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Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

Cordero P, Das R - PLoS Comput. Biol. (2015)

Bottom Line: In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments.An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations.These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Informatics Program, Stanford University, Stanford, California, United States of America.

ABSTRACT
Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

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Landscape dissection of a segment of the V. Vulnificus add adenine riboswitch in 0 and 5 mM adenine.(a) M2 measurements in 0 and 5 mM adenine; (b) REEFFIT bootstrapped fits; (c) base pair probability matrices of the structures in the wild type landscape (upper triangles: base pair probability values using no data, lower triangles: REEFFIT calculated base pair probabilities); (d) structure mediods of the structural ensemble found by REEFFIT. In (a-b), The Shine-Dalgarno (SD) ribosome-binding sequence was observed to be SHAPE-reactive even in the absence of adenine; while consistent with this region's unpaired status without and with ligand in prior NMR studies, our studies indicate a more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere. See Supporting Tables H-I in S1 Text for RNA sequences and fit summaries.
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pcbi.1004473.g003: Landscape dissection of a segment of the V. Vulnificus add adenine riboswitch in 0 and 5 mM adenine.(a) M2 measurements in 0 and 5 mM adenine; (b) REEFFIT bootstrapped fits; (c) base pair probability matrices of the structures in the wild type landscape (upper triangles: base pair probability values using no data, lower triangles: REEFFIT calculated base pair probabilities); (d) structure mediods of the structural ensemble found by REEFFIT. In (a-b), The Shine-Dalgarno (SD) ribosome-binding sequence was observed to be SHAPE-reactive even in the absence of adenine; while consistent with this region's unpaired status without and with ligand in prior NMR studies, our studies indicate a more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere. See Supporting Tables H-I in S1 Text for RNA sequences and fit summaries.

Mentions: We then challenged M2-REEFFIT with a more complex test case: a Vibrio vulnificus adenosine deaminase (add) mRNA riboswitch (Fig 3a–3d) that, in response to the ligand adenine, exposes start site segments (Shine-Dalgarno sequence and/or AUG start codon) to promote mRNA translation. In a detailed NMR study, spectra in ligand-free conditions fit well to a model with two states, apoA (~30%, with helices P1, P2, P3, P4, and P5) and apoB (~70%, with P1B, P2B, P3, an extended P4 called P4B, and P5). Addition of adenine ligand resulted in spectra dominated by a state holo with P1, P2, P3, and P5 and perturbed chemical shifts consistent with adenine binding to the aptamer [17]. While three states gave the simplest model for the prior data, more complex multi-state models were consistent as well and would be generally predicted from RNA secondary structure calculations [12,13]. We focused on whether M2-REEFFIT could recover the base pairs of P1, P2, P1B, P2B, P3, P4, P4B, and P5, which were unambiguously determined through NOE spectroscopy and model construct comparisons. Even in the absence of adenine, our M2 measurements of the RNA suggested the presence of at least two distinct structures that protect or expose the mRNA start site (AUG at nts 120–122) and, in an anticorrelated manner, expose or protect segments in the aptamer region (e.g., nts 53–60 and 66–72), respectively (M2 data in Fig 3a). In addition to observing these different states upon mutation, addition of 5 mM adenine induced clear changes in the M2 data, including protections in the aptamer and consistent exposure of the AUG start codon in most mutants (M2 data in Fig 3a, bottom;). While consistent with this region’s unpaired status without and with ligand in prior NMR studies, our studies indicatea more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere.


Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

Cordero P, Das R - PLoS Comput. Biol. (2015)

Landscape dissection of a segment of the V. Vulnificus add adenine riboswitch in 0 and 5 mM adenine.(a) M2 measurements in 0 and 5 mM adenine; (b) REEFFIT bootstrapped fits; (c) base pair probability matrices of the structures in the wild type landscape (upper triangles: base pair probability values using no data, lower triangles: REEFFIT calculated base pair probabilities); (d) structure mediods of the structural ensemble found by REEFFIT. In (a-b), The Shine-Dalgarno (SD) ribosome-binding sequence was observed to be SHAPE-reactive even in the absence of adenine; while consistent with this region's unpaired status without and with ligand in prior NMR studies, our studies indicate a more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere. See Supporting Tables H-I in S1 Text for RNA sequences and fit summaries.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643908&req=5

pcbi.1004473.g003: Landscape dissection of a segment of the V. Vulnificus add adenine riboswitch in 0 and 5 mM adenine.(a) M2 measurements in 0 and 5 mM adenine; (b) REEFFIT bootstrapped fits; (c) base pair probability matrices of the structures in the wild type landscape (upper triangles: base pair probability values using no data, lower triangles: REEFFIT calculated base pair probabilities); (d) structure mediods of the structural ensemble found by REEFFIT. In (a-b), The Shine-Dalgarno (SD) ribosome-binding sequence was observed to be SHAPE-reactive even in the absence of adenine; while consistent with this region's unpaired status without and with ligand in prior NMR studies, our studies indicate a more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere. See Supporting Tables H-I in S1 Text for RNA sequences and fit summaries.
Mentions: We then challenged M2-REEFFIT with a more complex test case: a Vibrio vulnificus adenosine deaminase (add) mRNA riboswitch (Fig 3a–3d) that, in response to the ligand adenine, exposes start site segments (Shine-Dalgarno sequence and/or AUG start codon) to promote mRNA translation. In a detailed NMR study, spectra in ligand-free conditions fit well to a model with two states, apoA (~30%, with helices P1, P2, P3, P4, and P5) and apoB (~70%, with P1B, P2B, P3, an extended P4 called P4B, and P5). Addition of adenine ligand resulted in spectra dominated by a state holo with P1, P2, P3, and P5 and perturbed chemical shifts consistent with adenine binding to the aptamer [17]. While three states gave the simplest model for the prior data, more complex multi-state models were consistent as well and would be generally predicted from RNA secondary structure calculations [12,13]. We focused on whether M2-REEFFIT could recover the base pairs of P1, P2, P1B, P2B, P3, P4, P4B, and P5, which were unambiguously determined through NOE spectroscopy and model construct comparisons. Even in the absence of adenine, our M2 measurements of the RNA suggested the presence of at least two distinct structures that protect or expose the mRNA start site (AUG at nts 120–122) and, in an anticorrelated manner, expose or protect segments in the aptamer region (e.g., nts 53–60 and 66–72), respectively (M2 data in Fig 3a). In addition to observing these different states upon mutation, addition of 5 mM adenine induced clear changes in the M2 data, including protections in the aptamer and consistent exposure of the AUG start codon in most mutants (M2 data in Fig 3a, bottom;). While consistent with this region’s unpaired status without and with ligand in prior NMR studies, our studies indicatea more dramatic switch in this region for more complete add riboswitch constructs and will be reported elsewhere.

Bottom Line: In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments.An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations.These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Informatics Program, Stanford University, Stanford, California, United States of America.

ABSTRACT
Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

Show MeSH
Related in: MedlinePlus