Insights into the structure and RNA-binding specificity of Caenorhabditis elegans Dicer-related heli
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Li, Kuohan↗
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Results for “structured RNA”
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Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication
Abstract Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 3′ UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.
Akiyama, Benjamin M.↗
A Structural Perspective on the RNA Editing of Plant Respiratory Complexes
The last steps of respiration, a core energy-harvesting process, are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. Several essential subunits of the respiratory complexes are RNA-edited in plants, frequently leading to changes in the encoded amino acids. While the impact of RNA editing is clear at the sequence and phenotypic levels, the underlying biochemical explanations for these effects have remained obscure. Here, we used the structures of plant respiratory complex I, complex III2 and complex IV to analyze the impact of the amino acid changes of RNA editing in terms of their location and biochemical features. Through specific examples, we demonstrate how the structural information can explain the phenotypes of RNA-editing mutants. This work shows how the structural perspective can bridge the gap between sequence and phenotype and provides a framework for the continued analysis of RNA-editing mutants in plant mitochondria and, by extension, in chloroplasts.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser
Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg 2+ , and, more importantly, a putative third (site C) Mg 2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg 2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.
Science & Technology - Other Topics↗
RNA target highlights in CASP15 : Evaluation of predicted models by structure providers
Abstract The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA‐only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x‐ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA–protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.
59 BASIC BIOLOGICAL SCIENCES↗
A conserved viral RNA fold enables nuclease resistance across kingdoms of life
Abstract Viral exoribonuclease-resistant RNA (xrRNA) structures block cellular nucleases to produce subgenomic viral RNAs during infection. High sequence variability among xrRNAs from distantly related viruses raises questions about the shared molecular features that enable these RNAs to withstand the strong unwinding forces of exoribonucleases. Here, we present the first structure of a plant-virus xrRNA in its active conformation and uncover universal principles of xrRNA folding. Comparison with the structure of a human-pathogenic flavivirus xrRNA reveals that both share a core structural motif—a protective ring encircling the RNA’s 5′ end—despite lacking sequence similarity. Disrupting this core motif through targeted mutagenesis eliminates exoribonuclease-resistance and attenuates viral infection. We identify hundreds of related structures across multiple virus families, supporting the conservation of this mechanism. Our study demonstrates how distantly related RNA viruses have converged on a common structural strategy to inhibit cellular nucleases, with a universal ring topology as the defining feature of viral xrRNAs.
Biochemistry & Molecular Biology↗
Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9
Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA ( tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo–electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.
Science & Technology - Other Topics↗
Structure of the T. brucei kinetoplastid RNA editing substrate-binding complex core component, RESC5
Kinetoplastid protists such as Trypanosoma brucei undergo an unusual process of mitochondrial uridine (U) insertion and deletion editing termed kinetoplastid RNA editing (kRNA editing). This extensive form of editing, which is mediated by guide RNAs (gRNAs), can involve the insertion of hundreds of Us and deletion of tens of Us to form a functional mitochondrial mRNA transcript. kRNA editing is catalyzed by the 20 S editosome/RECC. However, gRNA directed, processive editing requires the RNA editing substrate binding complex (RESC), which is comprised of 6 core proteins, RESC1-RESC6. To date there are no structures of RESC proteins or complexes and because RESC proteins show no homology to proteins of known structure, their molecular architecture remains unknown. RESC5 is a key core component in forming the foundation of the RESC complex. To gain insight into the RESC5 protein we performed biochemical and structural studies. We show that RESC5 is monomeric and we report the T . brucei RESC5 crystal structure to 1.95 Å. RESC5 harbors a dimethylarginine dimethylaminohydrolase-like (DDAH) fold. DDAH enzymes hydrolyze methylated arginine residues produced during protein degradation. However, RESC5 is missing two key catalytic DDAH residues and does bind DDAH substrate or product. Implications of the fold for RESC5 function are discussed. This structure provides the first structural view of an RESC protein.
