Report
Mitochondrial Trafficking and Processing of Telomerase RNA TERC Graphical Abstract
Authors Ying Cheng, Peipei Liu, Qian Zheng, ..., Zhi Lu, Jisong Guan, Geng Wang
Correspondence
[email protected]
In Brief The functions of most mitochondrial RNAs imported from the cytosol are poorly understood. Cheng et al. provide evidence that the RNA component of mammalian telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. The cytosolic TERC-53 level serves as a potential indicator of mitochondrial functions.
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The RNA component of mammalian telomerase TERC is imported into mitochondria TERC RNA is processed to a shorter form, TERC-53 TERC-53 is exported from the mitochondria and remains in the cytosol Cytosolic TERC-53 levels respond to changes in mitochondrial functions
Cheng et al., 2018, Cell Reports 24, 2589–2595 September 4, 2018 ª 2018 The Author(s). https://doi.org/10.1016/j.celrep.2018.08.003
Cell Reports
Report Mitochondrial Trafficking and Processing of Telomerase RNA TERC Ying Cheng,1 Peipei Liu,1 Qian Zheng,1 Ge Gao,1 Jiapei Yuan,1 Pengfeng Wang,2 Jinliang Huang,1 Leiming Xie,1 Xinping Lu,1 Tanjun Tong,2,3 Jun Chen,2,3 Zhi Lu,1 Jisong Guan,1 and Geng Wang1,4,* 1MOE
Key Laboratory of Bioinformatics, Cell Biology and Development Center, School of Life Sciences, Tsinghua University, Beijing 100084, China 2Peking University Research Center on Aging, Beijing 100191, China 3Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100191, China 4Lead Contact *Correspondence:
[email protected] https://doi.org/10.1016/j.celrep.2018.08.003
SUMMARY
Mitochondrial dysfunctions play major roles in many diseases. However, how mitochondrial stresses are relayed to downstream responses remains unclear. Here we show that the RNA component of mammalian telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. We found that the import is regulated by PNPASE, and the processing is controlled by mitochondrion-localized RNASET2. Cytosolic TERC-53 levels respond to changes in mitochondrial functions but have no direct effect on these functions. These findings uncover a mitochondrial RNA trafficking pathway and provide a potential mechanism for mitochondria to relay their functional states to other cellular compartments. INTRODUCTION Human mitochondrial dysfunctions have long been implicated in the development of many health problems such as metabolic defects, neurodegenerative diseases, and self-immune diseases (Bishop et al., 2010; Lo´pez-Otı´n et al., 2016; Sun et al., 2016). Mitochondria also play an active role in cancer cell metabolism and dedifferentiation (Vyas et al., 2016). Mutations that alter mitochondrial bioenergetics and biosynthesis are common in cancer cells, promoting oncogenesis by modulating signaling pathways and gene expression through mitochondrial retrograde signaling (Guha and Avadhani, 2013; Wallace, 2012). Mitochondrial retrograde signaling relays the functional state of mitochondria to the nucleus and other cellular compartments, sometimes even leading to cell fate determination (Guha and Avadhani, 2013; Zhang et al., 2011). The two major known mitochondrial retrograde signals are calcium ion and reactive oxygen species (ROS) (Gottlieb and Bernstein, 2016; Kotiadis et al., 2014; Reczek and Chandel, 2015; Sullivan and Chandel, 2014). Both, however, lack specificity, and neither could fully explain how mitochondria regulate a cellular process specifically. Mitochondria import a wide array of non-coding RNAs, including tRNAs, rRNAs, microRNAs, and long non-coding
RNAs (lncRNAs) (Alfonzo and So¨ll, 2009; Chang and Clayton, 1989; Mercer et al., 2011; Wang et al., 2010; Zhang et al., 2014). The import of most of these RNAs depends on PNPASE, a mitochondrial IMS (intermembrane space) protein, in mammalian cells (Sato et al., 2018; Vedrenne et al., 2012; von Ameln et al., 2012; Wang et al., 2010). The functions of most imported RNAs in mitochondria, however, remain unclear. Initially, we were trying to investigate the import and the mitochondrial functions of some lncRNAs. This led to an unexpected finding that one of the RNAs, the RNA component of Telomerase TERC (Gall, 1990), is processed in the mitochondria and then exported out of mitochondria. Partial impairment of mitochondrial function caused an accumulation of the mitochondrion-processed RNA in the cytosol. These findings provide a potential mechanism on how specificity is achieved during mitochondrial retrograde signaling.
