Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Wilkinson, Kevin A.; Vasa, Suzy M.; Deigan, Katherine E.; Mortimer, Stefanie; Giddings, Morgan C.; Weeks, Kevin M.

doi:10.1261/rna.1536209

Cited by 80 publications

(87 citation statements)

References 29 publications

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“…Similar C-and U-rich loop regions in other RNAs have been found to have low reactivity toward SHAPE reagents (Legiewicz et al 2010), in part because of the slightly lower reactivity of C and U nucleotides compared with A and G (Wilkinson et al 2009). Our Tb 3+ data, however, exhibit the same protection of these nucleotides as 1M7.…”

Section: Amentioning

confidence: 90%

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Todd

Walter

2013

RNA

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Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb 3+ ions and the selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb 3+ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

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Section: Amentioning

confidence: 90%

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Todd

Walter

2013

RNA

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“…′ OH group of the ribose present in each nucleoside of an RNA in a manner that is largely independent of base identity (Wilkinson et al 2009), but depends on the ribose adopting specific conformations that are sampled by flexible regions of the RNA, but not by basepaired regions (Merino et al 2005;McGinnis et al 2012). After SHAPE probing, primer extension by reverse transcriptase will result in the synthesis of cDNAs that terminate at positions immediately before SHAPE adducts.…”

Section: Shape Reagents React With Thementioning

confidence: 99%

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Poulsen

Kielpinski

Salama

et al. 2015

RNA

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Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.

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“…1C; Wilkinson et al 2008;McGinnis et al 2009;Watts et al 2009). SHAPE reactivities correlate strongly with model-free measurements of molecular order and are largely independent of nucleotide type or solvent accessibility (Gherghe et al 2008;Wilkinson et al 2009;McGinnis et al 2012). SHAPE reactivity information has been used to develop RNA secondary structure models, to detect changes in RNA conformation, and to monitor interactions with proteins, ligands, and metal ions.…”

Section: Introductionmentioning

confidence: 99%

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

Karabiber

McGinnis

Favorov

et al. 2012

RNA

Self Cite

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248

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Chemical probing of RNA and DNA structure is a widely used and highly informative approach for examining nucleic acid structure and for evaluating interactions with protein and small-molecule ligands. Use of capillary electrophoresis to analyze chemical probing experiments yields hundreds of nucleotides of information per experiment and can be performed on automated instruments. Extraction of the information from capillary electrophoresis electropherograms is a computationally intensive multistep analytical process, and no current software provides rapid, automated, and accurate data analysis. To overcome this bottleneck, we developed a platform-independent, user-friendly software package, QuShape, that yields quantitatively accurate nucleotide reactivity information with minimal user supervision. QuShape incorporates newly developed algorithms for signal decay correction, alignment of time-varying signals within and across capillaries and relative to the RNA nucleotide sequence, and signal scaling across channels or experiments. An analysis-by-reference option enables multiple, related experiments to be fully analyzed in minutes. We illustrate the usefulness and robustness of QuShape by analysis of RNA SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) experiments.

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Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Cited by 80 publications

References 29 publications

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

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