application): The interaction of proteins with specific RNA sites is important in many basic biological processes and is only beginning to be understood at a structural level. The wide diversity of RNA structures involved, and the ability of RNAs to undergo substantial conformational rearrangements, make the problem particularly interesting and challenging. This proposal describes experiments aimed at understanding how one class of RNA-binding proteins, that containing an arginine-rich binding motif, recognizes RNA. The motif is found in proteins that participate in translation, RNA splicing, and RNA transport, and in key viral regulatory proteins. The ability to mimic protein-RNA interactions important for cell or virus growth may help in designing inhibitors of cancer cell or viral replication, including HIV. Experiments are proposed in which arginine-binding RNAs will be used as model systems to develop an RNA structure prediction algorithm and to learn more about the structural details of arginine-RNA interactions. A series of biochemical and mutagenesis experiments will be used to generate constraints on the structures, and a computed library of all possible structures will be searched to identify structures consistent with the experimental data. It is also proposed to examine recognition of RNA loops by arginine-rich peptides from bacteriophage antiterminator proteins. Peptide and RNA mutagenesis and chemical modification interference experiments will be performed to identify amino acids and nucleotides involved in binding. Circular dichroism and NMR spectroscopy will be used to characterize structural features of the complexes, including any changes in RNA structure. An additional aim describes studies using a bacterial antitermination reporter system to screen libraries for RNA-binding peptides with desired binding specificities. Such peptides may provide useful tools for studying biological functions of RNA elements by inhibiting their normal activities, and may help in designing therapeutics directed against specific RNA targets.
| Nakamura, Robert L; Landt, Stephen G; Mai, Emily et al. (2012) A cell-based method for screening RNA-protein interactions: identification of constitutive transport element-interacting proteins. PLoS One 7:e48194 |
| Pastuszak, Alexander W; Joachimiak, Marcin P; Blanchette, Marco et al. (2011) An SF1 affinity model to identify branch point sequences in human introns. Nucleic Acids Res 39:2344-56 |
| Mokdad, Ali; Frankel, Alan D (2008) ISFOLD: structure prediction of base pairs in non-helical RNA motifs from isostericity signatures in their sequence alignments. J Biomol Struct Dyn 25:467-72 |
