The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

The anticancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is an anticancer and anti-inflammatory compound and its active form, compound 2, directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-messenger RNA (pre-mRNA) 3' processing. Surprisingly, compound 2-mediated inhibition of pre-mRNA cleavage is sequence specific and the drug sensitivity is predominantly determined by sequences flanking the cleavage site (CS). Using massively parallel in vitro assays, we identified key sequence features that determine drug sensitivity. We trained a machine learning model that can predict poly(A) site (PAS) relative sensitivity to compound 2 and provide the molecular basis for understanding the impact of JTE-607 on PAS selection and transcription termination genome wide. We propose that CPSF73 and associated factors bind to the CS region in a sequence-dependent manner and the interaction affinity determines compound 2 sensitivity. These results have not only elucidated the mechanism of action of JTE-607, but also unveiled an evolutionarily conserved sequence specificity of the mRNA 3' processing machinery.

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 °C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 °C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2- mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. As the Compound 2-resistant CS sequences, characterized by U/A-rich motifs, are prevalent in PASs from yeast to human, the CS region sequence may have more fundamental functions beyond determining drug resistance. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown and evolutionarily conserved sequence-specificity of the mRNA 3' processing machinery.

Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch.

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer's P1 helix and the expression platform's transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

Riboswitches have challenged our understanding of biological regulation for almost two decades. The ability of small molecules to bind to RNA and control gene expression offers another layer of regulation and the potential for direct action by compounds in the environment. While some riboswitches have been well studied, we lack a general understanding of how changes in RNA structure switch genetic expression from "On" to "Off". In this study, the authors propose an elegant "strand displacement" model to explain how the RNA structure shifts between "On" and "Off" states as the concentration of small molecule ligand changes. These observations help us to understand how riboswitches enable genetic decision-making. The data provide a possible general mechanism for understanding how the competition between different strand displacement outcomes can influence RNA folding. Understanding RNA folding pathways could advance the successful design of drugs that target RNA.

Computationally reconstructing cotranscriptional RNA folding from experimental data reveals rearrangement of non-native folding intermediates.

The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data.

MOTIVATION: RNA molecules can undergo complex structural dynamics, especially during transcription, which influence their biological functions. Recently developed high-throughput chemical probing experiments that study RNA cotranscriptional folding generate nucleotide-resolution 'reactivities' for each length of a growing nascent RNA that reflect structural dynamics. However, the manual annotation and qualitative interpretation of reactivity across these large datasets can be nuanced, laborious, and difficult for new practitioners. We developed a quantitative and systematic approach to automatically detect RNA folding events from these datasets to reduce human bias/error, standardize event discovery and generate hypotheses about RNA folding trajectories for further analysis and experimental validation. RESULTS: Detection of Unknown Events with Tunable Thresholds (DUETT) identifies RNA structural transitions in cotranscriptional RNA chemical probing datasets. DUETT employs a feedback control-inspired method and a linear regression approach and relies on interpretable and independently tunable parameter thresholds to match qualitative user expectations with quantitatively identified folding events. We validate the approach by identifying known RNA structural transitions within the cotranscriptional folding pathways of the Escherichia coli signal recognition particle RNA and the Bacillus cereus crcB fluoride riboswitch. We identify previously overlooked features of these datasets such as heightened reactivity patterns in the signal recognition particle RNA about 12 nt lengths before base-pair rearrangement. We then apply a sensitivity analysis to identify tradeoffs when choosing parameter thresholds. Finally, we show that DUETT is tunable across a wide range of contexts, enabling flexible application to study broad classes of RNA folding mechanisms. AVAILABILITY AND IMPLEMENTATION: https://github.com/BagheriLab/DUETT. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SnapShot: RNA Structure Probing Technologies.

Chemical probing coupled to high-throughput sequencing offers a flexible approach to uncover many aspects of RNA structure relevant to its cellular function. With a wide variety of chemical probes available that each report on different features of RNA molecules, a broad toolkit exists for investigating in vivo and in vitro RNA structure and interactions with other molecules.

High-throughput determination of RNA structures.

RNA performs and regulates a diverse range of cellular processes, with new functional roles being uncovered at a rapid pace. Interest is growing in how these functions are linked to RNA structures that form in the complex cellular environment. A growing suite of technologies that use advances in RNA structural probes, high-throughput sequencing and new computational approaches to interrogate RNA structure at unprecedented throughput are beginning to provide insights into RNA structures at new spatial, temporal and cellular scales.

Cotranscriptional folding of a riboswitch at nucleotide resolution.

