A potential long-range RNA-RNA interaction in the HIV-1 RNA.

It is well-established that viral and cellular mRNAs alike harbour functional long-range intra-molecular RNA-RNA interactions. Despite the biological importance of such interactions, their identification and characterization remain challenging. Here we present a computational method for the identification of certain kinds of long-range intra-molecular RNA-RNA interactions involving the loop nucleotides of a hairpin loop. Using the computational method, we analysed 4272 HIV-1 genomic mRNAs. A potential long-range intra-molecular RNA-RNA interaction within the HIV-1 genomic RNA was identified. The long-range interaction is mediated by a kissing loop structure between two stem-loops of the previously reported SHAPE-based secondary structure of the entire HIV-1 genome. Structural modelling studies were carried out to show that the kissing loop structure not only is sterically feasible, but also contains a conserved RNA structural motif often found in compact RNA pseudoknots. The computational method should be generally applicable to the identification of potential long-range intra-molecular RNA-RNA interactions in any viral or cellular mRNA sequence.Communicated by Ramaswamy H. Sarma.

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of "multiple" functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

Assembly of Two-Dimensional DNA Arrays Could Influence the Formation of Their Component Tiles.

Tile-based DNA self-assembly is a powerful approach for nano-constructions. In this approach, individual DNA single strands first assemble into well-defined structural tiles, which, then, further associate with each other into final nanostructures. It is a general assumption that the lower-level structures (tiles) determine the higher-level, final structures. In this study, we present concrete experimental data to show that higher-level structures could, at least in the current example, also impact on the formation of lower-level structures. This study prompts questions such as: how general is this phenomenon in programmed DNA self-assembly and can we turn it into a useful tool for fine tuning DNA self-assembly?

Tolerance of Senecavirus A to Mutations in Its Kissing-Loop or Pseudoknot Structure Computationally Predicted in 3' Untranslated Region.

Senecavirus A (SVA) is an emerging virus that belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense and single-stranded RNA, containing two untranslated regions (UTRs). The 68-nt-long 3' UTR is computationally predicted to possess two higher-order RNA structures: a kissing-loop interaction and an H-type-like pseudoknot, both of which, however, cannot coexist in the 3' UTR. In this study, we constructed 17 full-length SVA cDNA clones (cD-1 to -17): the cD-1 to -7 contained different point mutations in a kissing-loop-forming motif (KLFM); the cD-8 to -17 harbored one single or multiple point mutations in a pseudoknot-forming motif (PFM). These 17 mutated cDNA clones were independently transfected into BSR-T7/5 cells for rescuing recombinant SVAs (rSVAs), named rSVA-1 to -17, corresponding to cD-1 to -17. The results showed that the rSVA-1, -2, -3, -4, -5, -6, -7, -9, -13, and -15 were successfully rescued from their individual cDNA clones. Moreover, all mutated motifs were genetically stable during 10 viral passages in vitro. This study unveiled viral abilities of tolerating mutations in the computationally predicted KLFM or PFMs. It can be concluded that the putative kissing-loop structure, even if present in the 3' UTR, is unnecessary for SVA replication. Alternatively, if the pseudoknot formation potentially occurs in the 3' UTR, its deformation would have a lethal effect on SVA propagation.

Structural Landscape of the Transition from an ssDNA Dumbbell Plus Its Complementary Hairpin to a dsDNA Microcircle Via a Kissing Loop Intermediate.

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

Dengue virus strain 2 capsid protein switches the annealing pathway and reduces intrinsic dynamics of the conserved 5' untranslated region.

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

Aminoacylation of short hairpin RNAs through kissing-loop interactions indicates evolutionary trend of RNA molecules.

The unique G3:U70 base pair in the acceptor stem of tRNAAla has been shown to be a critical recognition site by alanyl-tRNA synthetase (AlaRS). The base pair resides on one of the arms of the L-shaped structure of tRNA (minihelix) and the genetic code has likely evolved from a primordial tRNA-aaRS (aminoacyl-tRNA synthetase) system. In terms of the evolution of tRNA, incorporation of a G:U base pair in the structure would be important. Here, we found that two independent short hairpin RNAs change their conformation through kissing-loop interactions, finally forming a minihelix-like structure, in which the G3:U70 base pair is incorporated. The RNA system can be properly aminoacylated by the minimal Escherichia coli AlaRS variant with alanylation activity (AlaRS442N). Thus, characteristic structural features produced via kissing-loop interactions may provide important clues into the evolution of RNA.

Chromosome Territorial Organization Drives Efficient Protein Complex Formation: A Hypothesis.

In eukaryotes, chromosomes often form a transcriptional kissing loop during interphase. We propose that these kissing loops facilitate the formation of protein complexes. mRNA transcripts from these loops could cluster together into phase-separated nuclear granules. Their export into the ER could be ensured by guided diffusion through the inter-chromatin space followed by association with nuclear baskets and export factors. Inside the ER, these mRNAs would form a translation hub. Juxtaposed translation of these mRNAs would increase the cis/trans protein complex assembly among the nascent protein chains. Eukaryotes might employ this pathway to increase complex formation efficiency.

Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy.

Viroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg2+ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg2+ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.

Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation.

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, and , the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in , but not in , despite the absence of a substrate-binding site in . Loop-loop interactions of - and -variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.

Thermodynamic investigation of kissing-loop interactions.

