y48D binding mutant protein. My results suggest that the combination of a IIc stable U2 and the Y48D protein does not cause a significant growth defect, and that this binding interaction might not be strong enough to halt U2 snRNA refolding, and inhibit splicing progression.To further explore the binding interaction between Cus2 and U2 I tested the affinity between different U2 stem II mutants and Cus2 or the Y48D mutant protein. If Cus2 protein does contain two RNA binding sites that discriminate between either conformation of stem II, it could be that the mechanism behind refolding utilizes different affinities for U2 stem IIa and IIc at each site. I therefore measured the binding affinity of Cus2 and Cus2-Y48D to U2 stem II stabilized in either its IIa or IIc form. My data indicates that the Cus2 protein binds to U2 snRNA in a fashion largely dependent on the presence of its RRM and access of the RRM to its target.