RNA Structure-function Relationships Research Articles

Bioorthogonal cyclopropenones for investigating RNA structure.

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments. Existing tools primarily rely on electrophiles that are constitutively "on" or triggered by UV light, often resulting in high background reactivity. We developed an alternative, chemically triggered approach to crosslink RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes-electrophiles that can trap neighboring nucleophiles to forge covalent crosslinks. As proof-of-concept, we synthesized a panel of CpOs and appended them to thiazole orange (TO-1). The TO-1 conjugates bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, confirmed by fluorescence turn-on. After phosphine administration, covalent crosslinks were formed between the CpO probes and RNA. The degree of crosslinking was both time and dose-dependent. We further applied the chemically triggered tools to model RNAs in biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

RNAMotifProfile: a graph-based approach to build RNA structural motif profiles.

RNA structural motifs are the recurrent segments in RNA three-dimensional structures that play a crucial role in the functional diversity of RNAs. Understanding the similarities and variations within these recurrent motif groups is essential for gaining insights into RNA structure and function. While recurrent structural motifs are generally assumed to be composed of the same isosteric base interactions, this consistent pattern is not observed across all examples of these motifs. Existing methods for analyzing and comparing RNA structural motifs may overlook variations in base interactions and associated nucleotides. RNAMotifProfile is a novel profile-to-profile alignment algorithm that generates a comprehensive profile from a group of structural motifs, incorporating all base interactions and associated nucleotides at each position. By structurally aligning input motif instances using a guide-tree-based approach, RNAMotifProfile captures the similarities and variations within recurrent motif groups. Additionally, RNAMotifProfile can function as a motif search tool, enabling the identification of instances of a specific motif family by searching with the corresponding profile. The ability to generate accurate and comprehensive profiles for RNA structural motif families, and to search for these motifs, facilitates a deeper understanding of RNA structure-function relationships and potential applications in RNA engineering and therapeutic design.

Mapping the structural landscape of the yeast Ty3 retrotransposon RNA genome.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5' ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.

RNAvigate: efficient exploration of RNA chemical probing datasets.

Chemical probing technologies enable high-throughput examination of diverse structural features of RNA, including local nucleotide flexibility, RNA secondary structure, protein and ligand binding, through-space interaction networks, and multistate structural ensembles. Deep understanding of RNA structure-function relationships typically requires evaluating a system under structure- and function-altering conditions, linking these data with additional information, and visualizing multilayered relationships. Current platforms lack the broad accessibility, flexibility and efficiency needed to iterate on integrative analyses of these diverse, complex data. Here, we share the RNA visualization and graphical analysis toolset RNAvigate, a straightforward and flexible Python library that automatically parses 21 standard file formats (primary sequence annotations, per- and internucleotide data, and secondary and tertiary structures) and outputs 18 plot types. RNAvigate enables efficient exploration of nuanced relationships between multiple layers of RNA structure information and across multiple experimental conditions. Compatibility with Jupyter notebooks enables nonburdensome, reproducible, transparent and organized sharing of multistep analyses and data visualization strategies. RNAvigate simplifies and accelerates discovery and characterization of RNA-centric functions in biology.

RNA structure profiling at single-cell resolution reveals new determinants of cell identity.

RNA structure is critical for multiple steps in gene regulation. However, how the structures of transcripts differ both within and between individual cells is unknown. Here we develop a SHAPE-inspired method called single-cell structure probing of RNA transcripts that enables simultaneous determination of transcript secondary structure and abundance at single-cell resolution. We apply single-cell structure probing of RNA transcripts to human embryonic stem cells and differentiating neurons. Remarkably, RNA structure is more homogeneous in human embryonic stem cells compared with neurons, with the greatest homogeneity found in coding regions. More extensive heterogeneity is found within 3' untranslated regions and is determined by specific RNA-binding proteins. Overall RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. We further discover a cell-type variable region of 18S ribosomal RNA that is associated with cell cycle and translation control. Our method opens the door to the systematic characterization of RNA structure-function relationships at single-cell resolution.

Cellular roadmaps of viroid infection.

Viroids are single-stranded circular noncoding RNAs that infect plants. According to the International Committee on Taxonomy of Viruses, there are 44 viroids known to date. Notably, more than 20 000 distinct viroid-like RNA sequences have recently been identified in existing sequencing datasets, suggesting an unprecedented complexity in biological roles of viroids and viroid-like RNAs. Interestingly, a human pathogen, hepatitis delta virus (HDV), also replicates via a rolling circle mechanism like viroids. Therefore, knowledge of viroid infection is informative for research on HDV and other viroid-like RNAs reported from various organisms. Here, we summarize recent advancements in understanding viroid shuttling among subcellular compartments for completing replication cycles, emphasizing regulatory roles of RNA motifs and structural dynamics in diverse biological processes. We also compare the knowledge of viroid intracellular trafficking with known pathways governing cellular RNA movement in cells. Future investigations on regulatory RNA structures and cognate factors in regulating viroid subcellular trafficking and replication will likely provide new insights into RNA structure-function relationships and facilitate the development of strategies controlling RNA localization and function in cells.

