Structural investigation of RNA-RNA and RNA-protein interactions involving the pre-mRNA branch site region of the functional core of the spliceosome

The removal of introns from precursor-messenger (pre-m)RNA in eukaryotes is mediated by the spliceosome, a dynamic supramolecular complex comprising five small nuclear (sn)RNAs and numerous proteins, organized into small ribonucleoprotein (snRNP) particles. At the functional center of the spliceosome is the pairing of a highly conserved region of the RNA intron and the U2 snRNA, called the pre-mRNA branch site duplex, and two U2 snRNP proteins, p14 and SF3b155. The goal of the research described in this dissertation was to characterize the interaction between p14 and the branch site RNA, and determine whether SF3b155 impacts on that interaction.;In order to refine the structure of the pairing between the intron and pseudouridine (Psi)-modified U2 snRNA (M. I. Newby and N. L. Greenbaum, 2001 RNA 7; 833-845), Nuclear Magnetic Resonance (NMR) experiments were performed in supercooled water in order to decrease the temperature-dependent exchange of protons in RNA duplexes. NMR spectra of aqueous samples of RNA in bundles of narrow capillaries acquired at temperatures as low as -18°C reveal resonances of exchangeable protons not seen at higher temperatures. In particular, we detected the imino protons of terminal base pairs and the imino proton of a non-base paired Psi in the pre-mRNA branch site helix. Analysis of the temperature dependence of chemical shift changes (thermal coefficients) for imino protons corroborated hydrogen bonding patterns observed in the NMR derived structural model of the branch site helix. The ability to observe non-base paired imino protons of RNA loop and bulge regions is of significant value in refining the structure of RNA motifs containing non-base paired regions. Also, detection of the imino proton of the non-base paired Psi will help identify this resonance when p14 is added.;As measures of affinity between p14 and branch site RNA, membrane filtration and gel electrophoresis methods indicated that the U2 snRNP protein p14 binds a single-stranded RNA representing the pre-mRNA intron in solution; however, affinity is significantly greater when the intron is paired with a U2 snRNA strand modified by Psi in its phylogenetically conserved location opposite the branch site residue. The binding between p14 and its cognate RNA is enhanced further upon addition of a segment of the neighboring protein SF3b155 that contacts p14 but not the RNA. NMR spectra reveal that p14 undergoes significant changes upon complex formation with both the pre-mRNA branch site duplex and SF3b155, in contrast, the RNA does not undergo any marked change, and changes in SF3b155 are limited to specific regions. These data imply that the three components (pre-mRNA branch site duplex, p14, and SF3b155) interact with each other under equilibrium conditions, and that SF3b155 facilitates the p14-RNA interaction. These results demonstrate, for the first time, specific recognition of a double-stranded RNA motif by an RNA Recognition Motif (RRM) protein, and suggest a cooperative mechanism for assembly of the pre-mRNA, U2 snRNA, p14 and SF3b155.