DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA

DEAD-box proteins are nonprocessive RNA helicases and can function as RNA chaperones, but the mechanisms of their chaperone activity remain incompletely understood. The Neurospora crassa DEAD-box protein CYT-19 is a mitochondrial RNA chaperone that promotes group I intron splicing and has been shown to resolve misfolded group I intron structures, allowing them to refold. Building on previous results, here we use a series of tertiary contact mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19–mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stability. Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase activity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunities. The strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does not form a compact structure, and small-angle X-ray scattering indicates that ATPase activity tracks with ribozyme compactness. Further, deletion of three helices that are prominently exposed in the folded structure decreases the ATPase stimulation by the folded ribozyme. Together, these results lead to a model in which CYT-19, and likely related DEAD-box proteins, rearranges complex RNA structures by preferentially interacting with and unwinding exposed RNA secondary structure. Importantly, this mechanism could bias DEAD-box proteins to act on misfolded RNAs and ribonucleoproteins, which are likely to be less compact and more dynamic than their native counterparts.DEAD-box proteins constitute the largest family of RNA helicases and function in all stages of RNA metabolism (1, 2). In vivo, many DEAD-box proteins have been implicated in assembly and conformational rearrangements of large structured RNAs and ribonucleoproteins (RNPs), including the ribosome, spliceosome, and self-splicing introns (3). Thus, it is important to establish how these proteins use their basic mechanisms of RNA binding and helix unwinding to interact with and remodel higher-order RNA structures.Structural and mechanistic studies have elucidated the basic steps of the ATPase cycle of DEAD-box proteins and have provided an understanding of the coupling between ATPase and duplex unwinding activities (4–11). The conserved helicase core consists of two flexibly linked RecA-like domains that contain at least 12 conserved motifs, including the D-E-A-D sequence in the ATP-binding motif II (3, 12). Binding of ATP and double-stranded RNA to domains 1 and 2, respectively, induces domain closure, which completes the formation of an ATPase active site at the domain interface and introduces steric clashes in the RNA binding site, leading to the displacement of one of the RNA strands (6, 7). ATP hydrolysis and inorganic phosphate release are then thought to regenerate the open enzyme conformation (4, 8, 13). Unlike conventional helicases, DEAD-box proteins have not been found to translocate, limiting the unwinding activity to short helices that can be disrupted in a single cycle of ATP binding and hydrolysis (4, 8, 9, 14–16). This mechanism is compatible with the physiological roles of DEAD-box proteins, because cellular RNAs rarely contain continuous base-paired regions that are longer than one or two helical turns.The interactions of DEAD-box proteins with structured RNAs have been extensively studied using two homologous proteins that function as general RNA chaperones: CYT-19 from Neurospora crassa and Mss116 from Saccharomyces cerevisiae. In vivo, CYT-19 is required for efficient splicing of several mitochondrial group I introns and can promote splicing of group I and group II introns in yeast mutants that lack functional Mss116 (17, 18). Both proteins have been shown to act as general RNA chaperones during group I and group II intron folding in vitro and are thought to act primarily by reversing misfolding of the intron RNAs, although additional mechanisms may be used for some substrates (17–23). Importantly, the chaperone activities of these and other DEAD-box proteins correlate with their ATP-dependent helix unwinding activities, suggesting that DEAD-box proteins function by lowering the energy barriers for transitions between alternative structures that involve disruption of base pairs (24, 25).In vitro studies using the group I intron ribozyme from Tetrahymena thermophila have been instrumental in probing the chaperone mechanism of CYT-19 (17, 26–28). This ∼400-nt RNA folds into a compact, globular structure composed of a conserved core and a series of peripheral elements that encircle the core by forming long-range tertiary contacts (Fig. 1) (29–31). Upon addition of Mg²⁺ ions, the majority of the ribozyme population becomes trapped in a long-lived misfolded conformation, which then slowly refolds to the native state (32). The misfolded intermediate is remarkably similar to the native ribozyme, forming a complete native network of secondary and tertiary interactions and a globally compact fold (33, 34). Despite these similarities, refolding to the native state requires extensive unfolding, including disruption of all five peripheral tertiary contacts and the core helix P3 (33, 35). To explain these results, a topological error has been proposed, wherein two single-stranded joining elements are crossed incorrectly in the core of the misfolded ribozyme, and transient disruption of the surrounding native structure is required for refolding (33, 35).Open in a separate windowFig. 1.The Tetrahymena group I intron ribozyme. (A) Secondary structure and mutations. Peripheral elements are colored and thick arrows mark the long-range peripheral tertiary contacts. Paired regions (P) and loops that were mutated in this study (L) are labeled based on group I intron nomenclature in ref. 31. The mutated regions are enclosed in dashed boxes and labeled in bold, with sequence substitutions indicated nearby. Sequences that were deleted to construct the helix truncation mutants (Fig. 6) are enclosed in gray dashed boxes and the replacement nucleotides are shown in gray italic font. (B) Tertiary structure model of the ribozyme (31). Peripheral elements (colored surface) and the locations of the long-range peripheral tertiary contacts (circles) are highlighted using the same color scheme as in A. The ribozyme core is shown in silver. The block arrows indicate the approximate positions of tertiary contacts not visible in each respective view of the ribozyme. The figures were prepared using PyMOL.Given the structural similarity between the native and misfolded ribozyme, it is interesting that CYT-19 can accelerate refolding of the misfolded intermediate by at least an order of magnitude without detectably unfolding the native ribozyme (26). Insights into this apparent preference for the misfolded ribozyme came from studies of two ribozyme mutants in which the tertiary structure was destabilized, making the stability of the native ribozyme comparable to that of the misfolded intermediate (28). CYT-19 unfolded the native and misfolded conformers of these mutants with comparable efficiencies, suggesting that the efficiency of chaperone-mediated unfolding depended on the stability of ribozyme tertiary structure. However, the mutations studied were concentrated in one region of the ribozyme, leaving open the possibility that CYT-19 recognizes local disruptions rather than global stability.Here we investigate the roles of RNA stability in CYT-19-mediated unfolding of the Tetrahymena ribozyme by using a series of ribozyme mutants with disruptions of each of the five peripheral tertiary contacts. We observe a strong correlation between CYT-19 activity and global stability of ribozyme tertiary structure. Further, we find that the RNA-dependent ATPase activity of CYT-19 depends on the accessibility of secondary structure in the ribozyme. Our results lead to a general model for recognition and remodeling of unstable or incorrectly folded RNAs by a DEAD-box protein.