Telomeres, which are located at the chromosomal ends, act as protective caps that prevent chromosome loss and degradation. Telomeres had always been thought to be transcriptionally silent until the recent finding that they could be transcribed into RNA molecules with lengths ranging from 100 to 9000 nt (1-2). It has further been shown that telomeric-repeat-containing RNA (TERRA) perform various cellular regulatory functions, such as regulation of telomere length, inhibition of telomerase, telomeric heterochromatin formation, and telomere protection (3-7).

Our structural studies showed that human TERRA sequences formed propeller-type parallel-stranded RNA G-quadruplexes. We have determined the NMR-based solution structure of a dimeric propeller-type RNA G-quadruplex formed by the 12-nt human TERRA sequence r(UAGGGUUAGGGU). We also observed the stacking of two such propeller-type G-quadruplex blocks for the 10-nt human TERRA sequence r(GGGUUAGGGU) and a higher-order G-quadruplex structure for the 9-nt human TERRA sequence r(GGGUUAGGG).

Ribonuclease protection assay was used to investigate the structures formed by long human TERRA (9-10). We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e. 5’-5’, 3’-3’, 3’-5’, and 5’-3’, demonstrated stacking feasibility for all but the 5’-3’ arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5’-5’ stacking conformation. Based on the results, we propose a “beads-on-a-string”-like arrangement along human TERRA (11), whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively (10).

References

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