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RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA. DEAD-box proteins, including Mss116p, were recently found to assist folding of group I and group II introns in vitro and in vivo. The underlying mechanism(s) have been studied extensively to explore the contribution of ATP hydrolysis and duplex unwinding in helicase-stimulated intron splicing. Here we summarize the ongoing efforts to understand the novel role of DEAD-box proteins in RNA folding.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing.

RNA folding is the most essential process underlying RNA function. While significant progress has been made in understanding the forces driving RNA folding in vitro, exploring the rules governing intracellular RNA structure formation is still in its infancy. The cellular environment hosts a great diversity of factors that potentially influence RNA folding in vivo. For example, the nature of transcription and translation is known to shape the folding landscape of RNA molecules. Trans-acting factors such as proteins, RNAs and metabolites, among others, are also able to modulate the structure and thus the fate of an RNA. Here we summarize the ongoing efforts to uncover how RNA folds in living cells.

In yeast mitochondria the DEAD-box helicase Mss116p is essential for respiratory growth by acting as group I and group II intron splicing factor. Here we provide the first structure-based insights into how Mss116p assists RNA folding in vivo. Employing an in vivo chemical probing technique, we mapped the structure of the ai5γ group II intron in different genetic backgrounds to characterize its intracellular fold. While the intron adopts the native conformation in the wt yeast strain, we found that the intron is able to form most of its secondary structure, but lacks its tertiary fold in the absence of Mss116p. This suggests that ai5γ is largely unfolded in the mss116-knockout strain and requires the protein at an early step of folding. Notably, in this unfolded state misfolded substructures have not been observed. As most of the protein-induced conformational changes are located within domain D1, Mss116p appears to facilitate the formation of this largest domain, which is the scaffold for docking of other intron domains. These findings suggest that Mss116p assists the ordered assembly of the ai5γ intron in vivo.

An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5'-AAYAAYAA-3' was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs.

The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified. Employing time-resolved nucleotide analog interference mapping, we have identified a cluster of atoms within the D1 main stem that control the rate constant for D135 collapse. Functional groups within the kappa-zeta element are particularly important for this earliest stage of folding, which is intriguing given that this same motif also serves later as the docking site for catalytic domain 5. More important, the kappa-zeta element is shown to be a divalent ion binding pocket, indicating that this region is a Mg(2+)-dependent switch that initiates the cascade of D135 folding events. By measuring the Mg(2+) dependence of the compaction rate constant, we conclude that the actual rate-limiting step in D1 compaction involves the formation of an unstable folding intermediate that is captured by the binding of Mg(2+). This carefully orchestrated folding pathway, in which formation of an active-site docking region is early and rate limiting, ensures proper folding of the intron core and faithful splicing. It may represent an important paradigm for the folding of large, multidomain RNA molecules.

The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 degrees C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions. We observe that compaction of the RNA proceeds slowly to completion, even at low magnesium concentration (3 mM). Kinetic analysis shows that this compact species is a "near-native" intermediate state that is readily chased into the native state by the addition of high salt. Structural probing reveals that the near-native state represents a compact domain 1 scaffold that is not yet docked with the catalytic domains (D3 and D5). Interestingly, native ribozyme reverts to the near-native state upon reduction in magnesium concentration. Therefore, while the intron can sustain the intermediate state under physiological conditions, the native structure is not maintained and is likely to require stabilization by protein cofactors in vivo.

Group II introns are among the largest ribozymes in nature. They have a highly complex tertiary architecture that enables them to catalyze numerous processes, including self-splicing and transposition reactions that have probably contributed to the evolution of eukaryotic genomes. Biophysical analyses show that, despite their large size, these RNAs can fold to their native state through direct pathways that are populated by structurally defined intermediates. In addition, proteins have specific and important roles in this folding process. As a consequence, the study of the group II introns provides a valuable system for both exploring the driving forces behind the folding of multidomain RNA molecules and investigating ribonucleoprotein assembly.

Ribozymes derived from the group II intron ai5gamma collapse to a compact intermediate, folding to the native state through a slow, direct pathway that is unperturbed by kinetic traps. Molecular collapse of ribozyme D135 requires high magnesium concentrations and is thought to involve a structural element in domain 1 (D1). We used nucleotide analog interference mapping, in combination with nondenaturing gel electrophoresis, to identify RNA substructures and functional groups that are essential for D135 tertiary collapse. This revealed that the most crucial atoms for compaction are located within a small section of D1 that includes the kappa and zeta elements. This small substructure controls specific collapse of the molecule and, in later steps of the folding pathway, it forms the docking site for catalytic D5. In this way, the stage is set for proper active site formation during the earliest steps of ribozyme folding.

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25 degrees C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.

Group I introns consist of two major structural domains, the P4-P6 and P3-P9 domains, which assemble through interactions with peripheral extensions to fold into an active ribozyme. To assess group I intron folding in vivo, we probed the structure of td wild-type and mutant introns using dimethyl sulfate. The results suggest that the majority of the intron population is in the native state in accordance with the current structural model, which was refined to include two novel tertiary contacts. The importance of the loop E motif in the P7.1-P7.2 extension in assisting ribozyme folding was deduced from modeling and mutational analyses. Destabilization of stem P6 results in a deficiency in tertiary structure formation in both major domains, while weakening of stem P7 only interferes with folding of the P3-P9 domain. The different impact of mutations on the tertiary structure suggests that they interfere with folding at different stages. These results provide a first insight into the structure of folding intermediates and suggest a putative order of events in a hierarchical folding pathway in vivo.