RESULTS AND DISCUSSION Mitochondria import many nucleus-encoded non-coding RNAs, and the import process is regulated by mitochondrial IMS localized PNPASE in mammalian cells (Mercer et al., 2011; Wang et al., 2010). Some of these RNAs contain a distinct import signal, a small stem loop structure (Wang et al., 2010). By looking for both sequence and structure similarity, we identified a noncoding RNA with a similar stem loop, the RNA component of human telomerase hTERC (Gall, 1990) (Figure 1A). To examine whether it is localized in mitochondria, we designed two sets of primers that anneal to different parts of the polynucleotide, and performed RT-PCR using RNA isolated from total cell lysate or nuclease-treated mitoplasts from HEK293 or PNPASE-overexpressing cells (Figures 1B–1E). Mitoplasts with the mitochondrial outer membrane stripped away by digitonin treatment were used to ensure no cytosolic contamination (Figures 1D and 1E). Both sets of primers produced correspondent amplification products from the total RNA; however, only the pair that anneal to the middle of the hTERC yielded a clear band from the mitochondrial RNA (Figure 1E), indicating that hTERC might not be the full-length form within mitochondria. Overexpression of PNPASE did not have a clear effect on the level of hTERC in mitochondria, which we will explain later in this report (Figures 1C–1E).
Cell Reports 24, 2589–2595, September 4, 2018 ª 2018 The Author(s). 2589 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Figure 1. Telomerase RNA TERC Is Imported into Mitochondria and Processed to a Shorter Form within Mitochondria
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To further confirm hTERC’s mitochondrial localization and examine whether it is processed upon entering mitochondria, we performed the in vitro import assay using radiolabeled or biotin-labeled RNA and isolated yeast mitochondria or mammalian mitochondria. As expected, the RNA was imported into both yeast mitochondria and mammalian mitochondria, but not the control RNAs (tRNAT for yeast import and ND6 mRNA for mammalian import) (Figures 1F and 1G). The import into yeast mitochondria was increased by exogenous expression of human PNPASE. In mammalian cells, overexpression of PNPASE also led to an increase on the import level, which is different from the in vivo localization results and will again be explained later in the text. In both yeast and mammalian mitochondria, the imported RNA was processed into a shorter form (Figures 1F and 1G). A couple smaller bands that are possible processing and degradation intermediates were also detected. Import into rho0 yeast mitochondria was less efficient than the wild-type mitochondria, suggesting an active translocation instead of a passive delivery (Figures S1A and S1B). Drosophila cells do not have telomerase (Villasante et al., 2008). However, mitochondria iso2590 Cell Reports 24, 2589–2595, September 4, 2018
(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. b-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 (hTERCshort). 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 PNPASEexpressing (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.
lated from Drosophila S2 cells also imported hTERC, consistent with previous reports that mitochondrial RNA import is a universal process (Figure S1C) (Alfonzo and So¨ll, 2009; Chang and Clayton, 1987; Mercer et al., 2011; Wang et al., 2010; Zhang et al., 2014). Interestingly, import into S2 mitochondria produced bands of various sizes instead of a clear major band of about 200 nt as in human or yeast mitochondria (Figure S1C). To identify the exact processing sites, we ligated the RNA to either a 50 or 30 RNA adaptor. The products were then amplified using RT-PCR and sequenced. The majority of hTERC is processed at two sites within mitochondria: one between nucleotides 52 and 53 and the other between nucleotides 247 and 248, which yields a polynucleotide of 195 nt in length, consistent with the size of the major processed band in the in vitro import assay (Figures 1F–1H). Small variations of 1–2 nt at the 30 end were also observed. Deletion studies show that the first 52 nt are required for mitochondrial TERC import (Figures S1D and S1E). Surprisingly, deletion of the sequence similar to mitochondrial import signal in H1 RNA significantly increased TERC import efficiency instead of abolishing the import (Figures S1D and
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Figure 2. TERC Is Exported out of Mitochondria after Being Processed within Mitochondria
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(A) Immunoblots of different cellular fractions: total cell lysate (T), the nucleus (N), the cytosol (C), and mitochondria (M). Creb, b-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 PNPASEoverexpressing (PNP) cells. Two exposures were shown. 5S rRNA was used as a loading control for the two cells. (E) Quantification of the relative cytosolic hTERC53r 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: b-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 (D1-63) by RNASET2 (T2). (L) Northern blots of cytosolic hTERC-full (including hTERC and hTERCD1-63), hTERC-53 (including hTERC-53 and the possible processing product of hTERCD1-63) and 5S rRNA in HEK cells (con), hTERC-overexpressing cells (hTERC), or hTERCD1-63-overexpressing cells (D1-63). (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.
