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Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats.

Malgowska M, Gudanis D, Kierzek R, Wyszko E, Gabelica V, Gdaniec Z - Nucleic Acids Res. (2014)

Bottom Line: We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)2A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)2C and p(UGG)2U, respectively.Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding.Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K(+) ions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland.

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Imino region of the 1H NMR spectra of (A) p(UGG)2U and (B) p(UGG)4U at 25°C in the presence of (Aa, Ba, Ad, Bd) 50 mM KCl, 10 mM potassium phosphate and 0.1 mM EDTA, pH 6.8, (Ab, Bb) 150 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 6.8, (Ac, Bc) 150 mM NH4Cl, 10 mM Tris/HCl, 0.1 mM EDTA, pH 6.8 and (Ae, Be) HeLa cell extract. Asterisks indicate imino proton resonances of uridines. The poor S/N ratio observed for p(UGG)4U is due to polymorphism or/and the presence of higher order structures.
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Figure 6: Imino region of the 1H NMR spectra of (A) p(UGG)2U and (B) p(UGG)4U at 25°C in the presence of (Aa, Ba, Ad, Bd) 50 mM KCl, 10 mM potassium phosphate and 0.1 mM EDTA, pH 6.8, (Ab, Bb) 150 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 6.8, (Ac, Bc) 150 mM NH4Cl, 10 mM Tris/HCl, 0.1 mM EDTA, pH 6.8 and (Ae, Be) HeLa cell extract. Asterisks indicate imino proton resonances of uridines. The poor S/N ratio observed for p(UGG)4U is due to polymorphism or/and the presence of higher order structures.

Mentions: The 1H NMR spectra of p(UGG)2U obtained in solutions containing K+, Na+ and NH4+ (Figure 6A) provided additional evidence for a G-quadruplex formation. In the 1H NMR spectrum of p(UGG)2U recorded in the presence of K+ (Supplementary Figure S9), several resonances sharpened with the increase in temperature and were visible even at 80°C. Additionally, new resonances appeared at 3°C. To elucidate their origin and, in particular, to distinguish guanosine from uridine imino protons, we acquired a 1H –15N HSQC spectrum at 3°C. Four guanosine and three uridine imino protons were identified based on the chemical shifts of the attached nitrogen (Supplementary Figure S10). The appearance of uridine imino signals suggested the formation of U-tetrads (56). To verify whether the uridine residues constitute part of the G-quadruplex architecture, we assigned all guanosine and uridine imino protons based on the analysis of a 2D NOESY spectrum (data not shown). Each of the three observed uridine imino resonances exhibited a strong NOE to H5 and much weaker one to H6 protons (Supplementary Figure S11), a pattern that is distinctive of a U-tetrad motif (55). The 3′-end uridine imino proton at 11.08 ppm and that at 9.85 ppm, assigned to the internal uridine residue, sharpened with increasing temperature and were still observed at a high temperature, revealing the formation of two stable U-tetrads. Although there is no NMR structure known with an internal U-tetrad, a similar shift ∼9.5 ppm was observed for imino protons of the thymidine tetrad accommodated in the centre of the DNA G-quadruplex (13,57,58). The 5′-end uridine imino proton at 10.74 ppm was observed only below 25°C (Supplementary Figure S9). These results, together with ESI-MS data (four very stable cation binding sites and one additional, more labile binding site in the tetramer), indicate that p(UGG)2U folds into a symmetrical tetramolecular parallel G-quadruplex with four G-tetrads and three U-tetrads (Figure 4B, D and G).


Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats.

Malgowska M, Gudanis D, Kierzek R, Wyszko E, Gabelica V, Gdaniec Z - Nucleic Acids Res. (2014)

Imino region of the 1H NMR spectra of (A) p(UGG)2U and (B) p(UGG)4U at 25°C in the presence of (Aa, Ba, Ad, Bd) 50 mM KCl, 10 mM potassium phosphate and 0.1 mM EDTA, pH 6.8, (Ab, Bb) 150 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 6.8, (Ac, Bc) 150 mM NH4Cl, 10 mM Tris/HCl, 0.1 mM EDTA, pH 6.8 and (Ae, Be) HeLa cell extract. Asterisks indicate imino proton resonances of uridines. The poor S/N ratio observed for p(UGG)4U is due to polymorphism or/and the presence of higher order structures.
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Figure 6: Imino region of the 1H NMR spectra of (A) p(UGG)2U and (B) p(UGG)4U at 25°C in the presence of (Aa, Ba, Ad, Bd) 50 mM KCl, 10 mM potassium phosphate and 0.1 mM EDTA, pH 6.8, (Ab, Bb) 150 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 6.8, (Ac, Bc) 150 mM NH4Cl, 10 mM Tris/HCl, 0.1 mM EDTA, pH 6.8 and (Ae, Be) HeLa cell extract. Asterisks indicate imino proton resonances of uridines. The poor S/N ratio observed for p(UGG)4U is due to polymorphism or/and the presence of higher order structures.
Mentions: The 1H NMR spectra of p(UGG)2U obtained in solutions containing K+, Na+ and NH4+ (Figure 6A) provided additional evidence for a G-quadruplex formation. In the 1H NMR spectrum of p(UGG)2U recorded in the presence of K+ (Supplementary Figure S9), several resonances sharpened with the increase in temperature and were visible even at 80°C. Additionally, new resonances appeared at 3°C. To elucidate their origin and, in particular, to distinguish guanosine from uridine imino protons, we acquired a 1H –15N HSQC spectrum at 3°C. Four guanosine and three uridine imino protons were identified based on the chemical shifts of the attached nitrogen (Supplementary Figure S10). The appearance of uridine imino signals suggested the formation of U-tetrads (56). To verify whether the uridine residues constitute part of the G-quadruplex architecture, we assigned all guanosine and uridine imino protons based on the analysis of a 2D NOESY spectrum (data not shown). Each of the three observed uridine imino resonances exhibited a strong NOE to H5 and much weaker one to H6 protons (Supplementary Figure S11), a pattern that is distinctive of a U-tetrad motif (55). The 3′-end uridine imino proton at 11.08 ppm and that at 9.85 ppm, assigned to the internal uridine residue, sharpened with increasing temperature and were still observed at a high temperature, revealing the formation of two stable U-tetrads. Although there is no NMR structure known with an internal U-tetrad, a similar shift ∼9.5 ppm was observed for imino protons of the thymidine tetrad accommodated in the centre of the DNA G-quadruplex (13,57,58). The 5′-end uridine imino proton at 10.74 ppm was observed only below 25°C (Supplementary Figure S9). These results, together with ESI-MS data (four very stable cation binding sites and one additional, more labile binding site in the tetramer), indicate that p(UGG)2U folds into a symmetrical tetramolecular parallel G-quadruplex with four G-tetrads and three U-tetrads (Figure 4B, D and G).

Bottom Line: We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)2A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)2C and p(UGG)2U, respectively.Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding.Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K(+) ions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Noskowskiego 12/14, Poland.

Show MeSH
Related in: MedlinePlus