An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity

Abstract

Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease.

Figure 1

hTR overexpression protects from dexamethasone-induced apoptosis independent of telomerase activity. (A) Telomerase activity with hTR variant overexpression. Error bars show standard deviation of biological triplicates. (B) RNA levels of hTR overexpression represented as fold change over empty vector. Error bars show standard deviation of PCR triplicates. (C) Caspase-3/7 activity measured by luminescence. Luminescence normalized to background levels. Error bars represent standard error of the mean of biological triplicates. (D) Caspase-3/7 activity measured by luminescence when hTERT is knocked down. Error bars represent standard error of the mean of biological triplicates. **P < .001 assessed by 1-way ANOVA. Solid bars: no dexamethasone treatment; checkered bars: treatment with 1μM dexamethasone.

Figure 2

hTR knockdown induces Bim-mediated apoptosis. (A) Telomerase activity in CD4 T cells in culture with different lentiviral vectors. Representative example of 10 different experiments using cells from 8 different donors; each experiment was performed in triplicate. Error bars show standard deviation of biological triplicates. (B) hTERT mRNA and hTR RNA levels in CD4 T cells. Error bars show standard deviation of the mean of 2 experiments. (C) Live cell counts measured by trypan blue exclusion. Representative example from 10 different experiments using cells from 8 different donors. Error bars show standard deviation of biological triplicates. (D) Stages in cell cycle measured by DyeCycle Green. Error bars show standard deviation of biological triplicates. (E) Caspase-8, -9, -3/7 activity, measured by luminescence. Error bars represent standard deviation of biological triplicates. One-way ANOVA was performed for each caspase. *P < .05. (F) Western blot measuring Bim, Puma, Bad, Bid, and p53 in shScramble, shTERT, shTR1, and shTR2 cells. (G) Cell survival measured by trypan blue exclusion with Bim knockdown followed by hTR knockdown, compared with empty vector knockdown followed by hTR knockdown. Error bars represent standard deviation of biological duplicates. EV, empty vector; shScr, shScramble.

Figure 3

Telomerase knockdown does not induce significant telomere shortening or telomere DNA damage-induced foci (TIFs) in the time frame of this experiment. (A) Cumulative frequency of telomere lengths measured by PNA intensity. (B) Telomeres detected per area. (C) TIFs detected per telomere measured by colocalization of γH2ax foci and PNA foci. (D) TIFs detected per γH2ax foci. (E) γH2ax foci detected per area. Statistical significance was assessed using 1-way ANOVA and Dunn’s multiple comparison test with a significance cutoff of P < .05 in Prism (GraphPad). NV, no virus.

Figure 4

hTERT overexpression increases telomerase activity and induces apoptosis. (A) Telomerase activity with overexpression of hTERT variants. Error bars show standard deviation of biological triplicates. (B) RNA levels with overexpression of hTERT variants. Error bars show standard deviation of PCR triplicates. (C) Live cell counts with overexpression of hTERT variants. Error bars represent standard error of the mean of biological triplicates. (D) Apoptosis measured by caspase-3/7. Error bars show standard deviation of biological triplicates. *P < .05 assessed by 1-way ANOVA.

Figure 5

Overexpression of catalytically inactive hTR mutants protects from hTERT-induced apoptosis. (A) Telomerase activity with hTR and hTERT cooverexpression. Error bars represent standard deviation of biological triplicates. (B) RNA levels with hTR and hTERT cooverexpression. Error bars represent standard deviation of PCR triplicates. (C) Live cell counts with hTR and hTERT cooverexpression. Error bars represent standard deviation of biological triplicates. (D) Caspase-3/7 activity measured by luminescence. Error bars represent standard deviation of biological triplicates. Significance assessed by unpaired Student t test. **P < .01; ***P < .001; ****P < .0001.

Figure 6

Two functions for hTR. hTR and hTERT complex to form catalytically active telomerase to maintain telomeres. In this catalytically active conformation, hTR complexes with hTERT and other factors to elongate telomeres. hTR also functions in a catalytically inactive state (shown here as unbound to hTERT with a disprupted pseudoknot) to prevent apoptosis. In a catalytically inactive state, hTR may be able to bind other factors to protect from apoptosis. Dyskerin is depicted because it is necessary for hTR accumulation, but some other binding partner might be involved with hTR to prevent apoptosis. Red lines: template; green lines: Δ96-7 hTR mutant; orange lines: P6.1 stem disrupted by G305A.