Global 5'-UTR RNA structure regulates translation of a SERPINA1 mRNA.

SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5'-untranslated region (5'-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5'-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5'-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5'-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5'-UTR structure. However, those mutations that disrupted the helix that anchors the 5'-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5'-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5'-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.

SHAPE-enabled fragment-based ligand discovery for RNA.

The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.

Direct detection of RNA modifications and structure using single-molecule nanopore sequencing.

Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2'-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2'-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing.

Visualization of lncRNA and mRNA Structure Models Within the Integrative Genomics Viewer.

Every class of RNA forms base-paired structures that impact biological functions. Chemical probing of RNA structure, especially with the advent of strategies such as SHAPE-MaP, vastly expands the scale and quantitative accuracy over which RNA structure can be examined. These methods have enabled large-scale structural studies of mRNAs and lncRNAs, but the length and complexity of these RNAs makes interpretation of the data challenging. We have created modules available through the open-source Integrative Genomics Viewer (IGV) for straightforward visualization of RNA structures along with complementary experimental data. Here we present detailed and stepwise strategies for exploring and visualizing complex RNA structures in IGV. Individuals can use these instructions and supplied sample data to become adept at using IGV to visualize RNA structure models in conjunction with useful allied information.

SHAPE Probing Reveals Human rRNAs Are Largely Unfolded in Solution.

Chemical probing experiments, coupled with empirically determined free energy change relationships, can enable accurate modeling of the secondary structures of diverse and complex RNAs. A current frontier lies in modeling large and structurally heterogeneous transcripts, including complex eukaryotic RNAs. To validate and improve on experimentally driven approaches for modeling large transcripts, we obtained high-quality SHAPE data for the protein-free human 18S and 28S ribosomal RNAs (rRNAs). To our surprise, SHAPE-directed structure models for the human rRNAs poorly matched accepted structures. Analysis of predicted rRNA structures based on low-SHAPE and low-entropy (lowSS) metrics revealed that, whereas ∼75% of Escherichia coli rRNA sequences form well-determined lowSS secondary structure, only ∼40% of the human rRNAs do. Critically, regions of the human rRNAs that specifically fold into well-determined lowSS structures were modeled to high accuracy using SHAPE data. This work reveals that eukaryotic rRNAs are more unfolded than are those of prokaryotic rRNAs and indeed are largely unfolded overall, likely reflecting increased protein dependence for eukaryotic ribosome structure. In addition, those regions and substructures that are well-determined can be identified de novo and successfully modeled by SHAPE-directed folding.

Guidelines for SHAPE Reagent Choice and Detection Strategy for RNA Structure Probing Studies.

Chemical probing is an important tool for characterizing the complex folded structures of RNA molecules, many of which play key cellular roles. Electrophilic SHAPE reagents create adducts at the 2'-hydroxyl position on the RNA backbone of flexible ribonucleotides with relatively little dependence on nucleotide identity. Strategies for adduct detection such as mutational profiling (MaP) allow accurate, automated calculation of relative adduct frequencies for each nucleotide in a given RNA or group of RNAs. A number of alternative reagents and adduct detection strategies have been proposed, especially for use in living cells. Here we evaluate five SHAPE reagents: three previously well-validated reagents 1M7 (1-methyl-7-nitroisatoic anhydride), 1M6 (1-methyl-6-nitroisatoic anhydride), and NMIA ( N-methylisatoic anhydride), one more recently proposed NAI (2-methylnicotinic acid imidazolide), and one novel reagent 5NIA (5-nitroisatoic anhydride). We clarify the importance of carefully designed software in reading out SHAPE experiments using massively parallel sequencing approaches. We examine SHAPE modification in living cells in diverse cell lines, compare MaP and reverse transcription-truncation as SHAPE adduct detection strategies, make recommendations for SHAPE reagent choice, and outline areas for future development.

Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing.

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

The roles of five conserved lentiviral RNA structures in HIV-1 replication.

The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.

Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2.

