Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2
Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.
KEYWORDSmitochondria intermembrane space ribonuclease mtRNA RNA degradation decay RNASET2 RNase T2 inner membrane transport RNA trafficking
Mammalian mitochondria contain a small circular genome, which encodes 2 ribosomal RNAs, 22 tRNAs, and 13 essential protein subunits in the OXPHOS pathway (Anderson et al., 1981). The transcription and translation machineries in the mitochondrial matrix require both mitochondrion-encoded RNAs and nucleus encoded protein and RNA factors (Bonawitz et al., 2006; Rubio et al., 2008; Wang et al., 2010). Mitochondrial RNAs (mtRNAs) are first transcribed as polycistronic transcripts from both H-strand and L-strand, and the transcripts are dissected by RNA processing enzymes into individual rRNAs, tRNAs, and mRNAs (Hallberg and Larsson, 2014; Mercer et al., 2011). These rRNAs and tRNAs together with translational factors imported from cytosol then synthesize the OXPHOS proteins from the mRNAs (Hallberg and Larsson, 2014).
Much is understood about mtRNA synthesis and processing (Sanchez et al., 2011; Schafer et al., 2005). By contrast, less attention has been given to understanding how mtRNAs are degraded. Yet, mitochondrial RNA homeostasis is one of the key elements in regulating mitochondrial functions. Mitochondria have to quickly respond to external stimuli and are constantly going through fusion and fission, which is directly linked to their biosynthesis and requires fast changes of their gene expression (Mishra and Chan, 2016). RNA decay adds a new dimension to the regulation network by changing the abundance of transcripts and removing aberrant RNA molecules and intermediates of transcription and processing. The process must be tightly regulated and often is performed by multi-protein complexes (Szczesny et al., 2012).
In yeast, mtRNAs are degraded by a degradosome complex consisting of a helicase Suv3 and an RNase II like protein Dss1 (Dziembowski et al., 1998; Margossian et al., 1996; Szczesny et al., 2013). Suv3 unwinds and feeds RNA substrates to Dss1, which acts as a 3′ to 5′ exoribonuclease that yields nucleoside monophosphates and a four-nucleotide residual core (Malecki et al., 2010).
In comparison, the degradation machinery of mammalian mtRNAs is much less clear. Even though the role of hSuv3p helicase has been demonstrated, the ribonuclease partner has not been identified (Khidr et al., 2008). Human polynucleotide phosphorylase PNPASE has been shown to be involved in mtRNA degradation and appears to colocalize with Suv3 in distinct mitochondrial foci, but the protein is mainly localized in the mitochondrial intermembrane space (IMS) and functions in mitochondrial RNA import from cytosol (Borowski et al., 2013; Chen et al., 2006; Chujo et al., 2012; Sato et al., 2017; Vedrenne et al., 2012; von Ameln et al., 2012; Wang et al., 2010). Whether its function in mitochondrial RNA degradation is that of a ribonuclease has never been proven. Other ribonucleases such as LACTB2 and Endo G have also been identified in mitochondria (Cote and Ruiz-Carrillo, 1993; Levy et al., 2016; Ohsato et al., 2002; Zhou et al., 2016). However, the exact mechanisms of their involvement in mtRNA degradation have never been resolved. It has long been assumed that mitochondrial RNA decay would occur in the mitochondrial matrix as both yeast and human Suv3 proteins were identified in the matrix. Here we show that contrary to the assumption, mtRNA degradation happens in the mitochondrial intermembrane space (IMS) and IMS-localized RNASET2 is the ribonuclease that carries out the degradation.
