Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

18S rDNA sequence-structure phylogeny of the eukaryotes simultaneously inferred from sequences and their individual secondary structures.

OBJECTIVE: The eukaryotic tree of life has been subject of numerous studies ever since the nineteenth century, with more supergroups and their sister relations being decoded in the last years. In this study, we reconstructed the phylogeny of eukaryotes using complete 18S rDNA sequences and their individual secondary structures simultaneously. After the sequence-structure data was encoded, it was automatically aligned and analyzed using sequence-only as well as sequence-structure approaches. We present overall neighbor-joining trees of 211 eukaryotes as well as the respective profile neighbor-joining trees, which helped to resolve the basal branching pattern. A manually chosen subset was further inspected using neighbor-joining, maximum parsimony, and maximum likelihood analyses. Additionally, the 75 and 100 percent consensus structures of the subset were predicted. RESULTS: All sequence-structure approaches show improvements compared to the respective sequence-only approaches: the average bootstrap support per node of the sequence-structure profile neighbor-joining analyses with 90.3, was higher than the average bootstrap support of the sequence-only profile neighbor-joining analysis with 73.9. Also, the subset analyses using sequence-structure data were better supported. Furthermore, more subgroups of the supergroups were recovered as monophyletic and sister group relations were much more comparable to results as obtained by multi-marker analyses.

Synthetic molecular switches driven by DNA-modifying enzymes.

Taking inspiration from natural systems, in which molecular switches are ubiquitous in the biochemistry regulatory network, we aim to design and construct synthetic molecular switches driven by DNA-modifying enzymes, such as DNA polymerase and nicking endonuclease. The enzymatic treatments on our synthetic DNA constructs controllably switch ON or OFF the sticky end cohesion and in turn cascade to the structural association or disassociation. Here we showcase the concept in multiple DNA nanostructure systems with robust assembly/disassembly performance. The switch mechanisms are first illustrated in minimalist systems with a few DNA strands. Then the ON/OFF switches are realized in complex DNA lattice and origami systems with designated morphological changes responsive to the specific enzymatic treatments.

The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

Interaction of dinuclear Co(III) cylinders with higher-order DNA structures.

Alternative DNA structures play critical roles in fundamental biological processes linked to human diseases. Thus, targeting and stabilizing these structures by specific ligands could affect the progression of cancer and other diseases. Here, we describe, using methods of molecular biophysics, the interactions of two oxidatively locked [Co2L3]6+ cylinders, rac-2 and meso-1, with diverse alternative DNA structures, such as junctions, G quadruplexes, and bulges. This study was motivated by earlier results demonstrating that both Co(III) cylinders exhibit potent and selective activity against cancer cells, accumulate in the nucleus of cancer cells, and prove to be efficient DNA binders. The results show that the bigger cylinder rac-2 stabilizes all DNA structures, while the smaller cylinder meso-1 stabilizes just the Y-shaped three-way junctions. Collectively, the results of this study suggest that the stabilization of alternative DNA structures by Co(III) cylinders investigated in this work might contribute to the mechanism of their biological activity.

5'UTR G-quadruplex structure enhances translation in size dependent manner.

Translation initiation in bacteria is frequently regulated by various structures in the 5' untranslated region (5'UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5'UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5'UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5'UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

The 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor.

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn2+ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb2+-bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb2+ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg2+, Na+, and Pb2+ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb2+ cofactors, including the biologically relevant Mg2+ ion.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site.

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

Characterisation of the Arabidopsis thaliana telomerase TERT-TR complex.

Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

Minimizing Structural Heterogeneity in DNA Self-Assembled Dye Templating via DNA Origami-Tuned Conformations.

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs). Atomic force microscopy confirmed that the HJs are bound to the surface of DOC with tuned IDAs. Dye orientation distributions were determined by combining dipole imaging and super-resolution microscopy. All IDAs led to dye orientations having dispersed distributions along planes perpendicular to the HJ plane, suggesting that stacking occurred between the dye and the neighboring DNA bases. The dye-base stacking interpretation was supported by increasing the size of the core cavity. The narrowest IDA minimizes structural heterogeneity and suggests dye intercalation. A strong correlation is found between the IDA and the orientation of the dye along the HJ plane. These results show that the HJ imposes restrictions on the dye and that the dye-DNA interactions are always present regardless of global conformation. The implications of our results are discussed for the scalability of dye aggregates using DNA self-assembly. Our methodology provides an avenue for the solid-supported single-molecule characterization of molecular assemblies templated on biomolecules─such as DNA and protein templates involved in light-harvesting and catalysis─with tuned conformations and restricted in position and orientation.

Chemical Annealing Restructures RNA for Nanopore Detection.

RNA is a key biochemical marker, yet its chemical instability and complex secondary structure hamper its integration into DNA nanotechnology-based sensing platforms. Relying on the denaturation of the native RNA structure using urea, we show that restructured DNA/RNA hybrids can readily be prepared at room temperature. Using solid-state nanopore sensing, we demonstrate that the structures of our DNA/RNA hybrids conform to the design at the single-molecule level. Employing this chemical annealing procedure, we mitigate RNA self-cleavage, enabling the direct detection of restructured RNA molecules for biosensing applications.

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

RNA Secondary Structure Thermodynamics.

