Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing.

RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptome-wide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA.

Assaying RNA structure with LASER-Seq.

Chemical probing methods are crucial to our understanding of the structure and function of RNA molecules. The majority of chemical methods used to probe RNA structure report on Watson-Crick pairing, but tertiary structure parameters such as solvent accessibility can provide an additional layer of structural information, particularly in RNA-protein complexes. Herein we report the development of Light Activated Structural Examination of RNA by high-throughput sequencing, or LASER-Seq, for measuring RNA structure in cells with deep sequencing. LASER relies on a light-generated nicotinoyl nitrenium ion to form covalent adducts with the C8 position of adenosine and guanosine. Reactivity is governed by the accessibility of C8 to the light-generated probe. We compare structure probing by RT-stop and mutational profiling (MaP), demonstrating that LASER can be integrated with both platforms for RNA structure analyses. We find that LASER reactivity correlates with solvent accessibility across the entire ribosome, and that LASER can be used to rapidly survey for ligand binding sites in an unbiased fashion. LASER has a particular advantage in this last application, as it readily modifies paired nucleotides, enabling the identification of binding sites and conformational changes in highly structured RNA.

Light-activated chemical probing of nucleobase solvent accessibility inside cells.

The discovery of functional RNAs that are critical for normal and disease physiology continues to expand at a breakneck pace. Many RNA functions are controlled by the formation of specific structures, and an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, we present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes resulting from ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA-protein interactions and unique intracellular RNA structures. Our photoactivation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in many environments.

Light-activated chemical probing of nucleobase solvent accessibility inside cells.

This corrects the article DOI: 10.1038/nchembio.2548.

Facile synthesis and evaluation of a dual-functioning furoyl probe for in-cell SHAPE.

Recent analysis of transcriptomes has revealed that RNA molecules perform a myriad of functions beyond coding for proteins. RNA molecules can fold into complex secondary and tertiary structures, which are critical for regulating their function. Selective Hydroxyl Acylation analyzed by Primer Extension, or SHAPE is a common method for probing RNA structure in and outside of cells. Recent developments in SHAPE include the design of acyl imidazole acylating electrophiles with alkyl azides to enrich the sites of SHAPE adduct formation. Enrichment is key for next-generation sequencing experiments as it dramatically improves the signal. In a recent comparison of different structures of such reagents, we realized that furoyl acylating reagents form hyper-stable ester adducts with hydroxyls. This prompted us to design, synthesize and test a novel dual-functioning SHAPE probe (FAI-N3), which has the stable furoyl scaffold and the alkyl azide for enrichment. Herein we present the results that show FAI-N3 is a suitable probe for RNA structure analysis by SHAPE and that it can be used for enrichment of SHAPE adducts. These results strongly demonstrate that FAI-N3 is an ideal probe for structure probing in cells and will be very useful for sequencing-based analysis of SHAPE.

Measuring RNA structure transcriptome-wide with icSHAPE.

RNA molecules can be found at the heart of nearly every aspect of gene regulation: from gene expression to protein translation. The ability of RNA molecules to fold into intricate structures guides their function. Chemical methods to measure RNA structure have been part of the RNA biologists toolkit for several decades. These methods, although often cumbersome and difficult to perform on large RNAs, are notable for their accuracy and precision of structural measurements. Recent extension of these methods to transcriptome-wide analyses has opened the door to interrogating the structure of complete RNA molecules inside cells. Within this manuscript we describe the biochemical basis for the methodology behind a novel technology, icSHAPE, which measures RNA flexibility and single-strandedness in RNA. Novel methods such as icSHAPE have greatly expanded our understanding of RNA function and have paved the way to expansive analyses of large groups of RNA structures as they function inside the native environment of the cell.

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts.

RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2'-hydroxyl acylation can then be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2'-hydroxyl. Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment.

RNA structure: merging chemistry and genomics for a holistic perspective.

The advent of deep sequencing technology has unexpectedly advanced our structural understanding of molecules composed of nucleic acids. A significant amount of progress has been made recently extrapolating the chemical methods to probe RNA structure into sequencing methods. Herein we review some of the canonical methods to analyze RNA structure, and then we outline how these have been used to probe the structure of many RNAs in parallel. The key is the transformation of structural biology problems into sequencing problems, whereby sequencing power can be interpreted to understand nucleic acid proximity, nucleic acid conformation, or nucleic acid-protein interactions. Utilizing such technologies in this way has the promise to provide novel structural insights into the mechanisms that control normal cellular physiology and provide insight into how structure could be perturbed in disease.

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma.

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of an aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxoglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1α, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively affects the levels of N6-methyladenosine (m6A), while the levels of 5-formylcytosine (f5C) in mitochondrial tRNA are unaffected. This supports the hypothesis of a differential impact of fumarate accumulation on distinct RNA demethylases. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

Functional Conservation of LncRNA JPX Despite Sequence and Structural Divergence.

Long noncoding RNAs (lncRNAs) have been identified in all eukaryotes and are most abundant in the human genome. However, the functional importance and mechanisms of action for human lncRNAs are largely unknown. Using comparative sequence, structural, and functional analyses, we characterize the evolution and molecular function of human lncRNA JPX. We find that human JPX and its mouse homolog, lncRNA Jpx, have deep divergence in their nucleotide sequences and RNA secondary structures. Despite such differences, both lncRNAs demonstrate robust binding to CTCF, a protein that is central to Jpx's role in X chromosome inactivation. In addition, our functional rescue experiment using Jpx-deletion mutant cells shows that human JPX can functionally complement the loss of Jpx in mouse embryonic stem cells. Our findings support a model for functional conservation of lncRNAs independent from sequence and structural divergence. This study provides mechanistic insight into the evolution of lncRNA function.

Assaying RNA Structure Inside Living Cells with SHAPE.

RNA molecules have emerged as key players in nearly every facet of gene regulation. Such functions are governed by RNA's unique ability to fold into intricate secondary and tertiary structures. In order to understand, on the molecular level, how such structures from several chemical and enzymatic methods have been developed. One such method is RNA Selective Hydroxyl Acylation analyzed by Primer Extension, or SHAPE. SHAPE has emerged as a premier method of measuring RNA structure, with recent developments extending SHAPE into living cells. Here, we describe the use of SHAPE for measuring RNA structure inside living cells.

A screening protocol for identification of functional mutants of RNA editing adenosine deaminases.

Genetic screens can be used to evaluate a spectrum of mutations and thereby infer the function of particular residues within a protein. The Adenosine Deaminase Acting on RNA (ADAR) family of RNA-editing enzymes selectively deaminate adenosines (A) in double-helical RNA, generating inosine (I). The protocol described here exploits the editing activity of ADAR2 in a yeast-based screen by inserting an editing substrate sequence with a stop codon incorporated at the editing site upstream from the sequence encoding the reporter α-galactosidase. A-to-I editing changes the stop codon to a tryptophan codon, allowing normal expression of the reporter. This technique is particularly well-suited for screening ADAR and ADAR substrate mutant libraries for editing activity. Curr. Protoc. Chem. Biol. 4:357-369 © 2012 by John Wiley & Sons, Inc.