Toward a molecular understanding of RNA remodeling by DEAD-box proteins

Russell, Rick; Jarmoskaite, Inga; Lambowitz, Alan M.

doi:10.4161/rna.22210

Cited by 86 publications

(76 citation statements)

References 149 publications

(231 reference statements)

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“…An alternative model postulates that the two phases reflect a rapid internal equilibration of the chemical steps of splicing, followed by a slower, irreversible step. However, we do not favor this model because inefficient folding has been demonstrated for other group II intron RNAs under similar conditions (35) and because of additional results at lower Mg 2+ concentrations (discussed below). Together these two phases accounted for splicing of 70-80% of the precursor in different assays.…”

Section: Splicing Of Wild-type and Variant Intron Rnas At Different Mmentioning

confidence: 99%

“…For DV RNA in isolation or in early folding intermediates of the intron, the conformational change is likely to be rapid and reversible. However, upon global collapse of the intron and encapsulation by DI, the transition may become very slow and divide the RNA into active and inactive populations, which give rise to the multiple phases observed in group II intron splicing reactions at low Mg 2+ concentrations (35). By stabilizing the native, bent conformation of DV, the mutations could increase the fraction of the RNA that readily folds to the native state and gives activity.…”

Section: Reverse Splicing Of Wild-type and Variant Intron Rnas At Difmentioning

confidence: 99%

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Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Truong

Sidote

Russell

et al. 2013

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

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Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg 2+ concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg 2+ transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg 2+ concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg 2+ concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg 2+ binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg 2+ -dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg 2+ concentrations, with implications for evolution and potential applications in gene targeting. directed evolution | Mg 2+ transport | RNA structure M obile group II introns are retrotransposons that are found in prokaryotes and the mitochondrial and chloroplast DNAs of some eukaryotes and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retrotransposons, and retroviruses in higher organisms (1). They consist of two components-an autocatalytic intron RNA ("ribozyme") and an intron-encoded protein (IEP) with reverse transcriptase activity-that function together in a ribonucleoprotein (RNP) complex to promote RNA splicing and site-specific integration of the intron into new DNA sites in a process called retrohoming (1). Like spliceosomal introns, group II introns splice via two sequential transesterification reactions that yield an excised intron lariat RNA (2). For group II introns, the splicing reactions are catalyzed by the intron RNA with the assistance of the IEP, which binds specifically to the intron RNA and stabilizes the catalytically active RNA structure. The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes retrohoming via reverse splicing of the intron RNA directly into DNA sites followed by reverse transcription by the IEP. The resulting intron cDNA is integrated into the genome by host enzymes (3, 4). The ribozyme-based, site-specific DNA integration mechanism used by g...

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Section: Splicing Of Wild-type and Variant Intron Rnas At Different Mmentioning

confidence: 99%

Section: Reverse Splicing Of Wild-type and Variant Intron Rnas At Difmentioning

confidence: 99%

Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Truong

Sidote

Russell

et al. 2013

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

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“…ATP-dependent DEAD-box RNA helicases have been implicated in almost every aspect of RNA metabolism including transcription, mRNA and tRNA processing, protein synthesis, RNA nuclear export, and RNA degradation, and certain DEAD-box helicases have been shown to facilitate splicing and translation of a variety of RNAs in vivo and in vitro (Russell et al 2013;. DEAD-box proteins exhibit little substrate specificity in vitro, but are usually found in large macromolecular complexes in vivo, such as spliceosomes and degradosomes, which direct their activities to specific pathways.…”

Section: Introductionmentioning

confidence: 99%

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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“…In fact, a common property of this family of enzymes is that their ATPase activity is stimulated by RNA. 6 Indeed, many DEAD-box proteins have been shown to bind cooperatively RNA and adenosine nucleotides, which ultimately results in enhanced ATPase kinetics. 6 CONCR may act as an oncogene.…”

mentioning

confidence: 99%

“…6 Indeed, many DEAD-box proteins have been shown to bind cooperatively RNA and adenosine nucleotides, which ultimately results in enhanced ATPase kinetics. 6 CONCR may act as an oncogene. It is transcriptionally controlled by c-MYC and presents higher levels in tumor cells with inactive p53.…”

mentioning

confidence: 99%