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

Abstract

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.

Characterization of the mammalian DEAD-box protein DDX5 reveals functional conservation with S. cerevisiae ortholog Dbp2 in transcriptional control and glucose metabolism

“…DEAD-box helicases also facilitate duplex annealing, an activity proposed to assist with RNA chaperone activity in vivo (Jarmoskaite and Russell 2014;Ruminski et al 2016). To know whether DDX5, DDX5ΔCTE, and Dbp2 have different annealing activities, we performed annealing assays with the single-stranded components of the 16-bp duplex.…”

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

“…Additionally, the binding of other macromolecules such as proteins could be responsible for alleviating ribozyme inhibition in vivo . Chaperone proteins, for instance, can assist in refolding small self-cleaving ribozymes, contributing to their regulation. − Indeed, the highly variable P3 extension of twister ribozymes ,, could provide an access point for a protein to bind. Furthermore, the presence of metabolites could alter the stability and rate of self-cleavage of the ribozyme.…”

Zooming in on protein–RNA interactions: a multi-level workflow to identify interaction partners

“…Since chemical equilibriums are temperaturedependent, in vivo kinetics may thus significantly differ from those measured in vitro. The thermodynamics of binding can also reveal the energetic landscape of protein-RNA interactions [100][101][102]. It is therefore very important to measure the thermodynamics parameters directly, when experimentally possible, or to calculate them.…”