Templated 3' terminal fluorescent labeling of RNA using Klenow DNA polymerase.

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3' end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:•Klenow DNA polymerase adds an amino-modified nucleotide to the 3' end of the RNA, guided by the DNA oligonucleotide template.•An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.•For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structures. This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3' terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.

A Newly Identified Peripheral Duplex Anchors and Stabilizes the MALAT1 Triplex.

The accumulation of the 8-kb oncogenic long noncoding MALAT1 RNA in cells is dependent on the presence of a protective triple helix structure at the 3' terminus. While recent studies have examined the functional importance of numerous base triples within the triplex and its immediately adjacent base pairs, the functional importance of peripheral duplex elements has not been thoroughly investigated. To investigate the functional importance of a peripheral linker region that was previously described as unstructured, we employed a variety of assays including thermal melting, protection from exonucleolytic degradation by RNase R, small-angle X-ray scattering, biochemical ligation and binding assays, and computational modeling. Our results demonstrate the presence of a duplex within this linker that enhances the functional stability of the triplex in vitro, despite its location more than 40 Å from the 3' terminus. We present a full-length model of the MALAT1 triple helix-containing RNA having an extended rod-like structure and comprising 33 layers of coaxial stacking interactions. Taken together with recent research on a homologous triplex, our results demonstrate that peripheral elements anchor and stabilize triplexes in vitro. Such peripheral elements may also contribute to the formation and stability of some triple helices in vivo.

A highly ordered, nonprotective MALAT1 ENE structure is adopted prior to triplex formation.

The 3' end of the ∼7 kb lncRNA MALAT1 contains an evolutionarily and structurally conserved element for nuclear expression (ENE) which confers protection from cellular degradation pathways. Formation of an ENE triple helix is required to support transcript accumulation, leading to persistent oncogenic activity of MALAT1 in multiple cancer types. Though the specific mechanism of triplex-mediated protection remains unknown, the MALAT1 ENE triplex has been identified as a promising target for therapeutic intervention. Interestingly, a maturation step of the nascent lncRNA 3' end is required prior to triplex formation. We hypothesize that disruption of the maturation or folding process may be a viable mechanism of inhibition. To assess putative cotranscriptional ENE conformations prior to triplex formation, we perform microsecond MD simulations of a partially folded ENE conformation and the ENE triplex. We identify a highly ordered ENE structure prior to triplex formation. Extensive formation of U•U base pairs within the large U-rich internal loops produces a global rod-like architecture. We present a three-dimensional structure of the isolated ENE motif, the global features of which are consistent with small angle X-ray scattering (SAXS) experiments. Our structural model represents a nonprotective conformation of the MALAT1 ENE, providing a molecular description useful for future mechanistic and inhibition studies. We anticipate that targeting stretches of U•U pairs within the ENE motif will prove advantageous for the design of therapeutics targeting this oncogenic lncRNA.

Fluorescence-based investigations of RNA-small molecule interactions.

Interrogating non-coding RNA structures and functions with small molecules is an area of rapidly increasing interest among biochemists and chemical biologists. However, many biochemical approaches to monitoring RNA structures are time-consuming and low-throughput, and thereby are only of limited utility for RNA-small molecule studies. Fluorescence-based techniques are powerful tools for rapid investigation of RNA conformations, dynamics, and interactions with small molecules. Many fluorescence methods are amenable to high-throughput analysis, enabling library screening for small molecule binders. In this review, we summarize numerous fluorescence-based approaches for identifying and characterizing RNA-small molecule interactions. We describe in detail a high-information content dual-reporter FRET assay we developed to characterize small molecule-induced conformational and stability changes. Our assay is uniquely suited as a platform for both small molecule discovery and thorough characterization of RNA-small molecule binding mechanisms. Given the growing recognition of non-coding RNAs as attractive targets for therapeutic intervention, we anticipate our FRET assay and other fluorescence-based techniques will be indispensable for the development of potent and specific small molecule inhibitors targeting RNA.

Finely tuned conformational dynamics regulate the protective function of the lncRNA MALAT1 triple helix.

Nucleic acid triplexes may regulate many important biological processes. Persistent accumulation of the oncogenic 7-kb long noncoding RNA MALAT1 is dependent on an unusually long intramolecular triple helix. This triplex structure is positioned within a conserved ENE (element for nuclear expression) motif at the lncRNA 3' terminus and protects the entire transcript from degradation in a polyA-independent manner. A requisite 3' maturation step leads to triplex formation though the precise mechanism of triplex folding remains unclear. Furthermore, the contributions of several peripheral structural elements to triplex formation and protective function have not been determined. We evaluated the stability, conformational fluctuations, and function of this MALAT1 ENE triple helix (M1TH) protective element using in vitro mutational analyses coupled with biochemical and biophysical characterizations. Using fluorescence and UV melts, FRET, and an exonucleolytic decay assay we define a concerted mechanism for triplex formation and uncover a metastable, dynamic triplex population under near-physiological conditions. Structural elements surrounding the triplex regulate the dynamic M1TH conformational variability, but increased triplex dynamics lead to M1TH degradation. Taken together, we suggest that finely tuned dynamics may be a general mechanism regulating triplex-mediated functions.

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes.

The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.

Structures to the people!

A combination of 3D modeling and high-throughput sequencing may offer a faster way to determine the three-dimensional structures of RNA molecules.

Analysis of riboswitch structure and ligand binding using small-angle X-ray scattering (SAXS).

