FIG 3.

TERT RdRP generates short RNA strands de novo. The IP-RdRP assay was performed with HeLa cells treated with nocodazole, using [α-³²P]UTP (A to E) or [γ-³²P]ATP (F). Synthetic RNA (RNA 1) was used as a template. (A) IP-RdRP assay. All four types of ribonucleotides (control) or rUTP alone [rNTP (−)] was added. (B) Supplemented ribonucleotides in the IP-RdRP assay were replaced by 3′-dNTPs at the indicated concentrations. (C) Suppression of RNA synthesis by a telomerase inhibitor. β-Rubromycin was used as an inhibitor in the IP-RdRP assay. (D) Suppression of RNA synthesis but not telomerase activity by an RdRP inhibitor. HeLa cells treated with nocodazole were used for the IP-RdRP and IP-TRAP assays. VX-222 (100 μM) was used as an inhibitor. IC, internal control. (E) No effects of α-amanitin on RNA synthesis. α-Amanitin at the indicated concentrations was used for the IP-RdRP assay. (F) IP-RdRP assay using [γ-³²P]ATP. HeLa cells treated with nocodazole or DMSO were used. (G) RT-qPCR of TERC in HeLa cells treated with nocodazole. Total RNA was extracted from cell lysate (input; 0.13%) or TERT immune complexes (IP; 2%) prepared for the RdRP assay. RT (−), the reverse transcriptase reaction was performed without reverse transcriptase.