S1E), indicating the tertiary structure instead of a local secondary structure of the RNA is directly involved in the import. More studies are needed to understand the import process. To further confirm the existence and localization of this smaller form that we named hTREC-53, we performed northern blotting on RNA samples isolated from different cellular fractions. Overexpression and knockdown with an antisense hTERC-53r were performed to ensure the correct band was observed in the northern blots (Figures S2A and S2B). Different protein markers (Creb for the nucleus, b-Tubulin for the cytosol, and Mortalin for the mitochondrion) were used to check the purity of each fraction and ensure equal cell volume of each fraction was used (Figure 2A). Surprisingly, the majority of the hTERC-53 is in the cytosolic fraction, and only when more RNA was loaded and with
longer exposure time was a mitochondrial hTERC-53 band detected (Figures 2B and 2C). Overexpression of PNPASE led to a four to six times increase of hTERC-53 level in the cytosol, and knockdown of PNPASE in an immortalized mouse MEF cell line TM6 caused the cytosolic mTerc-53 to decrease to about half of its original level (Figures 2D and 2E; Figures S2C–S2E). The very selective cellular distribution of Terc-53 suggests that either there is a TERC processing activity in the cytosol, or that TERC processed within mitochondria is then exported out of mitochondria, even though the in vitro import assays and the northern blotting with PNPASE overexpression or knockdown favor the latter. To test these two possibilities, we isolated different cellular fractions and performed the in vitro RNA Cell Reports 24, 2589–2595, September 4, 2018 2591
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processing assay using biotin-labeled full-length hTERC. Only in the fraction isolated from mitochondria was the full-length hTERC processed to the short form of correct size (Figure 2F). To examine whether the activity was strictly inside the mitochondria or on the outer surface of mitochondrial outer membrane, we incubated hTERC with the isolated mitochondria in the isotonic buffer. Under the processing condition that does not have ATP or succinate, no import occurred (Figure S2F), and without the addition of RNase I, no degradation or processing of hTERC was observed (Figure S2G). Only when the mitochondrial outer membrane was ruptured with hypotonic treatment (mitoplasts [MP]) was hTERC processed to the short form of correct size (Figure S2G). We have previously identified a ribonuclease RNASET2 in mitochondria that degrades mitochondrial RNAs (Liu et al., 2017). In vitro processing assay showed that the purified RNASET2, but not RNase I, processed hTERC to yield hTERC-53 (Figures 2G and 2H). The processing seems to be substrate dependent, as a fragment of 16S rRNA similar in size to the full-length hTERC was completely degraded by RNASET2 (Figure S2H). The processing activity was inhibited by 10 mM Mg2+, consistent with the mitochondrial activity (Figure S2I). Overexpression of RNASET2 led to an increase of the cytosolic hTERC-53 level, and the knockdown had the opposite effect (Figures 2I and 2J). Deletion of nucleotides 1–63 (hTERCD1-63) rendered hTERC more sensitive to the degradation activity of RNASET2 (Figure 2K). In HEK293 cells expressing hTERCD163, even though hTERCD1-63 level is slightly higher than endogenous hTERC level, no significant accumulation of a processing product was observed, consistent with the in vitro assay (Figures 2L and 2M). The in vitro RNA processing assay results together 2592 Cell Reports 24, 2589–2595, September 4, 2018
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.
