The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

Abstract

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Shelterin complex and associated factors at human telomeres

“…36 In addition to their roles on the telomeric chromatin, TERRAs also exert competitive and non-competitive inhibition on telomerase itself: they can hybridize with hTR, the telomerase RNA, with high affinity and interact with the catalytic subunit, hTERT. 41 Therefore, the discovery of TERRAs has introduced an additional layer of complexity in the understanding of telomere function. Indeed, TERRAs were recently found to mediate interactions between specific accessory factors and TRF2, 42 as well as TRF1.…”

Promoting transcription of chromosome ends

“…TERRA-like short RNA oligonucleotides are able to inhibit telomerase activity in vitro, by base-pairing with the template region of telomerase RNA moiety. 5,28 Therefore, TERRA might repress telomerase-mediated extension of telomeres in vivo. It is also possible that the physical action of transcription of telomeres could influence telomere length, both in telomerase-positive and -negative cells, by stripping telomere length regulators off the telomeric sequence.…”

Structure and target interaction of a G-quadruplex RNA-aptamer

“…RNAs were desalted from remnant Na C or K C by denaturation in presence of LiOAc and subsequent ethanol precipitation, resuspended in ddH 2 O and prepared at 5 mM in 10 mM TrisHCl (pH 7.5) with or without salts added at different concentrations ranging from 100 nM to 100 mM (KCl, LiCl, BaCl 2 , NH 4 OAc or NaCl), heated to 80 C for 3 min and allowed to cool on ice for renaturation until measured. CD spectra were recorded using a Jasco J815 CD spectrometer.…”

“…Such examples comprise telomere maintenance by genomic DNA-GQs 1,2 or telomeric regulation by the counterpart transcript or telomeric RNAGQs (TERRA). [3][4][5] Furthermore, GQs hold regulative potential for gene expression either on the transcriptional level by being part of or close to promoters in genomic DNA (e.g. c-MYC or bcl2 promoter) 6,7 or as regulators of splicing 8 and translation 9 (inhibitory or enhancing) when present in mRNAs and finally as regulative elements by binding of certain effector proteins.…”