RNA folding in vivo is influenced by the cellular environment, the vectorial nature of transcription and translation, trans-acting factors and ion homeostasis. Specific RNA-binding proteins promote RNA folding by stabilizing the native structure or by guiding folding. In contrast, RNA chaperones, which are believed to interact nonspecifically with RNA, were proposed to resolve misfolded RNA structures and to promote intermolecular RNA-RNA annealing. Small trans-acting noncoding RNAs are thought to modulate mRNA structures, thereby regulating gene expression. So far, there is some evidence that in vitro and invivo RNA folding pathways share basic features. However, it is unclear whether the rules deduced from in vitro folding experiments generally apply to invivo conditions.

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One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

RNAs need to adopt a specific architecture to exert their task in cells. While significant progress has been made in describing RNA folding landscapes in vitro, understanding intracellular RNA structure formation is still in its infancy. This is in part due to the complex nature of the cellular environment but also to the limited availability of suitable methodologies. To assess the intracellular structure of large RNAs, we recently applied a chemical probing technique and a metal-induced cleavage assay in vivo. These methods are based on the fact that small molecules, like dimethyl sulfate (DMS), or metal ions, such as Pb(2+), penetrate and spread throughout the cell very fast. Hence, these chemicals are able to modify accessible RNA residues or to induce cleavage of the RNA strand in the vicinity of a metal ion in living cells. Mapping of these incidents allows inferring information on the intracellular conformation, metal ion binding sites or ligand-induced structural changes of the respective RNA molecule. Importantly, in vivo chemical probing can be easily adapted to study RNAs in different cell types.

RNAs have to adopt specific three-dimensional structures to fulfill their biological functions. Therefore exploring RNA structure is of interest to understand RNA-dependent processes. Chemical probing in vitro is a very powerful tool to investigate RNA molecules under a variety of conditions. Among the most frequently used chemical reagents are the nucleobase-specific probes dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) and β-ethoxy-α-ketobutyraldehyde (kethoxal). These chemical reagents modify nucleotides which are not involved in hydrogen bonding or protected by a ligand, such as proteins or metabolites. Upon performing modification reactions with all three chemicals the accessibility of all four nucleobases can be determined. With this fast and inexpensive method local changes in RNA secondary and tertiary structure, as well as the formation of contacts between RNA and its ligands can be detected independent of the RNA's length.

RNAs need to adopt a specific architecture to exert their task in cells. While significant progress has been made in describing RNA folding landscapes in vitro, understanding intracellular RNA structure formation is still in its infancy. This is in part due to the complex nature of the cellular environment but also to the limited availability of suitable methodologies. To assess the intracellular structure of large RNAs, we recently applied a chemical probing technique and a metal-induced cleavage assay in vivo. These methods are based on the fact that small molecules, like dimethyl sulfate (DMS), or metal ions, such as Pb(2+), penetrate and spread throughout the cell very fast. Hence, these chemicals are able to modify accessible RNA residues or to induce cleavage of the RNA strand in the vicinity of a metal ion in living cells. Mapping of these incidents allows inferring information on the intracellular conformation, metal ion binding sites or ligand-induced structural changes of the respective RNA molecule. Importantly, in vivo chemical probing can be easily adapted to study RNAs in different cell types.

RNA folding is the most fundamental process underlying RNA function. RNA structure and associated folding paradigms have been intensively studied in vitro. However, in vivo RNA structure formation has only been explored to a limited extent. To determine the influence of the cellular environment, which differs significantly from the in vitro refolding conditions, on RNA architecture, we have applied a chemical probing technique to assess the structure of catalytic RNAs in living cells. This method is based on the fact that chemicals like dimethyl sulfate readily penetrate cells and modify specific atoms of RNA bases (N1-A, N3-C), provided that these positions are solvent accessible. By mapping the modified residues, one gains substantial information on the architecture of the target RNA on the secondary and tertiary structure level. This method also allows exploration of interactions of the target RNA with ligands such as proteins, metabolites, or other RNA molecules and associated conformational changes. In brief, in vivo chemical probing is a powerful tool to investigate RNA structure in its natural environment and can be easily adapted to study RNAs in different cell types.

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding. RNA functional groups important for this process can be identified by physical isolation of the functional and the nonfunctional RNA molecules and subsequent mapping of the modified nucleotide positions in both RNA populations by iodine cleavage of the susceptible phosphorothioate linkage. This approach has been used to analyze a variety of aspects of RNA biochemistry, including RNA structure, catalysis and ligand interaction. Here, I describe how to set up a NAIM assay for studying RNA folding. This protocol can be readily adapted to study any RNAs and their properties. The time required to complete the experiment is dependent on the length of the RNA and the number of atomic modifications tested. In general, a single NAIM experiment can be completed in 1–2 weeks, but expect a time frame of several weeks to obtain reliable and statistically meaningful results.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as "RNA chaperone activity" and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

Efficient splicing of the td group I intron in vivo is dependent on the ribosome. In the absence of translation, the pre-mRNA is trapped in nonnative-splicing-incompetent conformations. Alternatively, folding of the pre-mRNA can be promoted by the RNA chaperone StpA or by the group I intron-specific splicing factor Cyt-18. To understand the mechanism of action of RNA chaperones, we probed the impact of StpA on the structure of the td intron in vivo. Our data suggest that StpA loosens tertiary interactions. The most prominent structural change was the opening of the base triples, which are involved in the correct orientation of the two major intron core domains. In line with the destabilizing activity of StpA, splicing of mutant introns with a reduced structural stability is sensitive to StpA. In contrast, Cyt-18 strengthens tertiary contacts, thereby rescuing splicing of structurally compromised td mutants in vivo. Our data provide direct evidence for protein-induced conformational changes within catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the overall compactness of the td intron in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA‐protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td ) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 59 and 39 splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 59 splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.