Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister ribozyme.

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study, we have shown how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a "push-pull" mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate interactions. To further understand the ion atmosphere of RNA in folded and partially folded states results from atomistic umbrella sampling and oscillating chemical potential grand canonical Monte Carlo/molecular dynamics (GCMC/MD) simulations are used to obtain atomic-level details of the distributions of Mg2+ and K+ ions around Twister RNA. Results show the presence of 100 mM Mg2+ to lead to increased charge neutralization over that predicted by counterion condensation theory. Upon going from partially unfolded to folded states, overall charge neutralization increases at all studied ion concentrations that, while associated with an increase in the number of direct ion-phosphate interactions, is fully accounted for by the monovalent K+ ions. Furthermore, K+ preferentially interacts with purine N7 atoms of helical regions in partially unfolded states, thereby potentially stabilizing the helical regions. Thus, both secondary helical structures and formation of tertiary structures leads to increased counterion condensation, thereby stabilizing those structural features of Twister. Notably, it is shown that K+ can act as a surrogate for Mg2+ by participating in specific interactions with nonsequential phosphate pairs that occur in the folded state, explaining the ability of Twister to self-cleave at submillimolar Mg2+ concentrations.

Structure Validation of G-Rich RNAs in Noncoding Regions of the Human Genome.

We present the rapid biophysical characterization of six previously reported putative G-quadruplex-forming RNAs from the 5'-untranslated region (5'-UTR) of silvestrol-sensitive transcripts for investigation of their secondary structures. By NMR and CD spectroscopic analysis, we found that only a single sequence-[AGG]2 [CGG]2 C-folds into a single well-defined G-quadruplex structure. Sequences with longer poly-G strands form unspecific aggregates, whereas CGG-repeat-containing sequences exhibit a temperature-dependent equilibrium between a hairpin and a G-quadruplex structure. The applied experimental strategy is fast and provides robust readout for G-quadruplex-forming capacities of RNA oligomers.

Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent.

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg2+ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg2+ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1 riboswitches in E.coli.

For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands' in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

RNA Cloaking by Reversible Acylation.

We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mm) yields several 2'-OH acylations per RNA strand in as little as 10 min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.

Structural signatures of thermal adaptation of bacterial ribosomal RNA, transfer RNA, and messenger RNA.

Temperature adaptation of bacterial RNAs is a subject of both fundamental and practical interest because it will allow a better understanding of molecular mechanism of RNA folding with potential industrial application of functional thermophilic or psychrophilic RNAs. Here, we performed a comprehensive study of rRNA, tRNA, and mRNA of more than 200 bacterial species with optimal growth temperatures (OGT) ranging from 4°C to 95°C. We investigated temperature adaptation at primary, secondary and tertiary structure levels. We showed that unlike mRNA, tRNA and rRNA were optimized for their structures at compositional levels with significant tertiary structural features even for their corresponding randomly permutated sequences. tRNA and rRNA are more exposed to solvent but remain structured for hyperthermophiles with nearly OGT-independent fluctuation of solvent accessible surface area within a single RNA chain. mRNA in hyperthermophiles is essentially the same as random sequences without tertiary structures although many mRNA in mesophiles and psychrophiles have well-defined tertiary structures based on their low overall solvent exposure with clear separation of deeply buried from partly exposed bases as in tRNA and rRNA. These results provide new insight into temperature adaptation of different RNAs.

Divalent ion competition reveals reorganization of an RNA ion atmosphere upon folding.

Although RNA interactions with K+ and Mg2+ have been studied extensively, much less is known about the third most abundant cation in bacterial cells, putrescine2+, and how RNA folding might be influenced by the three ions in combination. In a new approach, we have observed the competition between Mg2+ and putrescine2+ (in a background of K+) with native, partially unfolded and highly extended conformations of an adenine riboswitch aptamer. With the native state, putrescine2+ is a weak competitor when the ratio of the excess Mg2+ (which neutralizes phosphate charge) to RNA is very low, but becomes much more effective at replacing Mg2+ as the excess Mg2+ in the RNA ion atmosphere increases. Putrescine2+ is even more effective in competing Mg2+ from the extended conformation, independent of the Mg2+ excess. To account for these and other results, we propose that both ions closely approach the surface of RNA secondary structure, but the completely folded RNA tertiary structure develops small pockets of very negative electrostatic potential that are more accessible to the compact charge of Mg2+. The sensitivity of RNA folding to the combination of Mg2+ and putrescine2+ found in vivo depends on the architectures of both the unfolded and native conformations.

Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg2+ concentrations typical of the cell's interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl2 and polyethylene glycol (PEG) concentrations. The resulting phase diagrams show that perturbations to folding by each variable do not overlap. A favorable enthalpy change drives the formation of compact, native-like structures, but requires Mg2+ ions at all temperatures studied (5-55°C). PEG reduces the entropic cost of helix assembly and increases correlations between RNA segments at all temperatures. The phase diagrams also revealed a semi-compact intermediate between the unfolded and folded ensemble that is locally more flexible than the unfolded state, as judged by SHAPE modification. These results suggest that environmental variables such as temperature and solute density will favor different types of RNA structures.

Visualizing the formation of an RNA folding intermediate through a fast highly modular secondary structure switch.

