A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM.

Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution.

Structures illustrate step-by-step mitochondrial transcription initiation.

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.

Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells.

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

The human SKI complex regulates channeling of ribosome-bound RNA to the exosome via an intrinsic gatekeeping mechanism.

The superkiller (SKI) complex is the cytoplasmic co-factor and regulator of the RNA-degrading exosome. In human cells, the SKI complex functions mainly in co-translational surveillance-decay pathways, and its malfunction is linked to a severe congenital disorder, the trichohepatoenteric syndrome. To obtain insights into the molecular mechanisms regulating the human SKI (hSKI) complex, we structurally characterized several of its functional states in the context of 80S ribosomes and substrate RNA. In a prehydrolytic ATP form, the hSKI complex exhibits a closed conformation with an inherent gating system that effectively traps the 80S-bound RNA into the hSKI2 helicase subunit. When active, hSKI switches to an open conformation in which the gating is released and the RNA 3' end exits the helicase. The emerging picture is that the gatekeeping mechanism and architectural remodeling of hSKI underpin a regulated RNA channeling system that is mechanistically conserved among the cytoplasmic and nuclear helicase-exosome complexes.

Nanobase.org: a repository for DNA and RNA nanostructures.

We introduce a new online database of nucleic acid nanostructures for the field of DNA and RNA nanotechnology. The database implements an upload interface, searching and database browsing. Each deposited nanostructures includes an image of the nanostructure, design file, an optional 3D view, and additional metadata such as experimental data, protocol or literature reference. The database accepts nanostructures in any preferred format used by the uploader for the nanostructure design. We further provide a set of conversion tools that encourage design file conversion into common formats (oxDNA and PDB) that can be used for setting up simulations, interactive editing or 3D visualization. The aim of the repository is to provide to the DNA/RNA nanotechnology community a resource for sharing their designs for further reuse in other systems and also to function as an archive of the designs that have been achieved in the field so far. Nanobase.org is available at https://nanobase.org/.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

Geometric deep learning of RNA structure.

RNA molecules adopt three-dimensional structures that are critical to their function and of interest in drug discovery. Few RNA structures are known, however, and predicting them computationally has proven challenging. We introduce a machine learning approach that enables identification of accurate structural models without assumptions about their defining characteristics, despite being trained with only 18 known RNA structures. The resulting scoring function, the Atomic Rotationally Equivariant Scorer (ARES), substantially outperforms previous methods and consistently produces the best results in community-wide blind RNA structure prediction challenges. By learning effectively even from a small amount of data, our approach overcomes a major limitation of standard deep neural networks. Because it uses only atomic coordinates as inputs and incorporates no RNA-specific information, this approach is applicable to diverse problems in structural biology, chemistry, materials science, and beyond.

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

Context-sensitivity of isosteric substitutions of non-Watson-Crick basepairs in recurrent RNA 3D motifs.

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils.

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

Structures of telomerase at several steps of telomere repeat synthesis.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.

Structure of human telomerase holoenzyme with bound telomeric DNA.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication1. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease2. A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture3, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.

Detecting protein and DNA/RNA structures in cryo-EM maps of intermediate resolution using deep learning.

An increasing number of density maps of macromolecular structures, including proteins and DNA/RNA complexes, have been determined by cryo-electron microscopy (cryo-EM). Although lately maps at a near-atomic resolution are routinely reported, there are still substantial fractions of maps determined at intermediate or low resolutions, where extracting structure information is not trivial. Here, we report a new computational method, Emap2sec+, which identifies DNA or RNA as well as the secondary structures of proteins in cryo-EM maps of 5 to 10 Å resolution. Emap2sec+ employs the deep Residual convolutional neural network. Emap2sec+ assigns structural labels with associated probabilities at each voxel in a cryo-EM map, which will help structure modeling in an EM map. Emap2sec+ showed stable and high assignment accuracy for nucleotides in low resolution maps and improved performance for protein secondary structure assignments than its earlier version when tested on simulated and experimental maps.

Dynamic Molecular Epidemiology Reveals Lineage-Associated Single-Nucleotide Variants That Alter RNA Structure in Chikungunya Virus.

Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.

Amino Acid Composition in Various Types of Nucleic Acid-Binding Proteins.

Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

A structurally conserved human and Tetrahymena telomerase catalytic core.

Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN-TRAP complex, can interact in a conserved manner to regulate telomere synthesis.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5'-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3'-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

In situ imaging of intracellular human telomerase RNA with molecular beacon-functionalized gold nanoparticles.

Since the expression level of human telomerase RNA (hTR) in tumor cells is much higher than that in normal cells, the determination of hTR is of prime importance in biological research of tumors. In this work, we report molecular beacon-functionalized gold nanoparticles for hTR imaging in live cells. The molecular beacon has a loop-and-stem structure with hTR recognition sequences and a red fluorophore Cy5. In the presence of hTR, the hTR sequence could be hybridized with the loop of molecular beacon to form a duplex DNA chain and thus the fluorescence state switched from "off" to "on". After co-incubation with cells, the probe could readily permeate into cells, leading to the in situ imaging of intracellular hTR. The proposed approach could be used to differentiate tumor cells from normal ones and assess hTR expression levels in different tumor cells. Furthermore, the proposed approach allowed us to dynamically monitor the expression level of hTR in live cells and holds great potential for application in tumor diagnosis and hTR-related drug delivery.

Genetic Characterization of Three Distinct Mechanisms Supporting RNA-Driven DNA Repair and Modification Reveals Major Role of DNA Polymerase ζ.

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.