Characterization of in organello mtRNA degradation
To identify the ribonuclease for mtRNA degradation, an in vitro assay is not sufficient, as multiple ribonucleases have been identified in mitochondria (Bruni et al., 2013; Cote and Ruiz-Carrillo, 1993; Levy et al., 2016; Zhou et al., 2016). The ribonuclease that functions directly in mtRNA degradation should have characteristics similar to those of mtRNA degradation. In order to find the ribonuclease, we designed a system that allows in organello characterization of mtRNA degradation and examined the dependency of mtRNA degradation on temperature, pH, ATP, and metal ions. Newly synthesized mtRNAs in isolated mitochondria were labeled with P32 or Biotin. Controls have been performed to show that the isolated mitochondria had no nuclear DNA contamination and the labeled RNAs were mtRNAs in the intact mitochondria as no labeling was observed in the rho0 mitochondria (Fig. S1A–E). Degradation of the newly synthesized mtRNAs was examined under various chasing conditions. Slower degradation was observed at 25°C than at 37°C (Fig. 1E), and lower pH had an inhibitory effect on the decay (pH 6.5 vs. pH 7.4) (Fig. 1F). The effects of ATP and di-valence metal ions were more complicated: Cu2+ had a strong inhibitory effect even at a low concentration (0.5 mmol/L) (Fig. 1G), while Mg2+ had little effect on the degradation at a low concentration (0.5 mmol/L) but an inhibitory effect at a higher concentration (20 mmol/L) (Fig. 1H). ATP had a similar effect as that of Mg2+, little effect at a low concentration (0.5 mmol/L) but inhibitory at a higher concentration (8 mmol/L) (Fig. 1I).
mtRNAs are degraded in the mitochondrial intermembrane space (IMS)
The IMS activity was further characterized and was shown to be inhibited by Cu2+ as the in organello mtRNA degradation was, but not by EDTA (Fig. 2D). The activity also degraded total mtRNAs with similar characteristics, and like the in organello mtRNA degradation, its inhibition by Mg2+ or ATP was also concentration dependent (Fig. 2E and 2F). In addition, it was inactivated by heating at 90°C, but not by proteinase K treatment (Fig. 2G).
Identification of an IMS-localized ribonuclease RNASET2
Since the IMS ribonuclease activity is insensitive to proteinase K treatment, we tested whether RNASET2 could be digested by proteinase K. Consistent with the IMS result, both the endogenous RNASET2 and the tagged version were resistant to proteinase K digestion, while TIM23 was readily degraded by the protease (Fig. 4E). The tagged version ran lower on SDS-PAGE after proteinase K treatment simply because the tag was digested by the protease as shown by a lack of immunoblotting signal for the tag after the treatment (Fig. 4E). Because RNASET2 was proteinase K insensitive, Mitoplasting and NaCl wash instead of the traditional Mitoplasting and protease treatment was performed to examine the sub-mitochondrial localization of the endogenous RNASET2. Endogenous RNASET2 showed a fractionation profile similar to that of an IMS protein DDP2 but different from those of a membrane protein BAP37 and a matrix protein Mortalin, suggesting an IMS localization of RNASET2 within mitochondria (Fig. 4F).
To rule out the possibility that the localization of RNASET2 in mitochondria we observed was due to contamination from other cellular compartments, we took advantage of a new technique called proximity-based labeling, which has been used to overcome the problem of fractionation contamination (Han et al., 2017; Kim and Roux, 2016; Williams et al., 2014). Ascorbate peroxidase (APEX) biotinylates proteins in close proximity when biotin-phenol and H2O2 are added to the medium of live cells (Jan et al., 2014). APEX was used to tag RNASET2 and a mitochondrial IMS protein MIA40. Subcellular fractionation and mitoplasting with or without proteinase K treatment was performed to ensure that APEX and other tags had no effect on the localization of the proteins (Fig. 4G). Biotinylation of mitochondrial IMS protein PNPASE was observed only when MIA40-APEX or RNASET2-APEX was expressed but not when cytosolic APEX was expressed at a similar level, further proving a mitochondrial localization of RNASET2 (Fig. 4H). This technique, however, lacks specificity; so another proximity labeling approach was adopted. Biotin ligase (BirA) biotinylates Avi-tagged proteins when they are in close proximity (Roux et al., 2012). BirA was used to tag a mitochondrial outer membrane protein TOM22 with its C-terminus facing the IMS and Avi was used to tag RNASET2 and MIA40. Subcellular fractionation and mitoplasting with or without proteinase K treatment was again performed to ensure that BirA, Avi, and other tags had no effect on the localization of the proteins and that BirA on the C-terminus of TOM22 is in the IMS (Fig. 4I). TOM22-BirA but not cytosolic BirA quickly biotinylated both MIA40-Avi and RNASET2-Avi, when Biotin was added to the medium, proving once again RNASET2 is localized in the mitochondrial IMS (Fig. 4J).
