Discovery of a trefoil knot in the RydC RNA: Challenging previous notions of RNA topology.

Knots are very common in polymers, including DNA and protein molecules. Yet, no genuine knot has been identified in natural RNA molecules to date. Upon re-examining experimentally determined RNA 3D structures, we discovered a trefoil knot 31, the most basic non-trivial knot, in the RydC RNA. This knotted RNA is a member of a small family of short bacterial RNAs, whose secondary structure is characterized by an H-type pseudoknot. Molecular dynamics simulations suggest a folding pathway of the RydC RNA that starts with a native twisted loop. Based on sequence analyses and computational RNA 3D structure predictions, we postulate that this trefoil knot is a conserved feature of all RydC-related RNAs. The first discovery of a knot in a natural RNA molecule introduces a novel perspective on RNA 3D structure formation and on fundamental research on the relationship between function and spatial structure of biopolymers.

RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.

3D feasibility of 2D RNA-RNA interaction paths by stepwise folding simulations.

The structure of an RNA, and even more so its interactions with other RNAs, provide valuable information about its function. Secondary structure-based tools for RNA-RNA interaction predictions provide a quick way to identify possible interaction targets and structures. However, these tools ignore the effect of steric hindrance on the tertiary (3D) structure level, and do not consider whether a suitable folding pathway exists to form the interaction. As a consequence, these tools often predict interactions that are unrealistically long and could be formed (in three dimensions) only by going through highly entangled intermediates. Here, we present a computational pipeline to assess whether a proposed secondary (2D) structure interaction is sterically feasible and reachable along a plausible folding pathway. To this end, we simulate the folding of a series of 3D structures along a given 2D folding path. To avoid the complexity of large-scale atomic resolution simulations, our pipeline uses coarse-grained 3D modeling and breaks up the folding path into small steps, each corresponding to the extension of the interaction by 1 or 2 bp. We apply our pipeline to analyze RNA-RNA interaction formation for three selected RNA-RNA complexes. We find that kissing hairpins, in contrast to interactions in the exterior loop, are difficult to extend and tend to get stuck at an interaction length of 6 bp. Our tool, including source code, documentation, and sample data, is available at www.github.com/irenekb/RRI-3D.

Dynamic RNA synthetic biology: new principles, practices and potential.

An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent advances in engineering dynamic RNA systems across the molecular, circuit and cellular scales for important societal-scale applications in environmental and human health, and bioproduction. For each scale, we introduce the core concepts of dynamic RNA folding and function at that scale, and then discuss technologies incorporating these concepts, covering new approaches to engineering riboswitches, ribozymes, RNA origami, RNA strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles and synthetic cells. Considering the dynamic nature of RNA within the engineering design process promises to spark the next wave of innovation that will expand the scope and impact of RNA biotechnologies.

A Hitchhiker's guide to RNA-RNA structure and interaction prediction tools.

RNA biology has risen to prominence after a remarkable discovery of diverse functions of noncoding RNA (ncRNA). Most untranslated transcripts often exert their regulatory functions into RNA-RNA complexes via base pairing with complementary sequences in other RNAs. An interplay between RNAs is essential, as it possesses various functional roles in human cells, including genetic translation, RNA splicing, editing, ribosomal RNA maturation, RNA degradation and the regulation of metabolic pathways/riboswitches. Moreover, the pervasive transcription of the human genome allows for the discovery of novel genomic functions via RNA interactome investigation. The advancement of experimental procedures has resulted in an explosion of documented data, necessitating the development of efficient and precise computational tools and algorithms. This review provides an extensive update on RNA-RNA interaction (RRI) analysis via thermodynamic- and comparative-based RNA secondary structure prediction (RSP) and RNA-RNA interaction prediction (RIP) tools and their general functions. We also highlighted the current knowledge of RRIs and the limitations of RNA interactome mapping via experimental data. Then, the gap between RSP and RIP, the importance of RNA homologues, the relationship between pseudoknots, and RNA folding thermodynamics are discussed. It is hoped that these emerging prediction tools will deepen the understanding of RNA-associated interactions in human diseases and hasten treatment processes.

Structural and dynamic mechanisms for coupled folding and tRNA recognition of a translational T-box riboswitch.

T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg2+-induced global folding and tRNA binding are cooperatively coupled. smFRET measurements reveal that high Mg2+ stabilizes IIA/B and its stacking on II, which drives the pre-docking of I and II into a competent conformation, subsequent tRNA binding promotes docking of I and II to form a high-affinity tRNA binding groove, of which the essentiality of IIA/B and S-turn in II is substantiated with mutational analysis. We highlight a delicate balance among Mg2+, the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.

Modified RNAs and predictions with the ViennaRNA Package.

MOTIVATION: In living organisms, many RNA molecules are modified post-transcriptionally. This turns the widely known four-letter RNA alphabet ACGU into a much larger one with currently more than 300 known distinct modified bases. The roles for the majority of modified bases remain uncertain, but many are already well-known for their ability to influence the preferred structures that an RNA may adopt. In fact, tRNAs sometimes require certain modifications to fold into their cloverleaf shaped structure. However, predicting the structure of RNAs with base modifications is still difficult due to the lack of efficient algorithms that can deal with the extended sequence alphabet, as well as missing parameter sets that account for the changes in stability induced by the modified bases. RESULTS: We present an approach to include sparse energy parameter data for modified bases into the ViennaRNA Package. Our method does not require any changes to the underlying efficient algorithms but instead uses a set of plug-in constraints that adapt the predictions in terms of loop evaluation at runtime. These adaptations are efficient in the sense that they are only performed for loops where additional parameters are actually available for. In addition, our approach also facilitates the inclusion of more modified bases as soon as further parameters become available. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are available at https://www.tbi.univie.ac.at/RNA.

Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.

Co-transcriptional assembly mechanisms of protein-RNA complexes.

This graphical review provides a mechanistic overview of different molecular processes that are tightly coupled and cooperate to achieve efficient and spatial-temporally regulated co-transcriptional protein-RNA complex assembly, including co-transcriptional RNA folding, processing, modification and the assembly in context of biomolecular condensates.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

Counting Distinguishable RNA Secondary Structures.

RNA secondary structures are essential abstractions for understanding spacial folding behaviors of those macromolecules. Many secondary structure algorithms involve a common dynamic programming setup to exploit the property that secondary structures can be decomposed into substructures. Dirks et al. noted that this setup cannot directly address an issue of distinguishability among secondary structures, which arises for classes of sequences that admit nontrivial symmetry. Circular sequences are among these. We examine the problem of counting distinguishable secondary structures. Drawing from elementary results in group theory, we identify useful subsets of secondary structures. We then extend an algorithm due to Hofacker et al. for computing the sizes of these subsets. This yields a cubic-time algorithm to count distinguishable structures compatible with a given circular sequence. Furthermore, this general approach may be used to solve similar problems for which the RNA structures of interest involve symmetries.

Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

DrForna: visualization of cotranscriptional folding.

MOTIVATION: Understanding RNA folding at the level of secondary structures can give important insights concerning the function of a molecule. We are interested to learn how secondary structures change dynamically during transcription, as well as whether particular secondary structures form already during or only after transcription. While different approaches exist to simulate cotranscriptional folding, the current strategies for visualization are lagging behind. New, more suitable approaches are necessary to help with exploring the generated data from cotranscriptional folding simulations. RESULTS: We present DrForna, an interactive visualization app for viewing the time course of a cotranscriptional RNA folding simulation. Specifically, users can scroll along the time axis and see the population of structures that are present at any particular time point. AVAILABILITY AND IMPLEMENTATION: DrForna is a JavaScript project available on Github at https://github.com/ViennaRNA/drforna and deployed at https://viennarna.github.io/drforna.

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life.

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Here, we detail next-generation sequencing (NGS) experiments performed in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Notably, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.

Local RNA folding revisited.

Most of the functional RNA elements located within large transcripts are local. Local folding therefore serves a practically useful approximation to global structure prediction. Due to the sensitivity of RNA secondary structure prediction to the exact definition of sequence ends, accuracy can be increased by averaging local structure predictions over multiple, overlapping sequence windows. These averages can be computed efficiently by dynamic programming. Here we revisit the local folding problem, present a concise mathematical formalization that generalizes previous approaches and show that correct Boltzmann samples can be obtained by local stochastic backtracing in McCaskill's algorithms but not from local folding recursions. Corresponding new features are implemented in the ViennaRNA package to improve the support of local folding. Applications include the computation of maximum expected accuracy structures from RNAplfold data and a mutual information measure to quantify the sensitivity of individual sequence positions.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

A transient intermediate RNA structure underlies the regulatory function of the E. coli thiB TPP translational riboswitch.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation by studying the Escherichia coli thiB thiamin pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the antisequestering stem that could mediate the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the antisequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the antisequestering stem break or bias thiB function according to whether the antisequestering stem or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

Using 5' 32 P-labeled Primer and Reverse Transcription to Probe RNA Structure.

RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. RNA chemical mapping strategies to elucidate RNA structural changes involve using chemical reagents that form adducts or cleave RNA. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) measures RNA flexibility by modification of the 2' hydroxyl groups of flexible nucleotides. These RNA adducts can be detected using 32 P-labeled primers and reverse transcription (RT) followed by PAGE analysis. This strategy can reveal the base-paired regions of the RNA and provide insight into tertiary structure and solvent accessibility. This protocol provides a method to interrogate RNA structure using furoyl acylimidazole (FAI). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Reverse transcription (RT) primer labeling with 32 P radionuclide Basic Protocol 2: Characterization of RNA structure with radiolabeled primer and reverse transcription (RT).

Pervasive RNA folding is crucial for narnavirus genome maintenance.

A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.

RNA folding pathways from all-atom simulations with a variationally improved history-dependent bias.

Atomically detailed simulations of RNA folding have proven very challenging in view of the difficulties of developing realistic force fields and the intrinsic computational complexity of sampling rare conformational transitions. As a step forward in tackling these issues, we extend to RNA an enhanced path-sampling method previously successfully applied to proteins. In this scheme, the information about the RNA's native structure is harnessed by a soft history-dependent biasing force promoting the generation of productive folding trajectories in an all-atom force field with explicit solvent. A rigorous variational principle is then applied to minimize the effect of the bias. Here, we report on an application of this method to RNA molecules from 20 to 47 nucleotides long and increasing topological complexity. By comparison with analog simulations performed on small proteins with similar size and architecture, we show that the RNA folding landscape is significantly more frustrated, even for relatively small chains with a simple topology. The predicted RNA folding mechanisms are found to be consistent with the available experiments and some of the existing coarse-grained models. Due to its computational performance, this scheme provides a promising platform to efficiently gather atomistic RNA folding trajectories, thus retain the information about the chemical composition of the sequence.