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

Siegfried, Nathan A.; Busan, Steven; Rice, Greggory M.; Nelson, Julie A.; Weeks, Kevin M.

doi:10.1038/nmeth.3029

Cited by 522 publications

(974 citation statements)

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“…1C). Shannon entropies are derived from a SHAPE-directed partition function (32) and report a measure of confidence in the predicted base-pairing pattern at each nucleotide (26,33). Low entropy also corresponds to high confidence in structure modeling.…”

Section: Resultsmentioning

confidence: 99%

“…S1-S3). This approach has been validated for RNAs of known structure (34) and for identification of novel functional elements in large RNAs (26). The secondary structure models are illustrated using arc plots, which capture both predicted base pairing and the degree of variability in the structural models.…”

Section: Resultsmentioning

confidence: 99%

“…Most existing structural data have been obtained using short RNA transcripts that may not fully recapitulate structures in the intact genome. Here we used selective 2′-hydroxyl acylation analyzed by primer extension, read out by mutational profiling (SHAPEMaP) using massively parallel sequencing (25,26), to model the structures of infectious HCV RNA genomes from genotypes 1a, 1b, and 2a. We developed a concise structure-first approach, based on SHAPE reactivity information, to identify structural features conserved across HCV genotypes.…”

mentioning

confidence: 99%

“…RNA was modified with 1M7 and SHAPE-MaP libraries prepared (26). Full SHAPE data and details regarding genomic subclones, SHAPE-MaP massively parallel sequencing, and RNA structure analysis are available in SI Methods and Datasets S1 and S2.…”

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confidence: 99%

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Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

119

190

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Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

119

190

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“…1). Chemical adducts were detected as sequence mutations based on their ability to induce efficient misreading of the template nucleotide by a reverse transcriptase enzyme, an approach called mutational profiling, or MaP (6). Singlemolecule probing data were used in two distinct ways: to detect correlated RNA modifications reflecting higher-order through-space interactions (Fig.…”

mentioning

confidence: 99%

Single-molecule correlated chemical probing of RNA

Homan¹,

Favorov²,

Lavender³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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144

272

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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 throughspace 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. These functions are mediated by tiered levels of information: The simplest is the primary sequence, and the most complex is the higher-order structure that governs interactions with ligands, proteins, and other RNAs (1, 2). Many RNAs can form more than one stable structure, and these distinct conformations often have different biological activities (3, 4). Currently, the rate of describing new RNA sequences vastly exceeds abilities to examine their structures.Here we characterize through-space interactions and multiple conformations in single RNAs by melding chemical probing and massively parallel sequencing. Because massively parallel sequencing reports the sequences of single templates, each read is fundamentally a single-molecule observation (5). We first modified RNA with a reagent that is sensitive to the underlying RNA structure and then detected multiple adducts in individual RNA strands (Fig. 1). Chemical adducts were detected as sequence mutations based on their ability to induce efficient misreading of the template nucleotide by a reverse transcriptase enzyme, an approach called mutational profiling, or MaP (6). Singlemolecule probing data were used in two distinct ways: to detect correlated RNA modifications reflecting higher-order through-space interactions (Fig. 1A) and to examine multiple conformations in single in-solution ensembles (Fig. 1B). Results and DiscussionMultisite Dimethyl Sulfate Reactivity with RNA. We used dimethyl sulfate (DMS) to probe the structures of three RNAs: the Escherichia coli thiamine pyrophosphate (TPP) riboswitch (79 nt) (7), the Tetrahymena group I intron P546 domain (160 nt) (8), and the Bacillus stearothermophilus RNase P catalytic domain (265 nt) (9). RNAs were selected to illustrate distinct RNA folding features and to emphasize increasingly difficult analysis challenges. The TPP riboswitch binds the...

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A Radiolabeling‐Free, qPCR‐Based Method for Locus‐Specific Pseudouridine Detection

Lei

2017

Angewandte Chemie

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

Cited by 522 publications

References 56 publications

Functionally conserved architecture of hepatitis C virus RNA genomes

Functionally conserved architecture of hepatitis C virus RNA genomes

Single-molecule correlated chemical probing of RNA

A Radiolabeling‐Free, qPCR‐Based Method for Locus‐Specific Pseudouridine Detection

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