RNA Internal Loops Research Articles

Knotify+: Toward the Prediction of RNA H-Type Pseudoknots, Including Bulges and Internal Loops.

The accurate "base pairing" in RNA molecules, which leads to the prediction of RNA secondary structures, is crucial in order to explain unknown biological operations. Recently, COVID-19, a widespread disease, has caused many deaths, affecting humanity in an unprecedented way. SARS-CoV-2, a single-stranded RNA virus, has shown the significance of analyzing these molecules and their structures. This paper aims to create a pioneering framework in the direction of predicting specific RNA structures, leveraging syntactic pattern recognition. The proposed framework, Knotify+, addresses the problem of predicting H-type pseudoknots, including bulges and internal loops, by featuring the power of context-free grammar (CFG). We combine the grammar's advantages with maximum base pairing and minimum free energy to tackle this ambiguous task in a performant way. Specifically, our proposed methodology, Knotify+, outperforms state-of-the-art frameworks with regards to its accuracy in core stems prediction. Additionally, it performs more accurately in small sequences and presents a comparable accuracy rate in larger ones, while it requires a smaller execution time compared to well-known platforms. The Knotify+ source code and implementation details are available as a public repository on GitHub.

A small-molecule fluorescence probe ANP77 for sensing RNA internal loop of C, U and A/CC motifs and their binding molecules.

Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam.

Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop.

U6 RNA contains a 1 × 2-nt internal loop that folds and unfold during spliceosomal assembly and activation. The 1 × 2 loop consists of a C67•A79 base pair that forms an additional hydrogen bond upon protonation, C67•A+79, and uracil (U80) that coordinates the catalytically essential magnesium ions. We designed a series of RNA and DNA constructs with a 1 × 2 loop sequence contained in the ISL, and its modifications, to measure the thermodynamic effects of protonation and magnesium binding using UV-visible thermal denaturation experiments. We show that the wild-type RNA construct gains 0.43 kcal/mol in 1 M KCl upon lowering the pH from 7.5 to 5.5; the presence of magnesium ions increases its stability by 2.17 kcal/mol at pH 7.5 over 1 M KCl. Modifications of the helix closing base pairs from C–G to U•G causes a loss in protonation-dependent stability and a decrease in stability in the presence of magnesium ions, especially in the C68U construct. A79G single-nucleotide bulge loop construct showed the largest gain in stability in the presence of magnesium ions. The DNA wild-type construct shows a smaller effect on stability upon lowering the pH and in the presence of magnesium ions, highlighting differences in RNA and DNA structures. A U6 RNA 1 × 2 loop sequence is rare in the databases examined.

Surprising Sequence Effects on GU Closure of Symmetric 2 × 2 Nucleotide RNA Internal Loops.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

The RNA “GAGU” duplex, (5′GACGAGUGUCA)2, contains the internal loop (5′-GAGU-3′)2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans-Watson–Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2′-endo conformation. This differs markedly from the internal loops, (5′-GAGC-3′)2, (5′-AAGU-3′)2, and (5′-UAGG-3′)2, which all have cis-Watson–Crick/Watson–Crick AG “imino” pairs flanked by cis-Watson–Crick/Watson–Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5′-GAGC-3′)2, (5′-AAGU-3′)2, and (5′-UAGG-3′)2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5′-UAGG-3′)2, equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5′-GAGU-3′)2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5′-GAGU-3′)2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed.

Nuclear Magnetic Resonance Structure of an 8 × 8 Nucleotide RNA Internal Loop Flanked on Each Side by Three Watson-Crick Pairs and Comparison to Three-Dimensional Predictions.

The prediction of RNA three-dimensional structure from sequence alone has been a long-standing goal. High-resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the nuclear magnetic resonance structure of the (5'CCAGAAACGGAUGGA)2 duplex, which contains an 8 × 8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5'AC/3'CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, the all-atom root-mean-square deviation for the loop, and the deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.

Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2′-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure.

Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics.

