Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p

Mallam, Anna L.; Campo, Mark Del; Gilman, Benjamin; Sidote, David J.; Lambowitz, Alan M.

doi:10.1038/nature11402

Cited by 113 publications

(188 citation statements)

References 41 publications

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“…We focused on Mss116p, a DEAD-box helicase found in yeast mitochondria that has been particularly well characterized in vitro (Huang et al 2005;Tijerina et al 2006;Del Campo et al 2007;Chen et al 2008;Liu et al 2008;Markov et al 2009;Karunatilaka et al 2010;Henn et al 2012;Mallam et al 2012;Russell et al 2013;Pan et al 2014). Our findings are entirely consistent with previous studies of the effects of Mss116p on RNA folding reactions in vitro and suggest that DEAD-box helicase activity might account for the unique discrepancy in the kinetics of secondary structure exchange exhibited between hairpin ribozyme pathways in vitro and in yeast.…”

Section: Introductionsupporting

confidence: 80%

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

Ruminski

Watson

Mahen

et al. 2016

RNA

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RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

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Section: Introductionsupporting

confidence: 80%

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

Ruminski

Watson

Mahen

et al. 2016

RNA

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“…Importantly, even with saturating ATP, the annealing rate constant was higher than that observed in the absence of Rok1 and the end point in helix formation was not affected. These data suggest that ATP changes the structure of Rok1, as observed with other DEADbox proteins, where ATP binding leads to closure of the two RecA domains (33,60,61; reviewed in refs. 1,3,5).…”

Section: A2 A2mentioning

confidence: 56%

“…In contrast, some have the ability to promote duplex formation and to dissociate RNA-protein complexes or function as ATP-dependent RNA-binding proteins (56)(57)(58)(59). The ability to unwind duplexes is thought to arise from the exclusion of dsRNAs when the two RecA domains are closed upon each other (33,60,61; reviewed in refs. 1,3,5).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, not all DEAD-box proteins appear to have unwinding activity (21,55). Unwinding is thought to arise from bending of the bound RNA, which leads to unsticking, and thus weakens the duplex (11,14), and steric exclusion of one strand, when RecA1 docks onto the duplex bound to RecA2 (60). RNA bending is stabilized by interactions with structures formed by the conserved motifs 1b and 1c (11,14), but it also appears to be forced by a loop on the opposite side of the RNA strand, which, otherwise, would be in steric conflict with the RNA (shown in red in SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown recently that the second RecA domain (RecA2) of Mss116 can bind dsRNA by itself (60). Because RecA2 contacts both strands of the duplex, it is expected to stabilize the duplex, thereby helping to explain the annealing activity.…”

Section: A2 A2mentioning

confidence: 99%

See 2 more Smart Citations

Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

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Structural Basis of Human Helicase DDX21 in RNA Binding, Unwinding, and Antiviral Signal Activation

Chen

et al. 2020

Advanced Science

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RNA helicase DDX21 plays vital roles in ribosomal RNA biogenesis, transcription, and the regulation of host innate immunity during virus infection. How DDX21 recognizes and unwinds RNA and how DDX21 interacts with virus remain poorly understood. Here, crystal structures of human DDX21 determined in three distinct states are reported, including the apo‐state, the AMPPNP plus single‐stranded RNA (ssRNA) bound pre‐hydrolysis state, and the ADP‐bound post‐hydrolysis state, revealing an open to closed conformational change upon RNA binding and unwinding. The core of the RNA unwinding machinery of DDX21 includes one wedge helix, one sensor motif V and the DEVD box, which links the binding pockets of ATP and ssRNA. The mutant D339H/E340G dramatically increases RNA binding activity. Moreover, Hill coefficient analysis reveals that DDX21 unwinds double‐stranded RNA (dsRNA) in a cooperative manner. Besides, the nonstructural (NS1) protein of influenza A inhibits the ATPase and unwinding activity of DDX21 via small RNAs, which cooperatively assemble with DDX21 and NS1. The structures illustrate the dynamic process of ATP hydrolysis and RNA unwinding for RNA helicases, and the RNA modulated interaction between NS1 and DDX21 generates a fresh perspective toward the virus–host interface. It would benefit in developing therapeutics to combat the influenza virus infection.

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Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p

Cited by 113 publications

References 41 publications

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

Cofactor-dependent specificity of a DEAD-box protein

Structural Basis of Human Helicase DDX21 in RNA Binding, Unwinding, and Antiviral Signal Activation

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