Telomeric repeat-containing RNA structure in living cells

Abstract

Telomeric repeat-containing RNA (referred to as TERRA), a noncoding RNA molecule, has recently been found in mammalian cells. The detailed structural features and function of the TERRA RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In the present studies, we investigated the structural features of human TERRA RNA in living cells. Using a light-switching pyrene probe, we found that human TERRA RNA forms a parallel G-quadruplex structure in living cells, providing the in vivo evidence for the presence of the G-quadruplex in human TERRA RNA. Furthermore, imaging experiments clearly show that TERRA RNA G-quadruplex localizes to telomere DNA at cell nuclei. These results provide valuable information to allow understanding of the structure and function of human TERRA RNA.

Fig. 1.

Schematic structure of human TERRA RNA G-quadruplex. A G-tetrad is formed by hydrogen bonds between adjacent guanines. Whether TERRA RNA G-quadruplexes exist in living cells is unknown.

Fig. 2.

Design and characterization of TERRA RNA G-quadruplex-specific pyrene excimer probe. (A) Use of the pyrene excimer to probe G-quadruplex structure. The pyrene molecule has monomer emission near ∼400 nm. G-quadruplex formation brings the pyrene molecules close to each other. Consequently, pyrene excimer (green) forms, and green light (∼480 nm) is emitted after photoexcitation. (B) Fluorescence spectra for pyrene-modified oligonucleotide probes. (C) Chemical structures of pyrene probes with different linker lengths (L and R), RNA chain sequences, and numbers of pyrenes. E/M is the excimer/monomer fluorescence intensity ratio (E at 480, M at 380). (D) Response of excimer probe 5 to KCl solution. (E) Fluorescence image of probe 5 without (Left) and with (Right) KCl after illumination with a UV lamp (365 nm).

Fig. 3.

TERRA RNA G-quadruplex formation in living cells. (A) Schematic for detection of RNA G-quadruplex formation in living cells using dual-pyrene probe 5. G-quadruplex formation in living cells will induce the excimer fluorescence. (B) Fluorescence microscopy images of live cells. (iv–vi) HeLa cells were incubated with excimer probe 5, (vii–ix) probe 7, or (i–iii) buffer as a negative control. (C) Real-time response of excimer probe 5 and two pyrene-labeled control probes, 2 and 7, to 200 mM KCl. Probe 2 is a 5′-end pyrene-labeled TERRA RNA; 7 is a random sequence with pyrene labeled at both ends. (D) Time-lapse imaging of G-quadruplex formation in live cells using excimer probe 5.

Fig. 4.

Intracellular localization of TERRA RNA G-quadruplex. (A) Localization of TERRA RNA G-quadruplex at cell nuclei was determined by costaining using probe 5. Green signals correspond to the probe 5 (green). Cell nuclei were stained with SYTO 25 dye (red). The merged panel indicates a colocalization event. (B) Telomere DNA FISH experiments were performed to demonstrate that TERRA RNA localizes to telomeric DNA. TERRA RNA G-quadruplexes signals are in green (probe 5), telomeric DNA signals are red that are shown by situ hybridization using Cy5-labeled peptide nucleic acid probe, colocalization of the two signals generates orange signals in the merged panel. (C) Localization of TERRA RNA G-quadruplex was determined by costaining using antibodies anti human TRF 2. TERRA RNA G-quadruplexes signals are in green (probe 5), TRF 2 signals in red, colocalization of the two signals generates orange signals in the merged panel. (D) Detection of TERRA RNA G-quadruplex at the ends of metaphase chromosome. TERRA RNA G-quadruplexes signals are in green (probe 5); chromosome DNA was stained by propidium iodide (red).