Bao HL et al. (2017), Characterization of human telomere RNA G-quadru...

XB-ART-54047

Nucleic Acids Res 2017 May 19;459:5501-5511. doi: 10.1093/nar/gkx109.

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Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy.

Bao HL , Ishizuka T , Sakamoto T , Fujimoto K , Uechi T , Kenmochi N , Xu Y .

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Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.

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Species referenced: Xenopus laevis
Genes referenced: acin1 bag3

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	Figure 1. Concept for the detection different RNA structures by a 19F label. Three 19F resonances of different chemical shifts are expected according to single-stranded, dimeric G-quadruplex and two-subunits stacked G-quadruplex.
	Figure 2. 19F NMR and 1H NMR spectra of 19F labeled RNA. (A) Chemical structure of 19F labeled telomere RNA bearing 3,5 bis(trifluoromethyl)phenyl group at the 5΄ terminal. (B) 19F NMR spectra of 19F labeled telomere RNA at different concentrations. The peaks of the dimeric and two-subunits stacked G-quadruplex are marked with red and green spots, respectively. Concentrations of RNA indicated on the right. (C)19F NMR of 19F labeled RNA at different temperatures. Red and green spots indicated dimer and two-subunits stacked G-quadruplex. The peaks of single strand are marked with black spots. Temperatures indicated on the right. Condition: 3 mM RNA in 50 mM KCl and 10 mM Tris-HCl buffer (pH 7.0). The sample is kept for 10 min at each temperature. (D) 19F NMR of 19F labeled RNA at different temperatures and concentrations. The peaks of dimer G-quadruplex are red spots. The peak of two-subunits stacked G-quadruplex is green spot. The peaks of single strand are marked with black spots. (E) 1H imino proton NMR of 19F labeled RNA corresponding to 19F NMR at different temperatures and concentrations. The peaks characteristic of the dimeric and two-subunits stacked G-quadruplex are marked with red and green spots, respectively.
	Figure 3. 19F NMR shift versus concentration and temperature profiles. (A) Profiles of the relative peak areas of the 19F resonance signals versus concentration. Dimer and two-subunits stacked G-quadruplex conversions followed by 19F NMR spectroscopy. (B) Profiles of the relative peak areas of the 19F resonance signals versus temperature. Two-subunits stacked G-quadruplex/dimer G-quadruplex/single strand conversions followed by 19F NMR spectroscopy. To obtain the relative peak signal of each conformation, the total value of the relative peak signal for three conformations was estimated to be 1.0. Plotting the values of relative peak signal against temperature results in two melting curves for the two-subunits stacked Gquadruplex and dimer G-quadruplex.
	Figure 4. In-cell 19F NMR of telomere RNA G-quadruplex. (A) Schematic overview of in-cell 19F NMR experiments. Xenopus laevis oocytes are sorted and collected for microinjections. For in-cell 19F NMR applications in Xenopus oocytes, telomere RNA sample was injected into the oocyte cells. Comparison with the position of reference in vitro spectrum provides a reliable determination of intracellular telomere RNA conformation. (B) Comparison of 19F NMR spectra of in vitro sample of telomere RNA (up) with Xenopus egg lysates (middle) and in Xenopus oocytes (bottom).
	Figure 5. 19F NMR of telomere RNA G-quadruplex in molecular crowding conditions. (A–D) 19F NMR spectra of 12-mer telomere RNA (0.5 mM) in crowded solutions induced by (A) ACN, (B) DMSO, (C) EtOH and (D) PEG 200. 19F NMR spectra were recorded under 30% (v/v), 40% (v/v) and 50% (v/v) of ACN, DMSO and PEG 200. The crowding condition of EtOH was only simulated by the addition of 30% (v/v) and 40% (v/v) EtOH, due to 50% (v/v) EtOH leading to a RNA precipitation. Red and green spots indicated the dimeric G-quadruplex and two-subunits stacked G-quadruplex. 19F NMR reference spectrum of 19F labeled 12-mer telomere RNA in dilute solution is shown in each case for comparison.
	Figure 6. A temperature-dependent experiment of telomere RNA G-quadruplex in molecular crowding conditions. NMR spectra were recorded in crowded solution simulated by 40% (v/v) and 50% (v/v) of ACN. Condition: 0.5 mM RNA in 50 mM KCl and 10 mM Tris-HCl buffer (pH 7.0). The sample is kept for 10 min at each temperature. Red and green spots indicated the dimeric G-quadruplex and two-subunits stacked G-quadruplex. The peaks of single strand are marked with black spots. Temperatures indicated on the right.
	Figure 7. Melting profiles of the relative peak areas of the 19F resonance signals versus temperature. The profiles were obtained by plotting the normalized two-subunits stacked G-quadruplex relative peak areas of the 19F resonance signals in NMR spectra (Figure 2C, Figure 6 and Supplementary Figure S14) as a function of temperature.

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