Kissing loop interactions (KLIs) are a common motif that is critical in retroviral dimerization, viroid replication, mRNA, and riboswitches. In addition, KLIs are currently used in a variety of biotechnology applications, such as in aptamer sensors, RNA scaffolds and to stabilize vaccines for therapeutics. Here we describe the thermodynamics of a basic intramolecular DNA capable of engaging in a KLI, consisting of two hairpins connected by a flexible linker. Each hairpin loop has a five-nucleotide complementary sequence theoretically capable of engaging in a KLI. On either side of each loop is two thymines which will not engage in kissing but are present to provide more flexibility and optimal KLI positioning. Our results suggest that the KLI occurs even at physiological salt levels, and that the KLI does not alter the thermodynamics and stability of the two stem structures. The KLI does not involve all five nucleotides, or at least each base-pair stack is not making full contact. Adding a second strand complementary to the bottom of the kissing complex removes flexibility and causes destabilization of the stems. The KLI of this less flexible complex is maintained but the TM is reduced, indicating an entopic penalty to its formation.

The 3' mRNA I-shaped structure of maize necrotic streak virus binds to eukaryotic translation factors for eIF4F-mediated translation initiation.

Unlike the mRNAs of their eukaryotic hosts, many RNAs of viruses lack a 5' m7GpppN cap and the 3' polyadenosine tail, and yet they are translated efficiently. Plant RNA viruses, in particular, have complex structures within their mRNA UTRs that allow them to bypass some cellular translation control steps. In the 3' UTR of maize necrotic streak virus (MNeSV), an I-shaped RNA structure (ISS) has been shown to bind eukaryotic initiation factor (eIF)4F and to mediate viral translation initiation. A 5'-3' RNA "kissing-loop" interaction is required for optimal translation. However, the details of how the 3' ISS mediates translation initiation are not well understood. Here, we studied the binding of the 3' ISS with eIFs. The eIF4A-eIF4B complex was found to increase binding affinity of eIF4F with the 3' ISS by 4-fold (from KD = 173 ± 34 nm to KD = 48 ± 11 nm). Pre-steady-state analysis indicated that the eIF4A-eIF4B complex increased the RNA association rate and decreased the dissociation rate in an ATP-independent manner. Furthermore, our findings suggest that eIF4F could promote binding of the 3' ISS with the MNeSV 5'UTR, enhancing the long-distance kissing-loop interaction. However, the association of the 5'UTR with the 3' ISS-eIF4F complex did not increase 40S ribosomal subunit binding affinity. These quantitative results suggest a stepwise model in which the first committed step is eIF4F binding to the 3' ISS, followed by an interaction with the 5'UTR and subsequent 40S ribosomal subunit binding.

Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid.

Apple hammerhead viroid-like RNA (AHVd RNA) has been reported in different apple cultivars and geographic regions and, considering the presence of hammerhead ribozymes in both polarity strands, suspected to be either a viroid of the family Avsunviroidae or a viroid-like satellite RNA. Here we report that dimeric head-to-tail in vitro transcripts of a 433-nt reference variant of AHVd RNA from cultivar "Pacific Gala" are infectious when mechanically inoculated to apple, thus showing that this RNA is a bona fide viroid for which we have kept the name apple hammerhead viroid (AHVd) until its pathogenicity, if any, is better assessed. By combining thermodynamics-based predictions with co-variation analyses of the natural genetic diversity found in AHVd we have inferred the most likely conformations for both AHVd polarity strands in vivo, with that of the (+) polarity strand being stabilized by a kissing loop-interaction similar to those reported in peach latent mosaic viroid and chrysathemum chlorotic mottle viroid, the two known members of the genus Pelamoviroid (family Avsunviroidae). Therefore, AHVd RNA fulfills the biological and molecular criteria to be allocated to this genus, the members of which, intriguingly, display low global sequence identity but high structural conservation.

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life.

R3C ligase ribozyme catalyzes the nucleophilic attack by a 3'-hydroxyl on a 5'-α-phosphorus of triphosphates to form a 3'-5'-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a "switch" to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world.

Structural basis for ligand binding to the guanidine-II riboswitch.

The guanidine-II riboswitch, also known as mini-ykkC, is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem-loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cations and functions as a translationally controlled on-switch. Here we report the structure of a P2 stem-loop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1.57 Å resolution. The hairpins dimerize via the conserved tetraloop, which also contains the binding pocket. Two guanidinium molecules bind near the dimerization interface, one in each tetraloop. The guanidinium cation is engaged in extensive hydrogen bonding to the RNA. Contacts include the Hoogsteen face of a guanine base and three nonbridging phosphate oxygens. Cation-π interactions and ionic interactions also stabilize ligand binding. The guanidine-II riboswitch utilizes the same recognition strategies as the guanidine-I riboswitch while adopting an entirely different and much smaller RNA fold.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

A trifunctional, triangular RNA-protein complex.

Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA-protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single triangular RNP via RNA kissing loop (KL) interactions. This RNP-based approach can be used as an alternative tool to produce unique, multifunctional molecules with customized dimensions, functions, and targets.

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

Installation of orthogonality to the interface that assembles two modular domains in the Tetrahymena group I ribozyme.

Two modular elements (P5abc and ΔP5) in the Tetrahymena group I ribozyme can be separated physically to generate a two-piece ribozyme derivative consisting of a separately prepared P5abc (P5 RNA) and the rest of the intron (ΔP5 RNA). Molecular recognition in the interface assembling P5 RNA and ΔP5 RNA is strong and specific, and the catalytic ability of the two-piece ribozyme is comparable to that of the parent unimolecular ribozyme. We designed alternative P14 (L5c-L2) interacting modules participating in the assembly of P5 and ΔP5 and investigated their ability in the context of complex formation of the two-piece ribozyme and in vivo splicing of the unimolecular intron ribozyme. Combined use of alternative P14 and L5b-P6 interacting modules provided robust orthogonality to the P5/ΔP5 assembly interface of the bimolecular complex.

Direct evidence for RNA-RNA interactions at the 3' end of the Hepatitis C virus genome using surface plasmon resonance.

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3' end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.