Mutational profiling (MaP) enables detection of sites of chemical modification in RNA as sequence changes during reverse transcription (RT), subsequently read out by massively parallel sequencing. We introduce ShapeMapper 2, which integrates careful handling of all classes of adduct-induced sequence changes, sequence variant correction, basecall quality filters, and quality-control warnings to now identify RNA adduct sites as accurately as achieved by careful manual analysis of electrophoresis data, the prior highest-accuracy standard. MaP and ShapeMapper 2 provide a robust, experimentally concise, and accurate approach for reading out nucleic acid chemical probing experiments.

Visualization of RNA structure models within the Integrative Genomics Viewer.

Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis.

Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

SHAPE analysis of small RNAs and riboswitches.

We describe structural analysis of small RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride, a reagent that detects local nucleotide flexibility; and N-methylisatoic anhydride and 1-methyl-6-nitroisatoic anhydride, reagents which together detect higher-order and noncanonical interactions. Chemical adducts are quantified as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding and to develop highly accurate models of RNA secondary structures.

Single-molecule correlated chemical probing of RNA.

Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP).

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, which makes it difficult to understand how RNA structure governs function. Here we describe selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2'-hydroxyl acylation by SHAPE are encoded as noncomplementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure, and we used it to define a new model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary-structure models, enables analysis of low-abundance RNAs, disentangles sequence polymorphisms in single experiments and will ultimately democratize RNA-structure analysis.

Role of context in RNA structure: flanking sequences reconfigure CAG motif folding in huntingtin exon 1 transcripts.

The length of the CAG-repeat region in the huntingtin mRNA is predictive of Huntington's disease. Structural studies of CAG-repeat-containing RNAs suggest that these sequences form simple hairpin structures; however, in the context of the full-length huntingtin mRNA, CAG repeats may form complex structures that could be targeted for therapeutic intervention. We examined the structures of transcripts spanning the first exon of the huntingtin mRNA with both healthy and disease-prone repeat lengths. In transcripts with 17-70 repeats, the CAG sequences base paired extensively with nucleotides in the 5' UTR and with conserved downstream sequences including a CCG-repeat region. In huntingtin transcripts with healthy numbers of repeats, the previously observed CAG hairpin was either absent or short. In contrast, in transcripts with disease-associated numbers of repeats, a CAG hairpin was present and extended from a three-helix junction. Our findings demonstrate the profound importance of sequence context in RNA folding and identify specific structural differences between healthy and disease-inducing huntingtin alleles that may be targets for therapeutic intervention.

Presynaptic inhibition of gamma-aminobutyric acid release in the bed nucleus of the stria terminalis by kappa opioid receptor signaling.

BACKGROUND: The kappa opioid receptor (KOR) and its endogenous agonist, the neuropeptide dynorphin, are a critical component of the central stress system. Both dynorphin and KOR are expressed in the bed nucleus of the stria terminalis (BNST), a brain region associated with anxiety and stress. This suggests that KOR activation in this region may play a role in the regulation of emotional behaviors. To date, however, there has been no investigation of the ability of KOR to modulate synaptic transmission in the BNST. METHODS: We used whole-cell patch-clamp recordings from acutely prepared mouse brain slices to examine the actions of KOR on inhibitory transmission in the BNST. Additionally, we used neurochemical and pathway-specific optogenetic manipulations to selectively stimulate gamma-aminobutyric acid (GABA)ergic fibers from the central nucleus of the amygdala (CeA) to the BNST. RESULTS: We found that activation of KOR reduced GABAergic transmission through a presynaptic mechanism. Furthermore, we examined the signal transduction pathways that mediate this inhibition and provide the first functional information implicating extracellular signal-regulated kinase in KOR-mediated presynaptic modulation. Moreover, we found that at KOR signaling robustly reduced inhibitory synaptic transmission in the CeA to BNST pathway. CONCLUSIONS: Together, these results demonstrate that KOR provides important inhibitory control over presynaptic GABAergic signaling within the BNST and provides the first direct functional demonstration of KOR-sensitive long-range GABAergic connections between the CeA and the BNST.