Mapping metal ion binding pockets in vivo

RNA plays essential roles in most processes in the cell. Although their tasks are very diverse, RNAs share their strict dependence on acquiring a specific three-dimensional fold in order to fulfill their biological function. Despite their importance for cell viability little is known about how these RNAs fold in vivo and how they interact with their targets. Therefore, it is of fundamental importance to gain insights into the forces driving RNA folding in vivo and to establish the contribution and impact of the cellular environment, in order to understand the basic mechanism of these RNA-dependent processes. Particularly, metal ions and proteins are important ligands interacting with RNA, thereby stabilizing their native, functional state. Catalytic RNAs, in particular group II introns, are the best-suited model system to study RNA folding in the living cell, as their structure and folding pathways are well characterized in vitro and native state formation can be measured as a function of catalysis. Here I propose to identify metal ion binding sites within RNA in vivo and to investigate how proteins influence such metal pockets. This is of interest as metal ion homeostasis was shown to be a crucial determinant for group II intron splicing in yeast mitochondria and because several specific RNA-binding proteins lower the Mg 2+- requirements for ribozyme function in vitro. Exploring these aspects of intracellular RNA structure formation will help us understand the driving forces of RNA folding in the living cell. Given my expertise in RNA folding both in vitro and in vivo, I am well prepared to successfully conduct this project, thereby providing a significant contribution and a new direction to the RNA field. Ultimately, this challenging project will advance us in exploiting the medical and biotechnological potential of catalytic RNA.

RNA plays essential roles in most processes in the cell. Although their tasks are very diverse, RNAs share their strict dependence on acquiring a specific three-dimensional fold in order to fulfill their biological function. Despite their importance for cell viability little is known about how these RNAs fold in vivo and how they interact with their targets. Therefore, it is of fundamental importance to gain insights into the forces driving RNA folding in vivo and to establish the contribution and impact of the cellular environment, in order to understand the basic mechanism of these RNA-dependent processes. Particularly, metal ions and proteins are important ligands interacting with RNA, thereby stabilizing their native, functional state. Catalytic RNAs, in particular group II introns, are the best-suited model system to study RNA folding in the living cell, as their structure and folding pathways are well characterized in vitro and native state formation can be measured as a function of catalysis. Here I propose to identify metal ion binding sites within RNA in vivo and to investigate how proteins influence such metal pockets. This is of interest as metal ion homeostasis was shown to be a crucial determinant for group II intron splicing in yeast mitochondria and because several specific RNA-binding proteins lower the Mg 2+- requirements for ribozyme function in vitro. Exploring these aspects of intracellular RNA structure formation will help us understand the driving forces of RNA folding in the living cell. Given my expertise in RNA folding both in vitro and in vivo, I am well prepared to successfully conduct this project, thereby providing a significant contribution and a new direction to the RNA field. Ultimately, this challenging project will advance us in exploiting the medical and biotechnological potential of catalytic RNA.