In vivo structure probing of RNA in Archaea: novel insights into the ribosome structure of Methanosarcina acetivorans.

Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.

Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP.

The functional roles of RNAs are often regulated by their structure. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) base reactivity can be employed to investigate the flexibility of nucleotides and correlate it to the constraints imparted by base-pairing and/or protein-binding. In vivo, a multitude of proteins could bind an RNA molecule, regulating its structure and function. Hence, to obtain a more comprehensive view of the RNA structure-function relationship in vivo, it may be required to characterize both the RNA structure and the RNA-protein interaction network. In this chapter, we describe methods for characterizing the in vivo nucleotide flexibility of RNA in cells by SHAPE-MaP (SHAPE by Mutational Profiling) and DMS-MaP. In another chapter, we will discuss the characterization of RNA-protein interaction network by RNP-MaP.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

Auto-DRRAFTER: Automated RNA Modeling Based on Cryo-EM Density.

RNA three-dimensional structures provide rich and vital information for understanding their functions. Recent advances in cryogenic electron microscopy (cryo-EM) allow structure determination of RNAs and ribonucleoprotein (RNP) complexes. However, limited global and local resolutions of RNA cryo-EM mapspose great challenges in tracing RNA coordinates. The Rosetta-based "auto-DRRAFTER" method builds RNA models into moderate-resolution RNA cryo-EM density as part of the Ribosolve pipeline. Here, we describe a step-by-step protocol for auto-DRRAFTER using a glycine riboswitch from Fusobacterium nucleatum as an example. Successful implementation of this protocol allows automated RNA modeling into RNA cryo-EM density, accelerating our understanding of RNA structure-function relationships. Input and output files are being made available at https://github.com/auto-DRRAFTER/springer-chapter .

A viral RNA uses conformational rearrangements of a dynamic tRNA-like structure to hijack cellular machinery

RNA viruses use multifunctional, structural elements in their untranslated regions (UTRs) to hijack cellular machinery, but detailed mechanisms are often difficult to understand due to the dynamic nature of RNA structure. We used cryo-EM to visualize a mysterious 55kDa tRNA-like structure (TLS) at the 3 end of the brome mosaic virus (BMV) genome that is recognized and aminoacylated by cellular tyrosyl-tRNA synthetase (TyrRS) and that plays critical functions in the viral life cycle. Although the BMV TLS has served as a model system of RNA structure-function relationships for decades, the structural nature of its tRNA-mimicry had remained elusive.

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library

Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale.

Structured RNA Contaminants in Bacterial Ribo-Seq.

Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, Ribo-Seq signal can be observed across RNAs that one would not expect to be bound by ribosomes. For example, Escherichia coli Ribo-Seq libraries also capture reads from most noncoding RNAs (ncRNAs). While some of these ncRNAs may overlap coding regions, this alone does not explain the majority of observed signal across ncRNAs. These fragments of ncRNAs in Ribo-Seq data pass all size selection steps of the Ribo-Seq protocol and survive hours of micrococcal nuclease (MNase) treatment. In this work, we specifically focus on Ribo-Seq signal across ncRNAs and provide evidence to suggest that RNA structure, as opposed to ribosome binding, protects them from degradation and allows them to persist in the Ribo-Seq sequencing library preparation. By inspecting these "contaminant reads" in bacterial Ribo-Seq, we show that data previously disregarded in bacterial Ribo-Seq experiments may, in fact, be used to gain partial information regarding the in vivo secondary structure of ncRNAs.IMPORTANCE Structured ncRNAs are pivotal mediators of bioregulation in bacteria, and their functions are often reliant on their specific structures. Here, we first inspect Ribo-Seq reads across noncoding regions, identifying contaminant reads in these libraries. We observe that contaminant reads in bacterial Ribo-Seq experiments that are often disregarded, in fact, strongly overlap with structured regions of ncRNAs. We then perform several bioinformatic analyses to determine why these contaminant reads may persist in Ribo-Seq libraries. Finally, we highlight some structured RNA contaminants in Ribo-Seq and support the hypothesis that structures in the RNA protect them from MNase digestion. We conclude that researchers should be cautious when interpreting Ribo-Seq signal as coding without considering signal distribution. These findings also may enable us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large scale and in vivo through the reanalysis of existing Ribo-Seq data sets.

