Mitochondrial Trafficking and Processing of Telomerase RNA TERC Ying Cheng, Peipei Liu, Qian Zheng, Ge Gao, Jiapei Yuan, Pengfeng Wang, Jinliang Huang, Leiming Xie, Xinping Lu, Tanjun Tong, Jun Chen, Zhi Lu, Jisong Guan, Geng Wang  Cell Reports  Volume 24, Issue 10, Pages 2589-2595 (September 2018) DOI: 10.1016/j.celrep.2018.08.003 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 24, 2589-2595DOI: (10.1016/j.celrep.2018.08.003) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Telomerase RNA TERC Is Imported into Mitochondria and Processed to a Shorter Form within Mitochondria (A) Alignment of the mitochondrial import signal of H1 RNA with a similar sequence in hTERC. (B) Primer design for amplification of different segments of hTERC. (C) PNPASE immunoblot of HEK293 (HEK) and PNPASE-overexpressing (PNP) cells. β-Tubulin was used as a loading control. (D) Immunoblots of cytosol (Cyto), mitochondria (Mito), and mitoplasts (MP) from HEK293 (HEK) and PNPASE-overexpressing (PNP) cells; Tubulin (cytosol), Mortalin (mitochondrial matrix), TOM40 (mitochondrial outer membrane), and TIM23 (mitochondrial inner membrane). (E) RNA isolated from total cell lysates or nuclease-treated mitoplasts of HEK293 (HEK) or PNPASE-overexpressing (PNP) cells were used as templates for RT-PCR with primers for hTERC: f-1 and r-1 (hTERC-long) and f-2 and r-2 (hTERC-short). GAPDH was used as a cytosolic marker and COX2 as a mitochondrial marker. (F) In vitro import of hTERC into yeast or human mitochondria. Upper panels: in vitro–transcribed hTERC, yeast tRNAT, or human ND6 RNA was incubated with mitochondria from the PNPASE-expressing (PNP) or control (Vec) yeast cells, or from PNPASE-overexpressing (PNP) or control (Vec) HEK293 (HEK) cells. Non-imported RNA was digested with nuclease. Lower panels: immunoblots of mitochondrial loading controls: Tom70 for yeast mitochondria and Mortalin for HEK293 mitochondria (HEK). (G) Quantification of the relative import level (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (H) hTerc sequence with the red arrows indicating the processing sites. Statistical comparisons are performed using unpaired t-tests; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are presented as mean ± SEM. Cell Reports 2018 24, 2589-2595DOI: (10.1016/j.celrep.2018.08.003) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 TERC Is Exported out of Mitochondria after Being Processed within Mitochondria (A) Immunoblots of different cellular fractions: total cell lysate (T), the nucleus (N), the cytosol (C), and mitochondria (M). Creb, β-tubulin, and mortalin were used as markers for the nucleus, the cytosol, and mitochondria, respectively. (B) Northern blots of hTERC-53 in equal cellular volume of nuclear, cytosolic, and mitochondrial fractions as shown in (A). Twice the amount of RNA was loaded in the lower panel compared with the upper panel. (C) Quantification of the relative hTERC-53 levels in different cellular fractions (n = 3). ∗∗∗p < 0.001. (D) Northern blots of hTERC-53 in different cellular fractions isolated from HEK293 (HEK) or PNPASE-overexpressing (PNP) cells. Two exposures were shown. 5S rRNA was used as a loading control for the two cells. (E) Quantification of the relative cytosolic hTERC-53r level in HEK- or PNPASE-overexpressing cells (n = 3). ∗∗∗∗p < 0.0001. (F) In vitro processing assay. Full-length hTERC (I as Input) was incubated with equal cellular volume of nuclear (N), cytosolic fractions (C), and IMS isolated from the mitochondrial fraction (M) as shown in (A). To compensate for the strong degradation in the IMS, we loaded five times the amount of final IMS mixture. (G) Full-length hTERC was incubated with purified RNASET2 (T2) or pulldown control from E. coli cells with empty vector (vec). Upper panel: RNA gel; lower panel: RNASET2 (T2) immunoblot. (H) Full-length hTERC was incubated with purified RNASET2 (T2) (1.5 ng, molecular weight [MW] 36 kDa) or RNase I (1 ng, MW 27 kDa). (I) Northern blots of cytosolic hTERC-53 and 5S rRNA in HEK cells (con), RNASET2-overexpressing cells (T2), or RNASET2 knockdown cells (KD) (top two panels), and immunoblots of the three cell lysates (T2: RNASET2; ActB: β-Actin) (bottom two panels). (J) Quantification of the relative cytosolic hTERC-53 levels in (I) (n = 3). ∗∗p < 0.01. (K) In vitro processing of hTERC or hTERC with a deletion from nucleotides 1–63 (Δ1-63) by RNASET2 (T2). (L) Northern blots of cytosolic hTERC-full (including hTERC and hTERCΔ1-63), hTERC-53 (including hTERC-53 and the possible processing product of hTERCΔ1-63) and 5S rRNA in HEK cells (con), hTERC-overexpressing cells (hTERC), or hTERCΔ1-63-overexpressing cells (Δ1-63). (M) Quantification of the relative cytosolic hTERC-full and hTERC-53 levels in (L) (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001. Statistical comparisons are performed using unpaired t-tests; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are presented as mean ± SEM. Cell Reports 2018 24, 2589-2595DOI: (10.1016/j.celrep.2018.08.003) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 In Vitro Export of TERC-53 from Mitochondria and Mitoplasts (A) In vitro export assay. After import of hTERC in HEK293 mitochondria, mitochondria were washed and resuspended in export buffer with (+) or without (−) the cytosol. Samples were taken out at different time points and spun to yield the mitochondrial pellet (P) and the supernatant (S). One set of samples was used for biotin detection (upper) of hTERC and the other for RNA isolation and RT-PCR to analyze the endogenous ND6 mRNA (lower). (B) Mitochondria (M) were treated with digitonin to wash away the outer membrane (mitoplast) or sonicated to rupture both outer and inner membranes (So). The membrane fraction or the remaining mitoplast was pelleted, and immunoblotting was performed with antibodies for Mortalin (Matrix), TIM23 (inner membrane), and DDP2 (IMS). (C) Export of hTERC from HEK mitoplast. After hTERC import, mitoplasting was performed and the in vitro export assay was performed with the mitoplasts. P, pellet; S, supernatant. (D) Comparison of hTERC export from HEK293 (HEK) mitochondria with that from PNPASE-overexpressing (PNP) mitochondria. Cell Reports 2018 24, 2589-2595DOI: (10.1016/j.celrep.2018.08.003) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Mitochondrial Function Impairment Affects Cytosolic TERC-53 Level, but Not Vice Versa (A) Northern blots of hTERC-53 in different cellular fractions (N: nucleus, C: cytosol, M: mitochondria) isolated from HEK293 cells with (+) or without (−) FCCP treatment (10 μM for 16 hr). 5S rRNA was used as a loading control for the treated and the untreated. (B) Quantification of the relative cytosolic hTERC-53 level in HEK cells with or without FCCP treatment (n = 3). ∗∗∗p < 0.001. (C) Northern blots of cytosolic hTERC-53 and 5S rRNA in HEK cells treated with different amounts of FCCP (top two panels), and immunoblots of mitochondrial lysates (T2: RNASET2) (bottom three panels). (D) Quantification of the relative cytosolic hTERC-53 level in (C) (n = 3). ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (E and F) OCR measurement of control cells (CON) and cells overexpressing hTERC-53 (53) or hTERC-53r (53R). Basal and spare respiratory capacity (E); proton leak and ATP production (F). Three concentrations (25,000, 30,000, and 35,000 cells/well) of cells were used. Statistical comparisons are performed using unpaired t-tests; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are presented as mean ± SEM. Cell Reports 2018 24, 2589-2595DOI: (10.1016/j.celrep.2018.08.003) Copyright © 2018 The Author(s) Terms and Conditions