Several different ways to predict RNA secondary structures have been suggested in the literature. Statistical methods, such as those that utilize stochastic context-free grammars (SCFGs), or approaches based on machine learning aim to predict the best representative structure for the underlying ensemble of possible conformations. Their parameters have therefore been trained on larger subsets of well-curated, known secondary structures. Physics-based methods, on the other hand, usually refrain from using optimized parameters. They model secondary structures from loops as individual building blocks which have been assigned a physical property instead: the free energy of the respective loop. Such free energies are either derived from experiments or from mathematical modeling. This rigorous use of physical properties then allows for the application of statistical mechanics to describe the entire state space of RNA secondary structures in terms of equilibrium probabilities. On that basis, and by using efficient algorithms, many more descriptors of the conformational state space of RNA molecules can be derived to investigate and explain the many functions of RNA molecules. Moreover, compared to other methods, physics-based models allow for a much easier extension with other properties that can be measured experimentally. For instance, small molecules or proteins can bind to an RNA and their binding affinity can be assessed experimentally. Under certain conditions, existing RNA secondary structure prediction tools can be used to model this RNA-ligand binding and to eventually shed light on its impact on structure formation and function.

Classified Dynamic Programming in RNA Structure Analysis.

Analysis of the folding space of RNA generally suffers from its exponential size. With classified Dynamic Programming algorithms, it is possible to alleviate this burden and to analyse the folding space of RNA in great depth. Key to classified DP is that the search space is partitioned into classes based on an on-the-fly computed feature. A class-wise evaluation is then used to compute class-wide properties, such as the lowest free energy structure for each class, or aggregate properties, such as the class' probability. In this paper we describe the well-known shape and hishape abstraction of RNA structures, their power to help better understand RNA function and related methods that are based on these abstractions.

Classification and Identification of Non-canonical Base Pairs and Structural Motifs.

The 3D structures of many ribonucleic acid (RNA) loops are characterized by highly organized networks of non-canonical interactions. Multiple computational methods have been developed to annotate structures with those interactions or automatically identify recurrent interaction networks. By contrast, the reverse problem that aims to retrieve the geometry of a look from its sequence or ensemble of interactions remains much less explored. In this chapter, we will describe how to retrieve and build families of conserved structural motifs using their underlying network of non-canonical interactions. Then, we will show how to assign sequence alignments to those families and use the software BayesPairing to build statistical models of structural motifs with their associated sequence alignments. From this model, we will apply BayesPairing to identify in new sequences regions where those loop geometries can occur.

LocARNA 2.0: Versatile Simultaneous Alignment and Folding of RNAs.

Generating accurate alignments of non-coding RNA sequences is indispensable in the quest for understanding RNA function. Nevertheless, aligning RNAs remains a challenging computational task. In the twilight-zone of RNA sequences with low sequence similarity, sequence homologies and compatible, favorable (a priori unknown) structures can be inferred only in dependency of each other. Thus, simultaneous alignment and folding (SA&F) remains the gold-standard of comparative RNA analysis, even if this method is computationally highly demanding. This text introduces to the recent release 2.0 of the software package LocARNA, focusing on its practical application. The package enables versatile, fast and accurate analysis of multiple RNAs. For this purpose, it implements SA&F algorithms in a specific, lightweight flavor that makes them routinely applicable in large scale. Its high performance is achieved by combining ensemble-based sparsification of the structure space and banding strategies. Probabilistic banding strongly improves the performance of LocARNA 2.0 even over previous releases, while simplifying its effective use. Enabling flexible application to various use cases, LocARNA provides tools to globally and locally compare, cluster, and multiply aligned RNAs based on optimization and probabilistic variants of SA&F, which optionally integrate prior knowledge, expressible by anchor and structure constraints.

VarGibbs Usage in the Optimization of Nearest-Neighbor Parameters and Prediction of Melting Temperature of RNA Duplexes.

The nearest-neighbor (NN) model is a general tool for the evaluation for oligonucleotide thermodynamic stability. It is primarily used for the prediction of melting temperatures but has also found use in RNA secondary structure prediction and theoretical models of hybridization kinetics. One of the key problems is to obtain the NN parameters from melting temperatures, and VarGibbs was designed to obtain those parameters directly from melting temperatures. Here we will describe the basic workflow from RNA melting temperatures to NN parameters with the use of VarGibbs. We start by a brief revision of the basic concepts of RNA hybridization and of the NN model and then show how to prepare the data files, run the parameter optimization, and interpret the results.

RNA Secondary Structure Modeling Following the IPANEMAP Workflow.

The structure of RNA molecules and their complexes are crucial for understanding biology at the molecular level. Resolving these structures holds the key to understanding their manifold structure-mediated functions ranging from regulating gene expression to catalyzing biochemical processes. Predicting RNA secondary structure is a prerequisite and a key step to accurately model their three dimensional structure. Although dedicated modelling software are making fast and significant progresses, predicting an accurate secondary structure from the sequence remains a challenge. Their performance can be significantly improved by the incorporation of experimental RNA structure probing data. Many different chemical and enzymatic probes have been developed; however, only one set of quantitative data can be incorporated as constraints for computer-assisted modelling. IPANEMAP is a recent workflow based on RNAfold that can take into account several quantitative or qualitative data sets to model RNA secondary structure. This chapter details the methods for popular chemical probing (DMS, CMCT, SHAPE-CE, and SHAPE-Map) and the subsequent analysis and structure prediction using IPANEMAP.