RNAs can begin to fold immediately as they emerge from RNA polymerase. During cotranscriptional folding, interactions between nascent RNAs and ligands are able to direct the formation of alternative RNA structures, a feature exploited by noncoding RNAs called riboswitches to make gene-regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extended selective 2'-hydroxyl acylation analyzed by primer-extension sequencing (SHAPE-seq) to measure structural information of nascent RNAs during transcription. Using cotranscriptional SHAPE-seq, we determined how the cotranscriptional folding pathway of the Bacillus cereus crcB fluoride riboswitch undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.

Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).

RNA molecules adopt a wide variety of structures that perform many cellular functions, including, among others, catalysis, small molecule sensing, and cellular defense. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally, we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

Nanopore sequencing in microgravity.

Rapid DNA sequencing and analysis has been a long-sought goal in remote research and point-of-care medicine. In microgravity, DNA sequencing can facilitate novel astrobiological research and close monitoring of crew health, but spaceflight places stringent restrictions on the mass and volume of instruments, crew operation time, and instrument functionality. The recent emergence of portable, nanopore-based tools with streamlined sample preparation protocols finally enables DNA sequencing on missions in microgravity. As a first step toward sequencing in space and aboard the International Space Station (ISS), we tested the Oxford Nanopore Technologies MinION during a parabolic flight to understand the effects of variable gravity on the instrument and data. In a successful proof-of-principle experiment, we found that the instrument generated DNA reads over the course of the flight, including the first ever sequenced in microgravity, and additional reads measured after the flight concluded its parabolas. Here we detail modifications to the sample-loading procedures to facilitate nanopore sequencing aboard the ISS and in other microgravity environments. We also evaluate existing analysis methods and outline two new approaches, the first based on a wave-fingerprint method and the second on entropy signal mapping. Computationally light analysis methods offer the potential for in situ species identification, but are limited by the error profiles (stays, skips, and mismatches) of older nanopore data. Higher accuracies attainable with modified sample processing methods and the latest version of flow cells will further enable the use of nanopore sequencers for diagnostics and research in space.

The SLE transcriptome exhibits evidence of chronic endotoxin exposure and has widespread dysregulation of non-coding and coding RNAs.

BACKGROUND: Gene expression studies of peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) have demonstrated a type I interferon signature and increased expression of inflammatory cytokine genes. Studies of patients with Aicardi Goutières syndrome, commonly cited as a single gene model for SLE, have suggested that accumulation of non-coding RNAs may drive some of the pathologic gene expression, however, no RNA sequencing studies of SLE patients have been performed. This study was designed to define altered expression of coding and non-coding RNAs and to detect globally altered RNA processing in SLE. METHODS: Purified monocytes from eight healthy age/gender matched controls and nine SLE patients (with low-moderate disease activity and lack of biologic drug use or immune suppressive treatment) were studied using RNA-seq. Quantitative RT-PCR was used to validate findings. Serum levels of endotoxin were measured by ELISA. RESULTS: We found that SLE patients had diminished expression of most endogenous retroviruses and small nucleolar RNAs, but exhibited increased expression of pri-miRNAs. Splicing patterns and polyadenylation were significantly altered. In addition, SLE monocytes expressed novel transcripts, an effect that was replicated by LPS treatment of control monocytes. We further identified increased circulating endotoxin in SLE patients. CONCLUSIONS: Monocytes from SLE patients exhibit globally dysregulated gene expression. The transcriptome is not simply altered by the transcriptional activation of a set of genes, but is qualitatively different in SLE. The identification of novel loci, inducible by LPS, suggests that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism.

Efficient digest of high-throughput sequencing data in a reproducible report.

BACKGROUND: High-throughput sequencing (HTS) technologies are spearheading the accelerated development of biomedical research. Processing and summarizing the large amount of data generated by HTS presents a non-trivial challenge to bioinformatics. A commonly adopted standard is to store sequencing reads aligned to a reference genome in SAM (Sequence Alignment/Map) or BAM (Binary Alignment/Map) files. Quality control of SAM/BAM files is a critical checkpoint before downstream analysis. The goal of the current project is to facilitate and standardize this process. RESULTS: We developed bamchop, a robust program to efficiently summarize key statistical metrics of HTS data stored in BAM files, and to visually present the results in a formatted report. The report documents information about various aspects of HTS data, such as sequencing quality, mapping to a reference genome, sequencing coverage, and base frequency. Bamchop uses the R language and Bioconductor packages to calculate statistical matrices and the Sweave utility and associated LaTeX markup for documentation. Bamchop's efficiency and robustness were tested on BAM files generated by local sequencing facilities and the 1000 Genomes Project. Source code, instruction and example reports of bamchop are freely available from https://github.com/CBMi-BiG/bamchop. CONCLUSIONS: Bamchop enables biomedical researchers to quickly and rigorously evaluate HTS data by providing a convenient synopsis and user-friendly reports.