RNASET2 functions in mtRNA degradation and indirectly affects mtRNA synthesis
Since RNASET2 is a protein with multiple subcellular localizations, the effects of overexpression and knockdown on mtRNA degradation could be indirect and be due to changes of its functions in other cellular compartments. To rule out the possibility, we fused an enzymatically dead mutant (C65R, C118R) without the N-terminal 24 amino acids to the targeting sequence of cytochrome C1 that targeted the protein to the IMS (C1ΔNT2M). Unlike the wild-type RNASET2, C1ΔNT2M localizes predominately in the mitochondria (Fig. 5J). This enzymatically dead mutant, when targeted to the IMS, acting like a dominant negative mutant, had similar effects on the in organello mtRNA synthesis and degradation as those of RNASET2 knockdown (Fig. 5K and 5L). Under in vivo condition that was inhibitory to mitochondrial RNA synthesis, C1ΔNT2M expressing mitochondria contained significantly higher levels of mitochondrial rRNAs and showed less structural defects (Figs. 5M and S4). The mild effect of C1ΔNT2M on mitochondrial mRNA levels is possibly due to the feedback down-regulation of mtRNA synthesis, as there is no efficient approach to quickly turn off mtRNA synthesis without dramatically affecting other cellular processes (Fig. 5K). Taken together, these data suggest the mitochondrial pool of RNASET2 indeed functions in mtRNA degradation and its ribonuclease activity is required.
Purified RNASET2 has characteristics similar to those of in organello mtRNA degradation
The response of purified RNASET2 to pH is of great interest. Protozoan RNASET2s are mostly acidic ribonucleases (Irie, 1999), but the human RNASET2 appears to favor more of a neutral pH (Fig. 6F). This change could be due to changes of the amino acid sequence or post-translational modification in mammalian cells. To find the reason behind the change, human RNASET2 was expressed in bacteria and the protein was purified under denaturing conditions (Fig. S5A). Again, the purified protein was insensitive to proteinase K treatment (Fig. S5B). The responses to temperature, pH, ATP, Cu2+ and Mg2+ were also identical to the protein purified from mammalian cells (Fig. S5C–F), suggesting the activity of human RNASET2 is intrinsically different from its protozoan counterparts.
Another IMS localized nuclease is Endo G (Cote and Ruiz-Carrillo, 1993; Ohsato et al., 2002; Zhou et al., 2016). To examine whether it has similar characteristics as in organello mitochondrial RNA degradation and can potentially be also involved in the process, we purified human Endo G from HEK mitochondria and E. coli (Fig. S6A–E). Unlike RNASET2, Endo G was sensitive to proteinase K and the responses to the conditions tested were vastly different from those of RNASET2 or in organello mitochondrial RNA degradation. Thus, a direct involvement of Endo G on mitochondrial RNA degradation seems unlikely. That the human RNASET2 purified from mammalian cells and bacteria showed responses to so many conditions almost identical to those of the in organello mtRNA degradation together with its IMS localization further verified it as the enzyme responsible for mtRNA degradation.
For decades, the identity of the ribonuclease responsible for degradation of mammalian mitochondrial RNAs (mtRNAs) had been a great mystery. Our attempt at unraveling this mystery uncovered a mitochondrial IMS-localized ribonuclease activity that degrades mtRNAs and is sensitive to many cellular conditions such as pH and Ca2+. We have done extensive studies to rule out the possibility that the action on the substrates was due to a cytosolic contamination or a mitochondrial leak. Localization of such a ribonuclease activity in the mitochondrial IMS guarantees a faster change of mitochondrial biogenesis and quick regulation of mitochondrial functions in response to signals outside of mitochondria.
