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A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat.

Duszczyk MM, Zanier K, Sattler M - Nucleic Acids Res. (2008)

Bottom Line: The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations.In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat.The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.

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Schematic structure and sequence of Xist RNA and its A-repeats (A) Xist RNA is a long (15 kb in mouse, 17 kb in human) noncoding RNA. The A-repeats located at the 5′-end are essential for silencing, while other regions are redundantly responsible for chromosome association. (B) The A-repeats consist of 7.5 copies of a conserved sequence predicted to fold into two hairpins, connected by long U-rich linkers. N = any nucleotide; Y = C/U. (C) The 26-mer A-repeat construct used containing both predicted hairpins. This construct is identical to the fifth human Xist RNA A-repeat apart from the reversed G4-C11 base pair as described in the Introduction section. (D) The 14-mer A-repeat construct used in this study, containing the first predicted hairpin with a novel tetraloop.
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Figure 1: Schematic structure and sequence of Xist RNA and its A-repeats (A) Xist RNA is a long (15 kb in mouse, 17 kb in human) noncoding RNA. The A-repeats located at the 5′-end are essential for silencing, while other regions are redundantly responsible for chromosome association. (B) The A-repeats consist of 7.5 copies of a conserved sequence predicted to fold into two hairpins, connected by long U-rich linkers. N = any nucleotide; Y = C/U. (C) The 26-mer A-repeat construct used containing both predicted hairpins. This construct is identical to the fifth human Xist RNA A-repeat apart from the reversed G4-C11 base pair as described in the Introduction section. (D) The 14-mer A-repeat construct used in this study, containing the first predicted hairpin with a novel tetraloop.

Mentions: Xist (X inactivation specific transcript) RNA is a large non-coding RNA essential for the initiation of X-inactivation in mammalian females (1). Early in embryonic development it is expressed from the X-chromosome that will be silenced and coats it in cis, which coincides with transcriptional shutdown through an unknown mechanism (2). The conserved so-called ‘A-repeats’ at the 5′-end of Xist are essential for its silencing function, while several other regions are redundantly responsible for chromosome association (3) (Figure 1A). In humans, the A-repeats are constituted of 7.5 copies of a 26 nt motif, connected by long U-rich linkers. A Mfold secondary structure prediction of a single A-repeat suggested a double hairpin structure where the two hairpins possibly stack on top of each other (3) (Figure 1B). As no structural information on the A-repeats is available, we started NMR studies on a single A-repeat (Figure 1C) with the goal to solve its atomic structure and to obtain molecular insight into X-Inactivation. The construct used in our study shown in Figure 1C is identical to the 5th human A-repeat, apart from switching the positions of G and C in the third G–C base-pair to facilitate chemical shift assignments. Previous studies have shown that altering the sequence of the stem in hairpin 1 does not influence Xist activity as long as base pairing is not disrupted (3). During our structural studies we encountered difficulties completing NMR assignments of the second predicted hairpin. Signals from this hairpin were broad, and sometimes doubled (data not shown), which indicated possible dynamics or sample heterogeneity, although native gel analysis of the 26-mer A-repeat RNA suggested a homogenous monomeric population (Supplementary Material). The strategy described in this article was essential to characterize and distinguish the intramolecular and intermolecular base pairs in monomeric/dimeric forms of RNA at sample conditions required for structural biology. The approach provided valuable insight into the possible architecture of the A-repeats.Figure 1.


A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat.

Duszczyk MM, Zanier K, Sattler M - Nucleic Acids Res. (2008)

Schematic structure and sequence of Xist RNA and its A-repeats (A) Xist RNA is a long (15 kb in mouse, 17 kb in human) noncoding RNA. The A-repeats located at the 5′-end are essential for silencing, while other regions are redundantly responsible for chromosome association. (B) The A-repeats consist of 7.5 copies of a conserved sequence predicted to fold into two hairpins, connected by long U-rich linkers. N = any nucleotide; Y = C/U. (C) The 26-mer A-repeat construct used containing both predicted hairpins. This construct is identical to the fifth human Xist RNA A-repeat apart from the reversed G4-C11 base pair as described in the Introduction section. (D) The 14-mer A-repeat construct used in this study, containing the first predicted hairpin with a novel tetraloop.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602763&req=5

Figure 1: Schematic structure and sequence of Xist RNA and its A-repeats (A) Xist RNA is a long (15 kb in mouse, 17 kb in human) noncoding RNA. The A-repeats located at the 5′-end are essential for silencing, while other regions are redundantly responsible for chromosome association. (B) The A-repeats consist of 7.5 copies of a conserved sequence predicted to fold into two hairpins, connected by long U-rich linkers. N = any nucleotide; Y = C/U. (C) The 26-mer A-repeat construct used containing both predicted hairpins. This construct is identical to the fifth human Xist RNA A-repeat apart from the reversed G4-C11 base pair as described in the Introduction section. (D) The 14-mer A-repeat construct used in this study, containing the first predicted hairpin with a novel tetraloop.
Mentions: Xist (X inactivation specific transcript) RNA is a large non-coding RNA essential for the initiation of X-inactivation in mammalian females (1). Early in embryonic development it is expressed from the X-chromosome that will be silenced and coats it in cis, which coincides with transcriptional shutdown through an unknown mechanism (2). The conserved so-called ‘A-repeats’ at the 5′-end of Xist are essential for its silencing function, while several other regions are redundantly responsible for chromosome association (3) (Figure 1A). In humans, the A-repeats are constituted of 7.5 copies of a 26 nt motif, connected by long U-rich linkers. A Mfold secondary structure prediction of a single A-repeat suggested a double hairpin structure where the two hairpins possibly stack on top of each other (3) (Figure 1B). As no structural information on the A-repeats is available, we started NMR studies on a single A-repeat (Figure 1C) with the goal to solve its atomic structure and to obtain molecular insight into X-Inactivation. The construct used in our study shown in Figure 1C is identical to the 5th human A-repeat, apart from switching the positions of G and C in the third G–C base-pair to facilitate chemical shift assignments. Previous studies have shown that altering the sequence of the stem in hairpin 1 does not influence Xist activity as long as base pairing is not disrupted (3). During our structural studies we encountered difficulties completing NMR assignments of the second predicted hairpin. Signals from this hairpin were broad, and sometimes doubled (data not shown), which indicated possible dynamics or sample heterogeneity, although native gel analysis of the 26-mer A-repeat RNA suggested a homogenous monomeric population (Supplementary Material). The strategy described in this article was essential to characterize and distinguish the intramolecular and intermolecular base pairs in monomeric/dimeric forms of RNA at sample conditions required for structural biology. The approach provided valuable insight into the possible architecture of the A-repeats.Figure 1.

Bottom Line: The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations.In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat.The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.

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