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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

The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.(A) Proximity maps of AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl with annotated helix elements (rounded rectangles) and tertiary hits (purple and pink circles). Red arrows indicate regions of proximity between J11-10 and helix P6 in the crystal structure. (B) Three MCM models with lowest Rosetta energy of AdoCbl riboswitch aptamer give an initial visualization of ligand-free ensemble (left); modeling included pseudo-energy constraints from 0 µM AdoCbl proximity map. The crystal structure is shown at right for comparison. (C–D) Mutation of A124 and A125 to U disrupts ligand-binding but not preformed tertiary structure. (C) Proximity maps of A124U/A125U mutant AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl, showing retention of tertiary proximity hits similar to the wild-type aptamer in 0 µM AdoCbl. (D) 1D chemical mapping reactivities of wild-type and A124U/A125U (‘Mut’) AdoCbl riboswitch aptamer. Green arrows in DMS reactivity lanes indicate locations of reduced reactivity in the presence of ligand in the wild type only. Residues 124 and 125 are unreactive to DMS in the A124U/A125U mutant because DMS does not modify U residues. Blue arrow in CMCT reactivity lanes indicates lack of protection of U124 and U125 in the mutant in the presence of ligand. Magenta arrows in SHAPE reactivity indicate locations of reduced/altered reactivity in the presence of ligand in the wild type but not in the mutant. All ligand-dependent protections are located in or near the AdoCbl-binding site in the riboswitch aptamer (Peselis & Serganov, 2012).DOI:http://dx.doi.org/10.7554/eLife.07600.021
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fig6: The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.(A) Proximity maps of AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl with annotated helix elements (rounded rectangles) and tertiary hits (purple and pink circles). Red arrows indicate regions of proximity between J11-10 and helix P6 in the crystal structure. (B) Three MCM models with lowest Rosetta energy of AdoCbl riboswitch aptamer give an initial visualization of ligand-free ensemble (left); modeling included pseudo-energy constraints from 0 µM AdoCbl proximity map. The crystal structure is shown at right for comparison. (C–D) Mutation of A124 and A125 to U disrupts ligand-binding but not preformed tertiary structure. (C) Proximity maps of A124U/A125U mutant AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl, showing retention of tertiary proximity hits similar to the wild-type aptamer in 0 µM AdoCbl. (D) 1D chemical mapping reactivities of wild-type and A124U/A125U (‘Mut’) AdoCbl riboswitch aptamer. Green arrows in DMS reactivity lanes indicate locations of reduced reactivity in the presence of ligand in the wild type only. Residues 124 and 125 are unreactive to DMS in the A124U/A125U mutant because DMS does not modify U residues. Blue arrow in CMCT reactivity lanes indicates lack of protection of U124 and U125 in the mutant in the presence of ligand. Magenta arrows in SHAPE reactivity indicate locations of reduced/altered reactivity in the presence of ligand in the wild type but not in the mutant. All ligand-dependent protections are located in or near the AdoCbl-binding site in the riboswitch aptamer (Peselis & Serganov, 2012).DOI:http://dx.doi.org/10.7554/eLife.07600.021

Mentions: Compared to the riboswitch aptamers above, much less was previously known about the AdoCbl riboswitch aptamer. Indeed, long-standing controversies in modeling this ncRNA raised the possibility of differences in both secondary and tertiary structure in states with and without ligand (Ravnum and Andersson, 2001; Nahvi et al., 2002; Vitreschak et al., 2003; Nahvi et al., 2004; Barrick and Breaker, 2007; Miao et al., 2015). Nevertheless, MCM revealed striking similarities between the states, most clearly visible in MOHCA-seq data (Figure 6A). In terms of secondary structure, features for six major helical stacks appeared identical with and without ligand. In terms of tertiary structure, the data without ligand showed reduction but not complete loss of strong tertiary proximities between helices that enclose the ligand-binding site (Figure 6A). As above, Rosetta modeling produced an initial visualization of a loose structural ensemble that samples, but does not stably remain in, the ligand-bound structure within the nanometer resolution of the MOHCA-seq method (Figure 6B and Table 2). Again, by rapidly resolving secondary and tertiary structure, this MCM analysis sets a foundation for further dissection of this RNA's ligand-free globule. As a first example, we found that the sole long-range tertiary interaction in the AdoCbl-binding site (an A-minor contact between J11-10 and helix P6) was necessary for ligand binding but not the global tertiary proximities measured by MOHCA-seq (Figure 6C,D). Like the data for the glycine riboswitch aptamers above, these data further suggest that the ligand-free proximities in the AdoCbl riboswitch aptamer arise not from direct tertiary contacts but from conformational preferences encoded in the riboswitch aptamer junctions.10.7554/eLife.07600.021Figure 6.The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.