Long Noncoding RNAs: Molecular Modalities to Organismal Functions.

We have known for decades that long noncoding RNAs (lncRNAs) can play essential functions across most forms of life. The maintenance of chromosome length requires an lncRNA (e.g., hTERC) and two lncRNAs in the ribosome that are required for protein synthesis. Thus, lncRNAs can represent powerful RNA machines. More recently, it has become clear that mammalian genomes encode thousands more lncRNAs. Thus, we raise the question: Which, if any, of these lncRNAs could also represent RNA-based machines? Here we synthesize studies that are beginning to address this question by investigating fundamental properties of lncRNA genes, revealing new insights into the RNA structure-function relationship, determining cis- and trans-acting lncRNAs in vivo, and generating new developments in high-throughput screening used to identify functional lncRNAs. Overall, these findings provide a context toward understanding the molecular grammar underlying lncRNA biology.

Statistical mechanical prediction of ligand perturbation to RNA secondary structure and application to riboswitches.

The realization that noncoding RNA is implicated in numerous cellular processes, makes it imperative to understand and predict RNA-folding. RNA secondary structure prediction is more tractable than tertiary structure or protein structure. Yet insights into RNA structure-function relationships are complicated by coupling between RNA-folding and ligand-binding. Here, perturbations to equilibrium secondary structure conformational distributions for two riboswitches are calculated in the presence of bound cognate ligands. This work incorporates a key factor coupling ligand binding to RNA conformation but not considered in most previous calculations: the differential affinity of the ligand for a range of RNA-folding intermediates. Significant shifts in the free energy landscape (FEL) due to the ligand occur for transcripts of lengths corresponding to the "decision window," following transcription of the so-called anti-terminator helix. The results suggest how ligand perturbation can stabilize the formation of an intermediate conformation, readily facilitating terminator hairpin formation in the full-length riboswitch.

VfoldLA: A web server for loop assembly-based prediction of putative 3D RNA structures

RNA three-dimensional (3D) structures are critical for RNA cellular functions. However, structure prediction for large and complex RNAs remains a challenge, which hampers our understanding of RNA structure-function relationship. We here report a new web server, the VfoldLA server (http://rna.physics.missouri.edu/vfoldLA), for the prediction of RNA 3D structures from nucleotide sequences and base-pair information (2D structure). This server is based on the recently developed VfoldLA, a model that classifies the single-stranded loops (junctions) into four different types and according to the loop-helix connections, assembles RNA 3D structures from the loop/junction templates. The VfoldLA web server provides a user-friendly online interface for a fully automated prediction of putative 3D RNA structures using VfoldLA. With a single-RNA or RNA-RNA complex sequence and 2D structure as input, the server generates structure(s) with the JSmol visualization along with a downloadable PDB file. The output result may serve as useful scaffolds for future structure refinement studies.

StructureFold2: Bringing chemical probing data into the computational fold of RNA structural analysis

The secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure-function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines. Herein, we present StructureFold2, a user-friendly set of analysis tools that makes precise data processing and detailed downstream analyses of such data sets both available and practical. StructureFold2 processes high-throughput reads sequenced from libraries prepared after experimental probing for reverse transcription (RT) stops generated by chemical modification of RNA at solvent accessible residues. This pipeline is able to analyze reads generated from a variety of structure-probing chemicals (e.g. DMS, glyoxal, SHAPE). Notably, StructureFold2 offers a new fully featured suite of utilities and tools to guide a user through multiple types of analyses. A particular emphasis is placed on analyzing the reactivity patterns of transcripts, complementing their use as folding restraints for predicting RNA secondary structure. StructureFold2 is hosted as a Github repository and is available at (https://github.com/StructureFold2/StructureFold2).

A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs

Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. However, when high G analogue:GTP ratios are used to bias G analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine (th G):GTP increased the percent incorporation and yield of 5'-th G-initiated precursor tRNA for a net ≈threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'-th G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

Charting the unknown epitranscriptome.

RNA modifications can alter RNA structure-function relationships and various cellular processes. However, the genomic distribution and biological roles of most RNA modifications remain uncharacterized. Here, we propose using phage display antibody technology and direct sequencing through nanopores to facilitate systematic interrogation of the distribution, location and dynamics of RNA modifications.

Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq.

Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNA structure-function relationships, RNA regulation of gene expression and RNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3' end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNAs are amplified by adapter-based PCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNA structures in ∼1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNA structure-function relationships in living organisms.