Intermediates play important roles in RNA folding but can be difficult to characterize when short-lived or not significantly populated. By combining (15)N relaxation dispersion NMR with chemical probing, we visualized a fast (kex=k1+k-1≈423 s(-1)) secondary structural switch directed towards a low-populated (∼3%) partially folded intermediate in tertiary folding of the P5abc subdomain of the 'Tetrahymena' group I intron ribozyme. The secondary structure switch changes the base-pairing register across the P5c hairpin, creating a native-like structure, and occurs at rates of more than two orders of magnitude faster than tertiary folding. The switch occurs robustly in the absence of tertiary interactions, Mg(2+) or even when the hairpin is excised from the three-way junction. Fast, highly modular secondary structural switches may be quite common during RNA tertiary folding where they may help smoothen the folding landscape by allowing folding to proceed efficiently via additional pathways.

SB203580 increases G-CSF production via a stem-loop destabilizing element in the 3' untranslated region in macrophages independently of its effect on p38 MAPK activity.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is a major regulator of the production and survival of neutrophils. Regulation of G-CSF expression is complex and occurs at both transcription and post-transcription levels. Two distinct types of cis-acting elements in the 3' untranslated region (3'UTR) of G-CSF mRNA have been identified as destabilizing elements; these consist of adenylate uridylate-rich elements (AUREs) and a stem-loop destabilizing element (SLDE). Regulation of the stability of mRNA by p38 mitogen-activated protein kinase (MAPK) has been indicated to be linked to AUREs in the 3'UTR. However, whether p38 MAPK is involved in the regulation of the stability of G-CSF mRNA has not been elucidated. This study investigated the effect of SB203580, an inhibitor of p38 MAPK, on the lipopolysaccharide-induced G-CSF expression in macrophages at the post-transcription level. RESULTS: Our study showed surprising results that SB203580 augmented the lipopolysaccharide-induced increase in the G-CSF mRNA levels in RAW264.7 mouse macrophages, mouse bone marrow-derived macrophages and in THP-1 human macrophages. This effect was also seen in p38α MAPK knockdown RAW264.7 cells, showing that it was not due to inhibition of p38 MAPK activity. In the presence of actinomycin D, the decay of G-CSF mRNA was slower in SB203580-treated cells than in control cells, showing that SB203580 increased the stability of G-CSF mRNA. Reporter genes containing luciferase with or without the 3'UTR of G-CSF were constructed and transfected into RAW264.7 cells and the results showed that the presence of the 3'UTR reduced the luciferase mRNA levels and luciferase activity. Furthermore, SB203580 increased the luciferase mRNA levels and activity in RAW264.7 cells transfected with the luciferase reporter containing the 3'UTR, but not in cells transfected with the luciferase reporter without the 3'UTR. Mutations of the highly conserved SLDE in the 3'UTR abolished these effects, showing that the SLDE was essential for the SB203580-induced increase in the stability of mRNA. CONCLUSIONS: SB203580 increases G-CSF expression in macrophages by increasing the stability of G-CSF mRNA via its 3'UTR, and the effect was not due to its inhibition of p38 MAPK activity. The results of this study also highlight a potential target for boosting endogenous production of G-CSF during neutropenia.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

Predicting 3D Structure, Flexibility, and Stability of RNA Hairpins in Monovalent and Divalent Ion Solutions.

A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility, and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we further develop the model by improving the implicit-salt electrostatic potential and including a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. The model presented here can predict 3D structures of RNA hairpins with bulges/internal loops (<77 nucleotides) from their sequences at the corresponding experimental ion conditions with an overall improved accuracy compared to the experimental data; the model also makes reliable predictions for the flexibility of RNA hairpins with bulge loops of different lengths at several divalent/monovalent ion conditions. In addition, the model successfully predicts the stability of RNA hairpins with various loops/stems in divalent/monovalent ion solutions.

Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer.

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

Mg(2+) shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection.

Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg(2+) and three ligands of distinct affinities. We show that, in the absence of Mg(2+), both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 µM Mg(2+) generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg(2+)-mediated RNA folding.

Role of lysine binding residues in the global folding of the lysC riboswitch.

Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.

Intramolecular phenotypic capacitance in a modular RNA molecule.

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA's ancient role in life suggests that phenotypic capacitance may have influenced evolution since life's origins.

A Cyclic Mimic of HIV Tat Differentiates Similar TAR RNAs on the Basis of Distinct Dynamic Behaviors.

Efforts toward the development of RNA-based drug leads have been challenging because of the complexity and dynamic nature of RNA structures as therapeutic targets. The transactivation response (TAR) RNA and cognate Tat protein of HIV have long been recognized as promising antiviral targets, and recent works have identified potentially potent inhibitors of the viral RNA-protein interaction. A new class of such inhibitors, conformationally constrained cyclic peptide mimetics of Tat, has been demonstrated to inhibit the HIV life cycle. We have previously probed the complexity and dynamics of TAR RNAs in their free states, as well as conformational shifting by various peptide and small molecule ligands. In this work, we have used an ultrafast dynamics approach to probe the interactions between TAR RNAs and one of the representatives of cyclic peptide inhibitors, L22. Our studies demonstrated that cyclic L22 specifically recognizes TAR RNAs with a unique single binding site compared to two binding sites for linear Tat protein. Although both Tat and L22 bind to the TAR RNAs as a ß-hairpin structure, cyclization in L22 allows it to be a more efficient ligand from a population shifting perspective. This study provided unique insights into drug design with desired properties to differentiate similar structures based on distinct dynamic behaviors.