RNA participates in a wide array of biological functions that require specifically folded secondary and tertiary molecular structures. RNA structures and the protein complexes they form are essential for a wide variety of processes in cells such as translational activity, regulation of transcription, replication of RNA viruses. RNA splicing, telomere synthesis and differentiation. We describe a study designed to characterize the secondary and tertiary structures of a set of model RNAs for a biologically important RNA and the interactions of these RNAs with a cellular protein that binds specifically to the native RNA. The sequence and predicted secondary structure of the native RNA, which consists of two directly linked stem loops, indicate that it may fold into a pseudoknot or a pair of coaxially stacked hairpins. A model in which the loop of one hairpin is docked into a helical groove of the stem of another hairpin is less likely, but will also be evaluated. An interesting possibility is that the native RNA may switch between conformations, such as the pseudoknot and coaxial helices, as part of its biological function. It should be emphasized that only a few biologically relevant and functionally active RNA structures, which are amenable to high resolution analysis, are known at this time. The results of this research will, thus, provide significant new general information on RNA structural units and their proteins, interactions. The Research plan is organized around three specific aims that will yield the RNA structures and define the structures necessary for protein interactions.
In Aim 1 we describe the design and preparation of the model RNAs to be used in a systematic analysis of the native RNA structure and its protein interactions.
Aim 2 involves a structural analysis of the model RNAs defined in aim 1, and involves three major experimental approaches; (i) chemical and enzymatic probing; (i) thermal melting and circular dichroism (CD); (iii) NMR and molecular modeling. The model RNAs will be reached with various chemical and enzymatic probes under a variety of conditions to investigate both secondary and tertiary structure interactions. The melting and CD studies will allow an analysis of the unfolding of structural units within the model RNAs with increasing temperature or changes in solution conditions. Analysis of the separated RNA hairpin units will allow assignment of the melting transitions in the native RNA to specific units and will help identify which transition(s) represent the unfolding of RNA tertiary structure. NMR analysis of the separated RNA hairpin units will provide detailed structural information and provide to base-pair mismatch information for the larger, native model RNAs. These studies will progress to high resolution analysis of some of the larger RNAs as our knowledge of the RNA systems and t heir tertiary structures increases in this project. All of the data described above will be organized through computer-based molecular models. Additional constraints in refinement of the model will be added as they are obtained.
Aim 3 involves evaluation of protein-RNA interactions. A cellular protein (p84) has been previously shown to form a specific complex with the RNA under study. The strength of the interactions of the (p84) protein with the model RNAs will be evaluated by the gel shift method. The half-life of complexes formed will also be determined. It is possible that protein binding induces a conformational shift in the RNA upon complex formation. We will monitor any changes in RNA structure induced by protein binding through the use of difference CD spectroscopy. Analysis of RNA mutants/variants that are forced in the direction of a specific conformation will also be used to determine whether the binding protein has a conformational preference. As the studies progress, selected mutations that affect RNA structure will be tested for function in vivo. The goal of this aim is to correlate the structural information obtained on the model RNAs with specific functional differences that occur as a result of sequence changes in the RNA.
