Two proteins co-opt to promote group I intron folding 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. Catalytic RNAs, like group I 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 determine the intracellular structure of the yeast mitochondrial bI5 group I intron, which depends on two nuclear-encoded proteins, Mss116p and Cbp2, for efficient splicing in vivo. In contrast, in vitro Cbp2 is required but also sufficient to promote intron folding at near-physiological conditions. While the bI5-Cbp2 complex has been well characterized in vitro, the function of Mss116p in bI5 folding remains enigmatic. By monitoring Mss116p- and Cbp2-induced conformational changes within the bI5 group I intron in vivo, we will provide the first mechanistic insights into how these two proteins co-opt to facilitate bI5 folding and in turn splicing in yeast mitochondria. In addition, we will complement the in vivo analyses by investigating the assembly pathway of the bI5-Cbp2- Mss116p complex in vitro. In brief, the aim of this research proposal is to explore the principles underlying the interplay of RNA folding and RNP assembly. Given my expertise in RNA folding both in vitro and in vivo, we are well prepared to successfully conduct this project, thereby providing a significant contribution to the RNA field. Ultimately, this challenging project will advance us in exploiting the medical and biotechnological potential of catalytic RNA.

In the past years it became increasingly evident that the macromolecule RNA plays a central role in virtually all cellular processes. Even though these RNAs differ in their composition and function, they share the requirement for adopting a highly specific three-dimensional structure in order to exert their tasks in the cell. The process of RNA folding describes the pathway of how RNA folds from an unstructured, disordered state into the native, functional conformation. Exploring such RNA folding pathways is essential for gaining fundamental insights into the mechanism of these RNA-dependent processes. My research project aims at providing groundbreaking insights into RNA folding in the living cell a new innovative direction in RNA research. As RNA also plays a crucial role in the development of genetic diseases and cancer as well as in the virulence of pathogenic bacteria and the spread of viral infections, my research goal is of vital medical importance. The development of novel therapeutic strategies, e.g. the design of new, potent antibiotics, necessitates that we gain insights into the complex nature of intracellular RNA structure and folding. By monitoring Mss116p and Cbp2-induced conformational changes within the bI5 group I intron in vivo as a model system, we provided the first mechanistic insights into how these two proteins co-opt to facilitate bI5 folding and in turn splicing in yeast mitochondria. In addition, we complemented the in vivo analyses by investigating the assembly pathway of the bI5-Cbp2-Mss116p complex in vitro. This allowed us to explore the principles underlying the interplay of RNA folding and RNP assembly.