An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA

Abstract

Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage-hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP.

Figure 1. hTERT and RMRP interact

a, Detection of RMRP and hTERC. RNA species associated with TAP-hTERT complexes from a single immunoprecipitation were isolated and subjected to RT-PCR. RT (−) indicates the absence of reverse transcriptase. Right panel shows the levels of TAP-hTERT. b, hTERT interacts endogenous RMRP. hTERT complexes from 293T and HeLa cells were isolated with an anti-hTERT antibody and associated RNAs were subjected to RT-PCR. c, RNAs purified from hTERT complexes isolated from HeLa-S cells expressing TAP-hTERT or a control vector or 293T cells were subjected to Northern blotting.

Figure 2. hTERT and RMRP exhibit RdRP activity

a, Predicted RNA products produced by RdRP or TT activity. b, RNA products produced by the RdRP activity derived from recombinant hTERT and RMRP. c,d Treatment of RNA products with RNase T1 (c) or bacterial RNase III (d). e, RdRP assay performed in the presence of ribonucleotides (middle) or in the absence of adenine (left lane) or guanine (right lane) ribonucleotides. A and G are present within the first 5 nt of the predicted complementary strand of RMRP. f, DN-hTERT binds RMRP but lacks RdRP activity. TERT immune complexes were isolated from 293T cells expressing FLAG-tagged hTERT or FLAG-tagged DN hTERT. RdRP activity is shown in lower panel. g, Northern blotting to detect complementary sequence of RMRP. h, Time course of RdRP activity. i, RNA products produced by recombinant hTERT and truncation mutants of RMRP transcribed in vitro. Faint signals at the 200 nt, 120 nt and 60 nt are hTERT TT products. j, RNA products produced by the RdRP activity derived from recombinant hTERT or DN hTERT and total RNA. A limited pool of RNAs serve a templates for RdRP activity.

Figure 3. Identification of ds RNA synthesized by the hTERT-RMRP RdRP

a, Northern blotting to detect complementary sequence of RMRP in cell lines. (+) indicates samples treated with RNase. b, Northern blotting to detect the RMRP sense strand. c, hTERT expression correlates with the levels of antisense RMRP detected by RNase protection assay. Vector denotes cells infected with a control vector. d, hTERT expression correlates with the levels of the sense (S) + antisense (AS) RMRP products detected by Northern blotting. The bottom panel shows U2 RNA levels.

Figure 4. Effects of ds RNA produced by the hTERT-RMRP RdRP

a, Semi-quantitative RT-PCR for total RMRP and retrovirally delivered RMRP (ectopic) in cell lines expressing control or RMRP expression vectors. Promoters used to express RMRP are indicated. The relative intensity of RMRP is noted below each panel. See Supplementary Fig. 14. b, RT-PCR for total RMRP. See Supplementary Fig. 15. c, Effects of suppressing hTERT on RMRP levels. A control shRNA (sh-GFP) or 2 different hTERT-specific shRNAs were stably introduced into HeLa cells. d, Effects of RMRP mutants on RMRP levels. RT-PCR was used to detect RMRP levels in (c, d). e, Detection of small RNA species in human cells. Northern blotting to detect small RNAs (22 nt in length) using antisense (left panel) and sense (right panel) probes derived from nt 21–40 of RMRP. f, g, Analysis of the termini of the small RNA species identified in (e). Total RNA was incubated with the indicated enzyme (f), or oxidation-β-elimination reactions (g) were performed. Northern blotting was performed with antisense probe. CIP = calf intestinal phosphatase. PNK = polynucleotide kinase. ATP-indicates samples lacking ATP.

Figure 5. Production of RMRP-derived endogenous siRNAs depends on Dicer

a, Effect of suppressing Dicer on RMRP-derived small RNAs. Northern blotting was performed to detect [1] small RNAs using the antisense strand of RMRP as a probe in the indicated cells expressing control shRNA (sh-GFP) or Dicer-specific shRNAs (sh-Dicer #1 and sh-Dicer #2), [2] pre-miR-16 and mature miR-16 using a miR-16 specific probe, and [3] U6 RNA. See Supplementary Fig. 16. b, RT-PCR for total RMRP from cell lines expressing control shRNA or Dicer-specific shRNAs. The relative intensity of RMRP is noted at the bottom of the panel. c, RMRP-derived small RNAs are associated with Ago2. Human Ago2 immune complexes were isolated using anti-hAgo2-specific antisera or pre-immune sera, and small RNAs were detected by Northern blotting. Blotting of oligonucleotides (RMRP 20–41 and RMRP AS 41-20) is also shown.