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.

Figure 1.

Endogenous TERRA is bound to telomerase. (A) Immunoprecipitation and analysis by western blotting. Immunoprecipitation was done with antibodies against tubulin (Tub), hnRNPA1 (A1) and the Myc-epitope. Western blots were probed with α-hTERT antibody (upper) and α-hnRNPA1 antibody (lower). I, input; IP, protein extracted from the beads after the immunoprecipitation; B, beads only control. One-fifteenth of the input and 1/10 of the immunoprecipitates was loaded. (B) RT-PCR for XpYp TERRA and hTR. RNA extracted from input (I) or from the beads after immunoprecipitation was analyzed. Immunoprecipitations were done with nuclear extracts from 293T cells transfected either with pBS-U1-hTR and pCDNA6MychTERT or with empty vectors. −RT: negative control in which no RT was added to the reaction.

Figure 2.

TERRA base pairs with the hTR template and interacts with the catalytic subunit of telomerase hTERT. (A) Interaction with telomeric RNA and DNA oligonucleotides. Top panel: [³²P] 5′-end labeled 5′-(UUAGGG)₃-3′, 5′-(AACUUU)₃-3′ or rA₁₈ was incubated with in RRL translated [³⁵S] methionine-labeled Flag-hTERT or Myc-hTERT which were both previously incubated or not with full-length in vitro transcribed wild-type hTR (hTR wt) or hTR bearing a template sequence that was complementary to the wild-type sequence (hTR comp) or a mutated template region (hTR C2). After immunoprecipitation with α-Myc antibodies and five washes, samples were separated on 4–20% gradient protein gels. One percent of the radiolabeled oligonucleotides and 8% of the radiolabeled proteins were loaded in the input, whereas 100% were loaded for the IP fractions. [³⁵S] and [³²P] signals were revealed by analysis on a PhosphorImager. By placing a plastic film between the gel and the PhosphorImager screen, only the [³²P] signal was detected (lower part). (A) Bottom panel: same experiment as in (A) top panel except that the experiment was done with DNA instead of RNA oligonucleotides: (TTAGGG)₃ and dA₁₈. (B) Interaction with oligoA containing telomeric RNA and DNA oligonucleotides. Top panel: [³²P] 5′-end labeled 5′-(UUAGGG)₃-3′ and 5′-(UUAGGG)₃A₆-3′. Bottom panel: 5′-(TTAGGG)₃-3′ and 5′-(TTAGGG)₃A₆-3′. (C) Quantification of oligonucleotides co-precipitated with Myc-hTERT based on the gels in panels (A) and (B) and one additional experiment. The values were normalized by the immunoprecipitation efficiency of the individual polypeptides and background signals present in the lane Flag-hTERT were subtracted from the values.

Figure 3.

TERRA inhibits telomerase activity without perturbing repeat addition processivity. (A) Telospot assay with 5′-(TTAGGG)₃-3′ as a substrate and serial titration of 5′-(UUAGGG)₃-3′. Each reaction was done three times (rows) and spotted in triplicate. EDTA (25 mM) was used as a positive control for complete inhibition. (B) Determination of IC₅₀. The graph shows the quantification of the data obtained in (A). The mean of the three spot intensities was used to calculate the IC₅₀ by fitting the data to a sigmoidal dose response curve. The indicated IC₅₀ value was calculated from nine independent experiments. (C) Direct telomerase activity assay in presence of 35 nM 5′-biotinylated 5′-(TTAGGG)₃-3′ primer and dATP, dTTP and ³²P-α-dGTP. Concentrations and identity of competitor oligonucleotides are indicated on the top. Reaction products were purified via the biotin-tag with streptavidin containing magnetic beads and resolved on an 8% polyacylamide sequencing gel. B-10mer (a 5′-radiolabeled and 3′-biotinylated 10-mer oligonucleotide) was used as recovery control and was added before DNA purification. The numbers on the left of the gel indicate the number of nucleotides added to the 3′ end of the primer. (D) Effects on repeat addition processivity. Signal intensities for each telomeric repeat seen in (C) were measured, corrected for the number of radiolabeled nucleotides incorporated and then plotted. Increased amounts of 5′-(UUAGGG)₃-3′ resulted in lines with the same slopes indicating no change in repeat addition processivity. However, increased amounts of BIBR1532 resulted in lines with steeper slopes indicating a decrease in processivity.

Figure 4.

TERRA is a mixed-type inhibitor of telomerase activity. (A) Telospot assay with increasing amounts of inhibitor. Telomerase activity was measured in presence of different concentrations (0–648 nM) of the TS primer (5′-AATCCGTCGAGCAGAGTT-3′). The total DNA oligonucleotide concentration was kept constant in each reaction through addition of dA₁₈ (from 648 to 0 nM). rA₁₈, (UUAGGG)₃ and BIBR1532 were added as indicated on the right side. Each reaction was spotted in triplicate. (B) Quantification of telomerase activity from (A) as a function of TS primer in presence of increasing amounts of (UUAGGG)₃. (C) Table with kinetic constants for two different primers and reaction constants for inhibitor binding. A simple reaction scheme (right side) was assumed in which the inhibitor (I) would be able to bind either to the free telomerase enzyme (E) or the enzyme–substrate (ES) complex. The constants were calculated from the following equations: ν₀ = V_max × [S]/(αK_M+α′[S]); ν₀ is telomerase activity measured on the membrane; [S] is the primer concentration; α=1 + [I]/K_I and α′ = 1 + [I]/K′_I; [I] is the inhibitor concentration. Results were obtained from three independent experiments for the inhibition with 5′-(UUAGGG)₃-3′ and from one experiment for the previously characterized telomerase inhibitor BIBR1532.

Figure 5.

Telomerase inhibition by TERRA is largely independent of its 3′ end register. (A) Direct telomerase activity assays were performed with 35 nM of 5′-biotinylated (TTAGGG)₃-3′ primer in the presence of dATP, dTTP and ³²P-α-dGTP, and increasing concentrations (indicated in nM) of rA₁₈, 5′-(UUAGGG)₃-3′ or permutations thereof as indicated, or 5′-(UUAGGG)₃A₆-3′. Reaction products were purified as in Figure 3B. B-15mer (a 5′-radiolabeled and 3′-biotinylated 15-mer oligonucleotide) was used as recovery control and was added before DNA purification. (B) Direct telomerase activity assay as in (A) but in presence of DNA oligonucleotide inhibitors as indicated. (C) Relative IC₅₀ values for permutations of 5′-(UUAGGG)₃-3′ or 5′-(UUAGGG)₃A₆- determined by the Telospot assay (three independent experiments; spotting data not shown).

Figure 6.

Proposed modes for telomerase sequestration by TERRA. TERRA (red line) base pairs with the telomerase RNA template (U-shaped blue line) and it interacts with the TERT polypeptide (dark-blue rounded rectangle). Three scenarios are modeled. (1) TERRA may be released from the telomere and bind and inhibit telomere-proximal telomerase molecules. (2) Telomere-bound TERRA may bind and sequester telomerase and prevent its access to the telomeric 3′ end. It is unknown how TERRA is bound to telomeric chromatin; telomeric chromatin binding of TERRA is modeled with the gray oval. (3) TERRA may bind to telomeric chromatin bound telomerase and prevents it from accessing the telomeric 3′ end. It is unknown how telomerase is bound to telomeric chromatin; telomeric chromatin binding of telomerase is modeled with the green oval. Human telomerase may be a dimer (see text), but for simplicity it is modeled here as a monomer.