Structural analysis of protein-facilitated cooperative folding in the bI3 RNP

Most large RNAs require protein facilitators to achieve their active structures. Despite the importance of RNA structure for understanding the functions of associated proteins, initial and intermediate structures of the RNA are often assumed or not fully characterized. This gap in structural knowledge has lead to an over-simplification of the roles of many proteins in ribonucleoprotein complexes. Protein facilitators are currently categorized into two broad classes, cofactors and chaperones. Cofactors bind tightly an RNA to stabilize tertiary structures, while chaperones interact transiently with an RNA to facilitate acquisition of the most stable secondary structure. In this research, I comprehensively interrogate the structure of an RNA throughout the stages of protein-facilitated folding. In principle, the yeast bI3 group I intron has to potential to fold into the active secondary structure conserved among group I introns. The bI3 RNA requires two proteins (the bI3 maturase and Mrs1) for splicing. These proteins seem to have characteristics of cofactors and were not expected to affect the secondary structure. In contrast, by developing a high-throughput SHAPE experiment, I find that approximately half the bI3 RNA is not in the catalytically active secondary structure prior to protein binding. I develop and test a structural model for the misfolded RNA using SHAPE analysis of point mutations. I find that three conserved elements form stable, extensively mispaired, non-native structures. Solvent accessibility experiments show that these nonnative structures are incapable of forming native group I intron tertiary interactions. I next demonstrate that binding by either the Mrs1 or maturase protein alone promotes the formation of distinct tertiary structures. However, neither individual protein binds to or affects the non-native secondary structures in the misfolded RNA. Strikingly, simultaneous binding of both proteins enables the RNA to achieve its active conformation. These results highlight a large-scale cooperative folding process between the bI3 RNA and the Mrs1 and maturase proteins in which the final RNA structure is drastically different than the sum of individual protein-bound states. The bI3 RNP thus represents a new category of ribonucleoprotein assembly mechanism in which binding by multiple proteins drives non-hierarchical RNA folding.