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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.

Applying SHAPE-seq to analyze cotranscriptional folding of the B. cereus crcB fluoride riboswitch at nucleotide resolution shows that the folding pathway undergoes a ligand-dependent bifurcation that influences terminator formation via coordinated structural transitions. 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.

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. 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. 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. 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.

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. DNA sequencing in space: Nanopores ready for liftoff Results from the first DNA sequencing experiments performed in microgravity reveal a promising future for portable 'nanopore' devices in space missions. Identifying health issues in outer space is challenging because of a lack of supplies and medical professionals. Researchers led by Christopher Mason from Weill Cornell Medicine in the U.S.A. are creating ways to rapidly monitor astronaut well-being and station health using nucleic acid sequencing. The team sent smartphone-sized sequencers, which detect DNA using current fluctuations as strands pass through protein-based nanopores, onto a low orbit, parabolic flight to evaluate their suitability for upcoming use on the International Space Station (ISS) and for extraterrestrial missions. They found that even under after multiple changes in gravity (G-forces), the nanopores sequenced DNA in microgravity as effectively as an Earth-based device. The authors also detailed two new approaches to DNA fingerprinting and sequence analysis using signal processing algorithms similar to Shazam for finding music. Their experiments show the nanopore-based sequencing can now be applied in missions for DNA diagnostics and detection on the ISS, planetary missions, and beyond.

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. Here the authors characterize the sequence-specific effect of an anticancer compound on mRNA cleavage, providing insights into the mechanism of mRNA 3′ processing.

RNAs perform diverse cellular functions that are mediated at least in part by their structure. However, how RNA structure changes throughout the RNA lifecycle and how these changes affect RNA function remain incompletely understood. A detailed in vivo characterization of RNA structure in various cellular subcompartments now provides insights into how RNA structural changes influence translation, RNA decay, protein binding and RNA modification.

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.

JTE-607 is an anti-cancer and anti-inflammatory compound and its active form Compound 2 directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in 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 series of RNA folding events that occur during transcription can influence the functional roles of cellular RNAs. However, few computational methods generate high-resolution models with the capability of testing mechanistic hypotheses of this ubiquitous process that is important for gene expression and macromolecular assembly. Additionally, cotranscriptional RNA folding experimental data are indirect measurements of the RNA’s structural conformation, at inadequate time scale resolution to capture all structural conformation changes, and/or require experimental manipulation that does not reflect the true process inside of a cell. Despite these technical difficulties, cotranscriptional RNA folding experimental data has elucidated differences in structure and function of several RNAs. The complexity and stochasticity of cotranscriptional RNA folding necessitates reliable and accurate reconstruction of these folding pathways to understand potential cotranscriptional structure underpinnings of RNA function and processing. Merging experimental and computational techniques in developing methods provides an avenue to achieve higher resolution and more accurate structural models. One such experimental method is cotranscriptional SHAPE-seq which collapses 3D structural information and temporal information into a 2D matrix of experimental measurements. Each row of the 2D matrix contains 3D structural information and corresponds to a different intermediate RNA length in the synthesis of the RNA. RNA folding pathways from cotranscriptional SHAPE-seq data had only previously been inferred by human inspection of the experimental data. Therefore, systematic computational methods are necessary to remove human biases as well as discover detailed structural information that is difficult to achieve manually. This thesis details work done towards achieving these goals through development of new computational tools and theory that taken together systematically extract signal from these complex experimental datasets and provide multiscale structural details of cotranscriptional RNA folding pathways, which led to discovery of mechanisms that could be general principles of RNA folding and processing. Overall, these methods provide a powerful strategy for utilizing experimental data to gain deeper insights into cotranscriptional RNA folding.

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 Goutieres 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.