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

Abstract

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.RNA folding | RNA misfolding | RNA tertiary structure | RNA unwinding | superfamily 2 helicase D EAD-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 higherorder 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 doublestranded 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 pro...

DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

“…Thus, this mechanism allows DEAD-box proteins to sense RNA stability, leading to preferential action on less stable misfolded intermediates, regardless of specific structural features in the misfolded RNAs, while minimizing activity upon stable, natively folded RNA. Consistent with this view, CYT-19 is activated for ATPase activity to a lower extent by the natively folded wild-type Tetrahymena ribozyme than by less stable mutants, suggesting fewer productive interactions with the more stable structure [47] . A corollary of the model is that groups of cellular RNAs that lack stable tertiary structure, such as mRNAs, are potentially subject to unfolding by DEAD-box proteins.…”

Division of Labor in an Oligomer of the DEAD-Box RNA Helicase Ded1p

“…DEAD-box helicases load directly to the duplex and pry apart a limited number of basepairs in an ATP-dependent fashion (Jarmoskaite et al, 2014; Mallam et al, 2012; Pan et al, 2014; Tijerina et al, 2006; Yang et al, 2007a). The remaining basepairs dissociate non-enzymatically (Chen et al, 2008; Rogers et al, 1999; Yang and Jankowsky, 2006).…”

Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

“…CYT-19 can then unwind the captured helix, further disrupting local structure and exposing more helices for binding. This result was bolstered by the finding that ribozyme mutations that decrease tertiary stability and lead to less compact structures also increase stimulation of CYT-19’s ATPase activity (38). The dependence of CYT-19 activity on exposed duplex structures suggests that DEAD-box proteins act preferentially on nonnative structures that lack the extensive compaction and tertiary contacts found in natively folded RNAs.…”