with the in vitro import assay results strongly indicate that TERC-53 outside of mitochondria is originated in the mitochondria and is the processing product of mitochondrial RNASET2, and that there is indeed an export of RNA from mitochondria. The lack of the processing activity in isolated cytosol or on the outside of the isolated mitochondria also makes it unlikely that the cytosolic TERC-53 observed was a result of subcellular fractionation (Figures 2F and S2G). To further prove and study the trafficking process, we set up an in vitro export assay and found that a certain amount of cytosolic fraction in the buffer is required (Figure 3A). Without the cytosolic fraction, little RNA was exported even after 15 min, whereas with the addition of the cytosolic fraction, the majority of the RNA was exported at 15 min (Figure 3A). Endogenous ND6 mRNA that is encoded in the mitochondrial genome was also examined to ensure that the cytosol did not have any negative effect on the mitochondrial integrity (Figure 3A). Mitoplasting was performed after import, and export of the processed hTERC-53 from the mitoplasts was also observed, suggesting hTERC was indeed imported into the mitochondrial matrix and then exported (Figures 3B and 3C). We next examined whether PNPASE overexpression has any effect on the export. Within 6 min after the addition of the cytosol, the majority of the mitochondrial hTERC-53 was exported with or without PNPASE overexpression (Figure 3D). Even though PNPASE-overexpressing mitochondria originally imported more hTERC, a similar amount was retained within mitochondria after 12 min. These results are consistent with the northern blot results showing more hTERC-53 in the cytosol in PNPASE-overexpressing cells and provide an explanation for the inconsistency between the RT-PCR results and the in vitro import data
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in Figure 1. Overexpression of PNPASE increases hTERC import, but export is a more efficient process, and what remains in the mitochondria is possibly determined by the amount of interacting factors in the mitochondria. As a net result, more hTERC53 accumulates in the cytosol instead of mitochondria in the PNPASE-overexpressing cells (Figures 2D and 3D). Next, we asked what the biological significance of cytosolic TERC-53 and the whole process of mitochondrial hTERC import, processing, and export is. One possibility is that the process is a way of communication between mitochondria and other cellular compartments. If that is true, a change of mitochondrial function could have an observable effect on TERC-53 level. HEK293 cells were treated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (10 mM) for 16 hr, and the distribution of hTERC-53 was examined in different cellular fractions. As expected, the cytosolic hTERC-53 level changed, and a 2-fold increase of the cytosolic level was observed in the FCCP-treated cells compared with the control cells (Figures 4A and 4B). These results suggest that cytosolic TERC-53 level could be an indicator of mitochondrial functions. A gradient of FCCP concentrations was used, and the cytosolic hTERC-53 level was examined. An increase of the level was observed as the FCCP concentration rose from 0 to 5 mM; the level dropped when FCCP was further increased to 10 mM; and a dramatic increase of the level
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 mM for 16 hr). 5S rRNA was used as a loading control for the treated and the untreated. (B) Quantification of the relative cytosolic hTERC53 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 hTERC53 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.
was observed when FCCP concentration reached 100 mM (Figures 4C and 4D). The increase at 100 mM was possibly due to compromise of mitochondrial outer membrane integrity and release of the processing activity as shown by a decrease of mitochondrial PNPASE and RNASET2 levels (Figure 4C). Rotenone also showed concentration-dependent effects on cytosolic hTERC-53 level (Figures S3A and S3B). The cytosolic TERC-53 level appeared to only respond to mitochondrial functions because general DNA damage by UV treatment or endoplasmic reticulum (ER) stress by tunicamycin treatment did not have much effect on the cytosolic hTERC-53 level (Figures S3C–S3F). To address whether the effect of mitochondrial functions on cytosolic TERC-53 level is a one-way process, we overexpressed hTERC-53 and its antisense RNA hTERC-53r in HEK cells (Figures S2A and S4A). The cells were analyzed for respiration and glycolysis. Oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), which approximate glycolysis from lactate production, were measured using a XF24 extracellular Flux Analyzer. No significant changes of OCR or ECAR were observed between cells overexpressing hTERC-53 or hTERC-53r and the control cells (Figures 4E, 4F, S4B, and S4C), suggesting the cytosolic TERC-53 does not directly regulate mitochondrial functions in return. One of the key findings of this study is the mitochondrial import of TERC and the export of TERC-53 to the cytosol. Mitochondrial RNA import has been well established, with IMS localized protein PNPASE as a possible receptor (Mercer et al., 2011; Sato et al., Cell Reports 24, 2589–2595, September 4, 2018 2593
