Among proteins that bind RNA, the most common structural motif is the alpha/beta sandwich global fold found in the RNA Binding Domain (RBDs). There are now more than 500 examples of RBDs identified in proteins involved in pre-mRNA splicing, mRNA cap binding, nuclear transport, mRNA stability, polyadenylation, and translation. The molecular mechanism of RNA recognition by RBDs is therefore a fundamental problem in the RNA world. The U1A protein is a component of the U1 snRNP (small nuclear RiboNucleoprotein Particle), where it specifically recognizes StemLoop II of U1 snRNA through interactions of its N-terminal RBD). It also recognizes RNA sequences in the 3' untranslated region (UTR) of its own pre-mRNA, where it regulates polyadenylation, apparently through interaction with poly(A) polymerase. The association of RBD1, with SLII, has been extensively studied, but despite the attention it has received, the complex mechanics of its recognition process are not sufficiently well understood to allow predictions of its behavior. The experiments described here use U1A RBD1:RNA thermodynamics and dynamical properties as probes for its RNA binding function, focusing on the wild type RBD1 and two isomorphic mutants. Using tSN NMR relaxation measurements and molecular dynamics simulations, the dynamics of U1A RBD1 free and bound to SLII will be compared, to determine how intrinsic protein dynamic networks are altered upon complex formation. 13CNMR relaxation and fluorescence will be used to probe analogous RNA properties. Binding energetics of the two RBD1 mutants studied by NMR will be measured, and the partitioning of energetic driving forces will be compared to the wild type protein. Thermodynamic and dynamic data will be combined to synthesize a model of the molecular mechanism of recognition of this RBD. Finally, in Drosophila melanogaster, the SNF protein takes the place of U1A in U1 and snRNP, but is also found in U2 snRNP, as a consequence of mutations that alter its binding affinity and specificity. A comparison of SNF and U1A binding properties and structures will show how RBD function has been modulated.
| Hall, Kathleen B (2012) Spectroscopic probes of RNA structure and dynamics. Methods Mol Biol 875:67-84 |
| Hall, Kathleen B (2008) RNA in motion. Curr Opin Chem Biol 12:612-8 |
| Clerte, C; Hall, K B (2000) Spatial orientation and dynamics of the U1A proteins in the U1A-UTR complex. Biochemistry 39:7320-9 |
