Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq

Watters, Kyle E.; Abbott, Timothy R.; Lucks, Julius B.

doi:10.1093/nar/gkv879

Cited by 79 publications

(136 citation statements)

References 63 publications

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“…However, recent reports have suggested that 1M7, an isatoic anhydride reagent related to NMIA, was able to robustly modify both ribosomal RNA as well as mRNAs inside both mammalian and bacterial cells (McGinnis et al 2015;Smola et al 2015;Takahashi et al 2016;Watters et al 2016). These apparently contradictory results stimulated us to further investigate the differences between the two types of SHAPE reagents.…”

Section: Resultsmentioning

confidence: 99%

“…In vivo CLICK SHAPE and SHAPE and mutational profiling (Siegfried et al 2014) are two recently developed methods that use SHAPE chemistry to read out RNA structure information. Collectively, these new sequencingbased SHAPE technologies have opened the door for scientists to make single-nucleotide resolution measurements of RNA structure across entire transcriptomes and full-length RNA molecules (Siegfried et al 2014;Spitale et al 2015;Watters et al 2016). However, they differ at many key levels.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of SHAPE reagents for mapping RNA structures inside living cells

Lee

Flynn

Kadina

et al. 2016

RNA

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Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N 3 and 1M7 reagents, respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N 3 give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of SHAPE reagents for mapping RNA structures inside living cells

Lee

Flynn

Kadina

et al. 2016

RNA

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“…Although not statistically significant, this correlation suggests that the most highly conserved regions appear to undergo the most significant conformational rearrangements. How cellular conditions impact RNA secondary structure is an active area of research (Ding et al 2014;Rouskin et al 2014;Spitale et al 2015;Smola et al 2016;Watters et al 2016a), and conservation may be key to understanding just how the environment affects RNA structures.…”

Section: Environmentally Dependent Mrna Structuresmentioning

confidence: 99%

“…The SHAPE-MaP approach improves the signal-to-noise by sequencing deeper and rigorously defines differences between protein-bound mRNA structures and in vitro mRNA folding. The extent of RNA structure change between cellular and in vitro conditions has been controversial and varies widely between studies and RNAs (Ding et al 2014;Rouskin et al 2014;Spitale et al 2015;Watters et al 2016a). We do identify multiple regions that are highly sensitive to environmental conditions ( Watters et al 2016b).…”

Section: ))mentioning

confidence: 99%

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

Lackey

Coria

Woods

et al. 2018

RNA

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The precise impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next generation sequencing to obtain experimental structure data, such as SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension and Mutational Profiling), can reveal structural effects of mutations especially when this data is rigorously incorporated in RNA structural modeling. Here we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single nucleotide mutations. We find that allele-specific sorting greatly improved the detection ability of SHAPE-MaP. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of non-coding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1. Combined with rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble we identified a subset of somatic and Lackey 2 inherited mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified acting as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures are on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both TPT1 and LCP1 have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro are likely to exist under biological conditions. Overall, these data reveal a remarkably robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with a benchmark for further algorithmic development.

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“…Antisense/attenuator binding initiates as a kissing hairpin interaction that proceeds to a more extensively paired state, shown schematically with interaction lines (Brantl and Wagner 2000). (B) In-cell SHAPE-Seq (Watters et al 2016) overview. In-cell SHAPE-Seq characterizes cellular RNA structures using a SHAPE chemical probe that preferentially modifies nucleotides in flexible regions of the RNA.…”

Section: Introductionmentioning

confidence: 99%

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Takahashi

Watters

Gasper

et al. 2016

RNA

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Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.

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Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq

Cited by 79 publications

References 63 publications

Comparison of SHAPE reagents for mapping RNA structures inside living cells

Comparison of SHAPE reagents for mapping RNA structures inside living cells

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

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