59 BASIC BIOLOGICAL SCIENCES↗
Small-Angle X-ray Scattering (SAXS) Combined with SAXS-Driven Molecular Dynamics for Structural Analysis of Multistranded RNA Assemblies
Not Available
97 MATHEMATICS AND COMPUTING↗
A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure
Viruses require multifunctional structured RNAs to hijack their host’s biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo–electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)–like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.
Science & Technology - Other Topics↗
Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9
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99 GENERAL AND MISCELLANEOUS↗
Structures of artificially designed discrete RNA nanoarchitectures at near-atomic resolution
Although advances in nanotechnology have enabled the construction of complex and functional synthetic nucleic acid–based nanoarchitectures, high-resolution discrete structures are lacking because of the difficulty in obtaining good diffracting crystals. Here, we report the design and construction of RNA nanostructures based on homooligomerizable one-stranded tiles for x-ray crystallographic determination. We solved three structures to near-atomic resolution: a 2D parallelogram, a 3D nanobracelet unexpectedly formed from an RNA designed for a nanocage, and, eventually, a bona fide 3D nanocage designed with the guidance of the two previous structures. Structural details of their constituent motifs, such as kissing loops, branched kissing loops, and T-junctions, that resemble natural RNA motifs and resisted x-ray determination are revealed, providing insights into those natural motifs. This work unveils the largely unexplored potential of crystallography in gaining high-resolution feedback for nanoarchitectural design and suggests a route to investigate RNA motif structures by configuring them into nanoarchitectures.
59 BASIC BIOLOGICAL SCIENCES↗
Did the exposure of coacervate droplets to rain make them the first stable protocells?
Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
A method for achieving complete microbial genomes and improving bins from metagenomics data
Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.
59 BASIC BIOLOGICAL SCIENCES↗
Crystal structure of a cap-independent translation enhancer RNA
In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV 2 ) RNA in complex with our structural chaperone, Fab BL3–6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV 2 tertiary architecture is conserved among these homologs.
59 BASIC BIOLOGICAL SCIENCES↗
RCSB protein data Bank: Next‐generation advanced search for exploration of experimental structures and computed structure models
Abstract The Protein Data Bank (PDB), established in 1971, is the primary global, open‐access archive for experimentally determined 3D macromolecular structures (proteins, RNA, DNA). The research‐focused RCSB.org web‐portal provides access to these data alongside more than one million machine‐learning‐predicted structure models, greatly expanding the available structural landscape. Rapid growth of both experimental and computational structures has increased the need for powerful yet accessible search tools that serve a broad and diverse scientific community. Herein, we describe a redesigned RCSB Protein Data Bank RCSB.org Advanced Search capability that supports intuitive discovery of 3D structures through a unified interface. This interface integrates annotation‐, sequence‐, and 3D structure‐based searches, embeds an interactive 3D viewer, and incorporates curated biological knowledge, such as catalytic site definitions from Mechanism and Catalytic Site Atlas and ligand‐guided structural motifs, for constructing geometry‐driven queries. A new Chemical Search tool allows definition of chemical queries via an integrated drawing tool or standard identifiers, seamlessly combining them with annotation filters. By allowing query definition directly within spatial and chemical contexts, these search interfaces reduce the need for detailed knowledge of residue numbering, chain identifiers, or external cheminformatics software. This capability enables efficient exploration of structures, chemical diversity, and structure–function relationships across all life domains. The redesigned interfaces can be accessed directly at rcsb.org/search/advanced for Advanced Search and rcsb.org/search/chemical for Chemical Search.
Rose, Yana [Research Collaboratory for Structural ↗
Cryo-$\mathrm{EM}$ reveals an entangled kinetic trap in the folding of a catalytic $\mathrm{RNA}$
Functional RNAs fold through complex pathways that can contain misfolded “kinetic traps.” A complete model of RNA folding requires understanding the formation of these misfolded states, but they are difficult to characterize because of their transient and potentially conformationally dynamic nature. We used cryo–electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophilagroup I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.
59 BASIC BIOLOGICAL SCIENCES↗