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)

The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.(A) Proximity maps of AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl with annotated helix elements (rounded rectangles) and tertiary hits (purple and pink circles). Red arrows indicate regions of proximity between J11-10 and helix P6 in the crystal structure. (B) Three MCM models with lowest Rosetta energy of AdoCbl riboswitch aptamer give an initial visualization of ligand-free ensemble (left); modeling included pseudo-energy constraints from 0 µM AdoCbl proximity map. The crystal structure is shown at right for comparison. (C–D) Mutation of A124 and A125 to U disrupts ligand-binding but not preformed tertiary structure. (C) Proximity maps of A124U/A125U mutant AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl, showing retention of tertiary proximity hits similar to the wild-type aptamer in 0 µM AdoCbl. (D) 1D chemical mapping reactivities of wild-type and A124U/A125U (‘Mut’) AdoCbl riboswitch aptamer. Green arrows in DMS reactivity lanes indicate locations of reduced reactivity in the presence of ligand in the wild type only. Residues 124 and 125 are unreactive to DMS in the A124U/A125U mutant because DMS does not modify U residues. Blue arrow in CMCT reactivity lanes indicates lack of protection of U124 and U125 in the mutant in the presence of ligand. Magenta arrows in SHAPE reactivity indicate locations of reduced/altered reactivity in the presence of ligand in the wild type but not in the mutant. All ligand-dependent protections are located in or near the AdoCbl-binding site in the riboswitch aptamer (Peselis & Serganov, 2012).DOI:http://dx.doi.org/10.7554/eLife.07600.021
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fig6: The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.(A) Proximity maps of AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl with annotated helix elements (rounded rectangles) and tertiary hits (purple and pink circles). Red arrows indicate regions of proximity between J11-10 and helix P6 in the crystal structure. (B) Three MCM models with lowest Rosetta energy of AdoCbl riboswitch aptamer give an initial visualization of ligand-free ensemble (left); modeling included pseudo-energy constraints from 0 µM AdoCbl proximity map. The crystal structure is shown at right for comparison. (C–D) Mutation of A124 and A125 to U disrupts ligand-binding but not preformed tertiary structure. (C) Proximity maps of A124U/A125U mutant AdoCbl riboswitch aptamer in 70 µM (left) or 0 µM (right) AdoCbl, showing retention of tertiary proximity hits similar to the wild-type aptamer in 0 µM AdoCbl. (D) 1D chemical mapping reactivities of wild-type and A124U/A125U (‘Mut’) AdoCbl riboswitch aptamer. Green arrows in DMS reactivity lanes indicate locations of reduced reactivity in the presence of ligand in the wild type only. Residues 124 and 125 are unreactive to DMS in the A124U/A125U mutant because DMS does not modify U residues. Blue arrow in CMCT reactivity lanes indicates lack of protection of U124 and U125 in the mutant in the presence of ligand. Magenta arrows in SHAPE reactivity indicate locations of reduced/altered reactivity in the presence of ligand in the wild type but not in the mutant. All ligand-dependent protections are located in or near the AdoCbl-binding site in the riboswitch aptamer (Peselis & Serganov, 2012).DOI:http://dx.doi.org/10.7554/eLife.07600.021
Mentions: Compared to the riboswitch aptamers above, much less was previously known about the AdoCbl riboswitch aptamer. Indeed, long-standing controversies in modeling this ncRNA raised the possibility of differences in both secondary and tertiary structure in states with and without ligand (Ravnum and Andersson, 2001; Nahvi et al., 2002; Vitreschak et al., 2003; Nahvi et al., 2004; Barrick and Breaker, 2007; Miao et al., 2015). Nevertheless, MCM revealed striking similarities between the states, most clearly visible in MOHCA-seq data (Figure 6A). In terms of secondary structure, features for six major helical stacks appeared identical with and without ligand. In terms of tertiary structure, the data without ligand showed reduction but not complete loss of strong tertiary proximities between helices that enclose the ligand-binding site (Figure 6A). As above, Rosetta modeling produced an initial visualization of a loose structural ensemble that samples, but does not stably remain in, the ligand-bound structure within the nanometer resolution of the MOHCA-seq method (Figure 6B and Table 2). Again, by rapidly resolving secondary and tertiary structure, this MCM analysis sets a foundation for further dissection of this RNA's ligand-free globule. As a first example, we found that the sole long-range tertiary interaction in the AdoCbl-binding site (an A-minor contact between J11-10 and helix P6) was necessary for ligand binding but not the global tertiary proximities measured by MOHCA-seq (Figure 6C,D). Like the data for the glycine riboswitch aptamers above, these data further suggest that the ligand-free proximities in the AdoCbl riboswitch aptamer arise not from direct tertiary contacts but from conformational preferences encoded in the riboswitch aptamer junctions.10.7554/eLife.07600.021Figure 6.The S. thermophilus adenosylcobalamin riboswitch aptamer retains preformed tertiary structure without binding ligand.

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