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Consistent global structures of complex RNA states through multidimensional chemical mapping.

Cheng CY, Chou FC, Kladwang W, Tian S, Cordero P, Das R - Elife (2015)

Bottom Line: Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis.Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions.This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, United States.

ABSTRACT
Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis. Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions. We report that integrating a new method called MOHCA-seq (Multiplexed •OH Cleavage Analysis with paired-end sequencing) with mutate-and-map secondary structure inference guides Rosetta 3D modeling to consistent 1-nm accuracy for intricately folded ncRNAs with lengths up to 188 nucleotides, including a blind RNA-puzzle challenge, the lariat-capping ribozyme. This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon. MCM offers a sequencing-based route to uncovering ncRNA 3D structure, applicable to functionally important but potentially heterogeneous states.

No MeSH data available.


Related in: MedlinePlus

M2 analysis of the S. thermophilum adenosylcobalamin riboswitch aptamer, performed during the sixth RNA-puzzles structure prediction trial.(A) M2 data set for 1M7 modification across 168 single mutations along the AdoCbl riboswitch aptamer sequence in the presence of 60 µM AdoCbl ligand. Mutants showing poor data quality are marked by red bars. (B) Z-score contact map extracted from (A). (C) Secondary structure prediction and (D) bootstrap support matrix using M2 data. In (B) and (D), the crystallographic secondary structure is overlaid as cyan circles.DOI:http://dx.doi.org/10.7554/eLife.07600.010
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fig3s1: M2 analysis of the S. thermophilum adenosylcobalamin riboswitch aptamer, performed during the sixth RNA-puzzles structure prediction trial.(A) M2 data set for 1M7 modification across 168 single mutations along the AdoCbl riboswitch aptamer sequence in the presence of 60 µM AdoCbl ligand. Mutants showing poor data quality are marked by red bars. (B) Z-score contact map extracted from (A). (C) Secondary structure prediction and (D) bootstrap support matrix using M2 data. In (B) and (D), the crystallographic secondary structure is overlaid as cyan circles.DOI:http://dx.doi.org/10.7554/eLife.07600.010

Mentions: In addition to generating accurate models, the full MCM pipeline enabled selection of single confident 1-nm resolution tertiary structures in each of the above cases, an achievement highlighted by the AdoCbl riboswitch aptamer test case. For this RNA-puzzle target, we previously collected and disseminated M2 data (Figure 3—figure supplement 1) that correctly discriminated between literature models of this ncRNA's secondary structure (Peselis & Serganov, 2012). However, these data did not disambiguate between a range of 3D structures, and our ten submitted models gave RMSDs from 12.1 Å to 32.8 Å to the subsequently released riboswitch aptamer crystal structure (Figure 3—figure supplement 3). With the developments in this study, we suspected that MOHCA-seq data would complement our M2/Rosetta method and permit confident selection and submission of a single model. Indeed, the full MCM pipeline resolved tertiary contacts between the peripheral and core regions of the ncRNA (Figure 3D), and automated modeling converged to a structure defined with 13.9 Å resolution (in situ precision estimate), which achieved 11.9 Å RMSD to the crystal structure (Figure 3D, Table 1, Figure 3—figure supplement 6, and ‘Materials and methods’), comparable to the accuracy of our best RNA-puzzle submission and clearly defining the global structure to helix resolution.


Consistent global structures of complex RNA states through multidimensional chemical mapping.

Cheng CY, Chou FC, Kladwang W, Tian S, Cordero P, Das R - Elife (2015)

M2 analysis of the S. thermophilum adenosylcobalamin riboswitch aptamer, performed during the sixth RNA-puzzles structure prediction trial.(A) M2 data set for 1M7 modification across 168 single mutations along the AdoCbl riboswitch aptamer sequence in the presence of 60 µM AdoCbl ligand. Mutants showing poor data quality are marked by red bars. (B) Z-score contact map extracted from (A). (C) Secondary structure prediction and (D) bootstrap support matrix using M2 data. In (B) and (D), the crystallographic secondary structure is overlaid as cyan circles.DOI:http://dx.doi.org/10.7554/eLife.07600.010
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495719&req=5

fig3s1: M2 analysis of the S. thermophilum adenosylcobalamin riboswitch aptamer, performed during the sixth RNA-puzzles structure prediction trial.(A) M2 data set for 1M7 modification across 168 single mutations along the AdoCbl riboswitch aptamer sequence in the presence of 60 µM AdoCbl ligand. Mutants showing poor data quality are marked by red bars. (B) Z-score contact map extracted from (A). (C) Secondary structure prediction and (D) bootstrap support matrix using M2 data. In (B) and (D), the crystallographic secondary structure is overlaid as cyan circles.DOI:http://dx.doi.org/10.7554/eLife.07600.010
Mentions: In addition to generating accurate models, the full MCM pipeline enabled selection of single confident 1-nm resolution tertiary structures in each of the above cases, an achievement highlighted by the AdoCbl riboswitch aptamer test case. For this RNA-puzzle target, we previously collected and disseminated M2 data (Figure 3—figure supplement 1) that correctly discriminated between literature models of this ncRNA's secondary structure (Peselis & Serganov, 2012). However, these data did not disambiguate between a range of 3D structures, and our ten submitted models gave RMSDs from 12.1 Å to 32.8 Å to the subsequently released riboswitch aptamer crystal structure (Figure 3—figure supplement 3). With the developments in this study, we suspected that MOHCA-seq data would complement our M2/Rosetta method and permit confident selection and submission of a single model. Indeed, the full MCM pipeline resolved tertiary contacts between the peripheral and core regions of the ncRNA (Figure 3D), and automated modeling converged to a structure defined with 13.9 Å resolution (in situ precision estimate), which achieved 11.9 Å RMSD to the crystal structure (Figure 3D, Table 1, Figure 3—figure supplement 6, and ‘Materials and methods’), comparable to the accuracy of our best RNA-puzzle submission and clearly defining the global structure to helix resolution.

Bottom Line: Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis.Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions.This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, United States.

ABSTRACT
Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis. Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions. We report that integrating a new method called MOHCA-seq (Multiplexed •OH Cleavage Analysis with paired-end sequencing) with mutate-and-map secondary structure inference guides Rosetta 3D modeling to consistent 1-nm accuracy for intricately folded ncRNAs with lengths up to 188 nucleotides, including a blind RNA-puzzle challenge, the lariat-capping ribozyme. This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon. MCM offers a sequencing-based route to uncovering ncRNA 3D structure, applicable to functionally important but potentially heterogeneous states.

No MeSH data available.


Related in: MedlinePlus