Small-angle X-ray scattering (SAXS) is a powerful tool for examining the global conformation of riboswitches in solution, and how this is modulated by binding of divalent cations and small molecule ligands. SAXS experiments, which typically require only minutes per sample, directly yield two quantities describing the size and shape of the RNA: the radius of gyration (Rg) and the maximum linear dimension (Dmax). Examination of these quantities can reveal if a riboswitch undergoes cation-induced compaction. Comparison of the Rg and Dmax values between samples containing different concentrations of ligand reveals the overall structural response of the riboswitch to ligand. The Kratky plot (a graphical representation that emphasizes the higher-resolution SAXS data) and the P(r) plot or pair-probability distribution (an indirect Fourier transform, or power spectrum of the data) can provide additional evidence of riboswitch conformational changes. Simulation methods have been developed for generating three-dimensional reconstructions consistent with the one-dimensional SAXS data. These low-resolution molecular envelopes can aid in deciphering the relative helical arrangement within the RNA.

Modulation of quaternary structure and enhancement of ligand binding by the K-turn of tandem glycine riboswitches.

Most known glycine riboswitches have two homologous aptamer domains arranged in tandem and separated by a short linker. The two aptamers associate through reciprocal "quaternary" interactions that have been proposed to result in cooperative glycine binding. Recently, the interaptamer linker was found to form helix P0 with a previously unrecognized segment 5' to the first aptamer domain. P0 was shown to increase glycine affinity, abolish cooperativity, and conform to the K-turn motif consensus. We examine the global thermodynamic and structural role of P0 using isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS), respectively. To evaluate the generality of P0 function, we prepared glycine riboswitch constructs lacking and including P0 from Bacillus subtilis, Fusobacterium nucleatum, and Vibrio cholerae. We find that P0 indeed folds into a K-turn, supports partial pre-folding of all three glycine-free RNAs, and is required for ITC observation of glycine binding under physiologic Mg(2+) concentrations. Except for the unusually small riboswitch from F. nucleatum, the K-turn is needed for maximally compacting the glycine-bound states of the RNAs. Formation of a ribonucleoprotein complex between the B. subtilis or the F. nucleatum RNA constructs and the bacterial K-turn binding protein YbxF promotes additional folding of the free riboswitch, and enhances glycine binding. Consistent with the previously reported loss of cooperativity, P0-containing B. subtilis and V. cholerae tandem aptamers bound no more than one glycine molecule per riboswitch. Our results indicate that the P0 K-turn helps organize the quaternary structure of tandem glycine riboswitches, thereby facilitating ligand binding under physiologic conditions.

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops.

The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined.

Discrete structure of an RNA folding intermediate revealed by cryo-electron microscopy.

RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.

Riboswitch function: flipping the switch or tuning the dimmer?

Riboswitches are structured mRNA elements involved in gene regulation that respond to the intracellular concentration of specific small molecules. Binding of their cognate ligand is thought to elicit a global conformational change of the riboswitch, in addition to modulating the fine structure of the binding site. X-ray crystallography has produced detailed descriptions of the three-dimensional structures of the ligand-bound conformations of several riboswitches. We have employed small-angle X-ray scattering (SAXS) to generate low-resolution reconstructions of the ligand-free states of the ligand-binding domains of riboswitches that respond to thiamine pyrophosphate (TPP), and cyclic diguanylate (c-di-GMP), a bacterial second messenger. Comparison of the SAXS reconstructions with the crystal structures of these two riboswitches demonstrates that the RNAs undergo dramatic ligand-induced global conformational changes. However, this is not an universal feature of riboswitches. SAXS analysis of the solution behavior of several other riboswitch ligand-binding domains demonstrates a broad spectrum of conformational switching behaviors, ranging from the unambiguous switching of the TPP and c-di-GMP riboswitches to complete lack of switching for the flavin mononucleotide (FMN) riboswitch. Moreover, the switching behavior varies between examples of the same riboswitch from different organisms. The range of observed behaviors suggests that in response to the evolutionary need for precise genetic regulation, riboswitches may be tuned to function more as dimmers or rheostats than binary on/off switches.

Extended structures in RNA folding intermediates are due to nonnative interactions rather than electrostatic repulsion.

RNA folding occurs via a series of transitions between metastable intermediate states for Mg(2+) concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.

Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis.

Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.

Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch.

The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

Folding of a universal ribozyme: the ribonuclease P RNA.

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.

Structural basis for altering the stability of homologous RNAs from a mesophilic and a thermophilic bacterium.

Tertiary RNA structures from thermophilic bacteria generally are more stable than their mesophilic homologs. To understand the structural basis of the increase in stability, we investigated equilibrium folding of the specificity domain (S-domain) of RNase P RNA from a mesophilic (Escherichia coli) and a thermophilic (Thermus thermophilus) bacterium. Equilibrium folding of both S-domains is described by a minimal, three-state folding scheme, U-to-I-to-N. In the I-to-N transition of the thermophilic S-domain, more structure forms and protections are stronger against T1 nuclease and hydroxyl radical reactions. Phylogenetic comparison in the context of the native structure reveals that among 39 nucleotide differences between these S-domains, 12 likely contribute to higher stability. These residues participate in extensive networks of hydrogen bonding, stacking, and metal ion coordination throughout the molecule. The thermophilic S-domain achieves higher stability by mutating strategic base pairs to G-C, decreasing surface accessibility of the native state, and increasing the amount of structure formation in the native folding transition. An E. coli S-domain mutant containing these 12 nt has the same stability and folding cooperativity as the T. thermophilus S-domain. E. coli S-domain mutants containing a subset of 4 or 6 nt have the same stability as the T. thermophilus S-domain but the same folding cooperativity as the E. coli S-domain. These results show that increasing stability can be accomplished by mutations within a local structure, but increasing folding cooperativity needs concerted changes among multiple structural units.

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding.

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.