2018; Vedrenne et al., 2012; von Ameln et al., 2012; Wang et al., 2010; Zhang et al., 2014). Interestingly, PNPASE overexpression led to an accumulation of cytosolic TERC-53, but not the mitochondrial TERC-53. This indicates that cytosolic TERC-53 originated within mitochondria and that mitochondrial export is the dominant driving force of TERC-53 localization. Mitochondrial RNA export has been suggested in other reports (BienertovaVasku et al., 2013; Duarte et al., 2015). For example, mitochondrion-derived microRNAs (MitomiRs) have been detected in the cytosol and proposed to function in post-transcriptional regulation of gene expression (Duarte et al., 2015). However, the export process itself has never been experimentally confirmed. Our in vitro export assay proves unequivocally that there is an active mitochondrial export and that cytosolic factors are required for the process. In addition to RNA, mitochondria also import and export DNA. Uptake of DNA by mitochondria has been reported in plant, mammalian cells, and yeast, and export of mitochondrial DNA promotes antiviral innate immune responses (Koulintchenko et al., 2003; Weber-Lotfi et al., 2009, 2015; West et al., 2015). The functions of these nucleic acids sometimes differ wildly from their established functions. For example, microRNAs enhance mitochondrial translation instead of repressing translation as in the cytosol, and exported mitochondrial DNA functions as signaling molecules by engaging DNA sensor cGAS (West et al., 2015; Zhang et al., 2014). Together, these reports paint a much broader picture of nucleic acids trafficking in and out of mitochondria. A key question that this study presages is how the mitochondrial function is manifested through the cytosolic level of TERC53. The cytosolic level is a result of the balance between import and export. Because the pH of mitochondrial IMS is lower than the matrix (Marreiros et al., 2016) and the RNA molecule is negatively charged, translocation across the mitochondrial inner membrane is down the electrical gradient during import and against the gradient during export. In addition, the import and export are both ATP dependent. The final output is likely determined by a fine balance among different mitochondrial activities, such as ATP production and membrane potential maintenance. Partial impairment of mitochondrial membrane potential with low dosage of FCCP and rotenone would favor the export when the cellular ATP level is not greatly affected, and leads to accumulation of TERC-53 in the cytosol. However, when the mitochondrial functions are further disrupted, both import and export are negatively affected. More severe damage of mitochondrial functions would lead to mitophagy or apoptosis, which could result in leakage of the RNA processing activity and/or ribonuclease activities and dramatic changes of cytosolic TERC-53 level. More studies are needed to understand the cellular functions and the physiological functions of the cytosolic TERC-53. EXPERIMENTAL PROCEDURES RNA Import Assay RNA import was performed as previously described (Wang et al., 2010). RNA Export Assay After RNA import, micrococcal nuclease (Thermo) was inactivated with 10 mM EGTA. Mitochondria were pelleted, washed with MitoPrep buffer, and then resuspended in 160 mL of prewarmed (30 C) import buffer. At a different time
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point, 50 mL of mixture was taken out and spun at 16 kg for 3 min at 4 C. The supernatant was transferred to a new tube. Both the supernatant and pellet were kept at 80 C for at least 15 min before further analysis. Statistical Methods Statistical analysis was performed with GraphPad’s Prism Software. Statistical comparisons were performed using unpaired t tests; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are presented as mean ± SEM. Additional information and resources are included in the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at https://doi.org/ 10.1016/j.celrep.2018.08.003. ACKNOWLEDGMENTS We thank Dr. Bing Zhou for providing us the yeast strains. This research was supported by the Priority Research Program of the Ministry of Science and Technology of the People’s Republic of China (grant 2017YFA0504600), the National Natural Science Foundation of the People’s Republic of China (grants 31371439 and 91649103), and the Ministry of Education of the People’s Republic of China 1000 Talents Youth program. AUTHOR CONTRIBUTIONS Y.C., Q.Z., and G.G. performed the northern blotting, the import and export assays, and identified the excision sites. P.L. and J.Y. performed the rest of northern blotting, OCR and ECAR measurement, and the processing activity characterization. P.W., L.X., J.H., and X.L. performed most of the immunoblotting. T.T., J.C., Z.L., J.G., and G.W. conceived the ideas, designed the experiments, and wrote the paper. DECLARATION OF INTERESTS The authors declare no competing interests. Received: January 17, 2017 Revised: July 17, 2018 Accepted: July 31, 2018 Published: September 4, 2018 REFERENCES Alfonzo, J.D., and So¨ll, D. (2009). Mitochondrial tRNA import—the challenge to understand has just begun. Biol. Chem. 390, 717–722. Bienertova-Vasku, J., Sana, J., and Slaby, O. (2013). The role of microRNAs in mitochondria in cancer. Cancer Lett. 336, 1–7. Bishop, N.A., Lu, T., and Yankner, B.A. (2010). Neural mechanisms of ageing and cognitive decline. Nature 464, 529–535. Chang, D.D., and Clayton, D.A. (1987). A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J. 6, 409–417. Chang, D.D., and Clayton, D.A. (1989). Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate. Cell 56, 131–139. Duarte, F.V., Palmeira, C.M., and Rolo, A.P. (2015). The emerging role of mitomiRs in the pathophysiology of human disease. Adv. Exp. Med. Biol. 888, 123–154. Gall, J.G. (1990). Telomerase RNA: tying up the loose ends. Nature 344, 108–109. Gottlieb, R.A., and Bernstein, D. (2016). Mitochondrial remodeling: rearranging, recycling, and reprogramming. Cell Calcium 60, 88–101.
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