The backbone dihedral parameters of the Amber RNA force field were improved by fitting using multiple linear regression to potential energies determined by quantum chemistry calculations. Five backbone and four glycosidic dihedral parameters were fit simultaneously to reproduce the potential energies determined by a high-level density functional theory calculation (B97D3 functional with the AUG-CC-PVTZ basis set). Umbrella sampling was used to determine conformational free energies along the dihedral angles, and these better agree with the population of conformations observed in the protein data bank for the new parameters than for the conventional parameters. Molecular dynamics simulations performed on a set of hairpin loops, duplexes and tetramers with the new parameter set show improved modeling for the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal loop of noncanonical pairs, as compared to the conventional parameters. For the tetramers, the new parameters largely avoid the incorrect intercalated structures that dominate the conformational samples from the conventional parameters. For the internal loop, the major conformation solved by NMR is stable with the new parameters, but not with the conventional parameters. The new force field performs similarly to the conventional parameters for the UUCG and GCAA hairpin loops and the [U(UA)6A]2 duplex.

Intrinsic Base-Pair Rearrangement in the Hairpin Ribozyme Directs RNA Conformational Sampling and Tertiary Interface Formation.

Dynamic fluctuations in RNA structure enable conformational changes that are required for catalysis and recognition. In the hairpin ribozyme, the catalytically active structure is formed as an intricate tertiary interface between two RNA internal loops. Substantial alterations in the structure of each loop are observed upon interface formation, or docking. The very slow on-rate for this relatively tight interaction has led us to hypothesize a double conformational capture mechanism for RNA-RNA recognition. We used extensive molecular dynamics simulations to assess conformational sampling in the undocked form of the loop domain containing the scissile phosphate (loop A). We observed several major accessible conformations with distinctive patterns of hydrogen bonding and base stacking interactions in the active-site internal loop. Several important conformational features characteristic of the docked state were observed in well-populated substates, consistent with the kinetic sampling of docking-competent states by isolated loop A. Our observations suggest a hybrid or multistage binding mechanism, in which initial conformational selection of a docking-competent state is followed by induced-fit adjustment to an in-line, chemically reactive state only after formation of the initial complex with loop B.

JAR3D Webserver: Scoring and aligning RNA loop sequences to known 3D motifs.

Many non-coding RNAs have been identified and may function by forming 2D and 3D structures. RNA hairpin and internal loops are often represented as unstructured on secondary structure diagrams, but RNA 3D structures show that most such loops are structured by non-Watson–Crick basepairs and base stacking. Moreover, different RNA sequences can form the same RNA 3D motif. JAR3D finds possible 3D geometries for hairpin and internal loops by matching loop sequences to motif groups from the RNA 3D Motif Atlas, by exact sequence match when possible, and by probabilistic scoring and edit distance for novel sequences. The scoring gauges the ability of the sequences to form the same pattern of interactions observed in 3D structures of the motif. The JAR3D webserver at http://rna.bgsu.edu/jar3d/ takes one or many sequences of a single loop as input, or else one or many sequences of longer RNAs with multiple loops. Each sequence is scored against all current motif groups. The output shows the ten best-matching motif groups. Users can align input sequences to each of the motif groups found by JAR3D. JAR3D will be updated with every release of the RNA 3D Motif Atlas, and so its performance is expected to improve over time.

Metal ion binding to an RNA internal loop

Studying the interaction of metal ions with RNA is challenging because of the fast dynamics of the system and the intricate interplay between structural and functional roles of metal ions. NMR spectroscopy is an exceptional tool to investigate such interactions in solution and allows for a detailed description of both metal ion binding sites and binding modes in complex and dynamic RNA structures. We recently applied heteronuclear NMR to study the metal ion binding properties of a three-way junction RNA (D1κζ) which plays an important role in group II intron splicing, and observed metal ion binding in both κ and ζ regions of the construct. Here we concentrate in more detail on the ζ region (D1ζ) using NMR to investigate the interaction with Mg(II), Cd(II) and cobalt(III)hexammine. Our data confirm Cd(II) induced macrochelate formation at the 5′-end triphosphate, suggest an overall similar behaviour for the two divalent metal ions, but with much clearer changes in chemical shifts upon Cd(II) addition, and reveal only little changes upon cobalt(III)hexammine addition, allowing to discriminate between inner- and outer-sphere binding. Moreover, we observed distinct differences when we titrated the sample with Cd(II) in the presence of either KCl or KClO4 as background monovalent salt.

Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA

Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

Computational investigation of RNA CUG repeats responsible for myotonic dystrophy 1.

Myotonic Dystrophy 1 (DM1) is a genetic disease caused by expansion of CTG repeats in DNA. Once transcribed, these repeats form RNA hairpins with repeating 1×1 nucleotide UU internal loop motifs, r(CUG)n, which attract muscleblind-like 1 (MBNL1) protein leading to the disease. In DM1 CUG can be repeated thousands of times, so these structures are intractable to characterization using structural biology. However, inhibition of MBNL1-r(CUG)n binding requires a detailed analysis of the 1×1 UU internal loops. In this contribution we employ regular and umbrella sampling molecular dynamics (MD) simulations to describe the structural and thermodynamic properties of 1×1 UU internal loops. Calculations were run on a reported crystal structure and a designed system, which mimics an infinitely long RNA molecule with continuous CUG repeats. Two-dimensional (2D) potential of mean force (PMF) surfaces were created by umbrella sampling, and the discrete path sampling (DPS) method was utilized to investigate the energy landscape of 1×1 UU RNA internal loops, revealing that 1×1 UU base pairs are dynamic and strongly prefer the anti–anti conformation. Two 2D PMF surfaces were calculated for the 1×1 UU base pairs, revealing several local minima and three syn–anti ↔ anti–anti transformation pathways. Although at room temperature the syn–anti ↔ anti–anti transformation is not observed on the MD time scale, one of these pathways dominates the dynamics of the 1×1 UU base pairs in temperature jump MD simulations. This mechanism has now been treated successfully using the DPS approach. Our results suggest that local minima predicted by umbrella sampling calculations could be stabilized by small molecules, which is of great interest for future drug design. Furthermore, distorted GC/CG conformations may be important in understanding how MBNL1 binds to RNA CUG repeats. Hence we provide new insight into the dynamic roles of RNA loops and their contributions to presently incurable diseases.

Identifying novel sequence variants of RNA 3D motifs.

Predicting RNA 3D structure from sequence is a major challenge in biophysics. An important sub-goal is accurately identifying recurrent 3D motifs from RNA internal and hairpin loop sequences extracted from secondary structure (2D) diagrams. We have developed and validated new probabilistic models for 3D motif sequences based on hybrid Stochastic Context-Free Grammars and Markov Random Fields (SCFG/MRF). The SCFG/MRF models are constructed using atomic-resolution RNA 3D structures. To parameterize each model, we use all instances of each motif found in the RNA 3D Motif Atlas and annotations of pairwise nucleotide interactions generated by the FR3D software. Isostericity relations between non-Watson–Crick basepairs are used in scoring sequence variants. SCFG techniques model nested pairs and insertions, while MRF ideas handle crossing interactions and base triples. We use test sets of randomly-generated sequences to set acceptance and rejection thresholds for each motif group and thus control the false positive rate. Validation was carried out by comparing results for four motif groups to RMDetect. The software developed for sequence scoring (JAR3D) is structured to automatically incorporate new motifs as they accumulate in the RNA 3D Motif Atlas when new structures are solved and is available free for download.

RNA dances to center stage.

RNA dances to center stage.

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle X-ray Scattering

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

Probing a 2-Aminobenzimidazole Library for Binding to RNA Internal Loops via Two-Dimensional Combinatorial Screening

There are many potential RNA drug targets in bacterial, viral, and human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Using modularly assembled ligands to bind RNA internal loops separated by different distances.

Looped in: Modular assembly provides a unique opportunity to tune the affinity and specificity of ligands for a target biomolecule. Herein, we describe our investigations into how the distance between bis-benzimidazole ligand modules (see graphic) affects affinity and specificity for RNA internal loops.