At first glance, degradation of mtRNA in the IMS instead of the matrix where transcription and translation occur seems a little radical. However, this is not the first time a nuclease is reported in the IMS. REXO2, a 3’ to 5’ exonuclease specific for small oligomers also has an IMS localization (Bruni et al., 2013). It could act on oligonucleotides yielded by RNASET2 and recycle the NTPs for new mtRNA synthesis. Another example is Endo G, a nuclease reported to primarily localize in mitochondrial IMS and function on degradation of both nuclear and mitochondrial DNA under special circumstances (Cote and Ruiz-Carrillo, 1993; Ohsato et al., 2002; Zhou et al., 2016). Proteinase K insensitivity of RNASET2 is also a surprising finding. However, it is not a unique feature. Same characteristic is shared by ribonucleases in Leishmania tarentolae mitochondria (Alfonzo et al., 1998; Simpson et al., 1992).
Degradation of mtRNAs in the mitochondrial IMS means there are active transports of RNAs from the matrix across the mitochondrial inner membrane. Both mitochondrial RNA import and export have been previously reported. Mitochondria import a wide array of RNAs from the cytosol, including tRNAs, 5S rRNA and other non-coding RNAs, and the import pathways have been partially characterized (Chang and Clayton, 1989; Duchene et al., 2009; Mercer et al., 2011; Noh et al., 2016; Smirnov et al., 2011; Wang et al., 2010; Zhang et al., 2014). Mitochondrion-derived MitomiRs could also be exported to cytosol to function in post-transcriptional regulation of gene expression (Bienertova-Vasku et al., 2013; Duarte et al., 2015). These existing import and export pathways could potentially be the pathways for substrate delivery to the IMS ribonuclease activity. More studies are needed to understand substrate selection and the transport pathways.
The mammalian IMS ribonuclease activity shown here is quite different from yeast mtRNA degradosome and bacterium degradosome (Dziembowski et al., 1998; Miczak et al., 1996; Szczesny et al., 2012). Yeast degradosome has its ribonuclease component Dss1 in the matrix, and bacterium degradosome ribonuclease PNPASE is absent in yeast (Miczak et al., 1996; Wang et al., 2010). Mammalian PNPASE resides in mitochondrial IMS. It has been reported to be involved in mtRNA degradation and processing (Clemente et al., 2015; Daoud et al., 2012), but based on our results, a direct role as mtRNA ribonuclease seems unlikely. We have shown that mammalian mitochondrial membranes have no apparent ribonuclease activity (Fig. 2A and 2B), and PNPASE, a mitochondrial membrane bound protein, has no ribonuclease activity (Fig. 1A–C). A known role for mammalian PNPASE is importing nucleus-encoded RNAs into mitochondria (Sato et al., 2017; Vedrenne et al., 2012; von Ameln et al., 2012; Wang et al., 2010), so it could have trafficking role in substrate transport for the IMS ribonuclease activity. Evidence that mammalian PNPASE is not the ribonuclease for mtRNA degradation but could be involved in their transport also comes from RNAi silencing of PNPASE expression. In some cases, a decrease of mtRNA level was observed; while in others, there were no significant changes (Slomovic and Schuster, 2008).
An important question that arose from this study is how the RNA degradation activity is coordinated with the transcriptional activity in mitochondria. Mitochondria appear to be capable of quickly up-regulate transcriptional activity when the IMS RNA degradation activity is up, and vice versa, hence maintaining relatively stable RNA levels (Fig. 5). A signaling pathway or pathways are clearly needed for such regulation. The responses of both RNASET2 activity and in organello mitochondrial RNA degradation to conditions such as ATP and pH also suggest coupling and a feedback circuit between mitochondrial RNA degradation and ETC activity/ATP synthesis.
Interestingly, RNASET2 has been shown to be involved in cancer suppression and loss of function mutation causes cystic leukoencephalopathy (Acquati et al., 2011; Henneke et al., 2009). An enzymatic dead mutant without the N-terminus also has tumor suppression activity (Nesiel-Nuttman et al., 2015). However, we have shown that truncation of the N-terminus led to mislocalization of the protein, so possible involvement of its ribonuclease activity in tumor suppression has not been rule out yet. Since mammalian RNASET2 has little ribonuclease activity at low pH, the function of lysosomal pool remains to be elucidated. More work is also needed to understand its physiological roles.
MATERIALS AND METHODS
Cell lines used include HEK293, HeLa, TM6, and stable cell lines generated on these cell lines. See Supplemental Experimental Procedures for details.
Plasmids used include PQCXIP-RNASET2-HAHisPC, PQCXIP-C1-ΔN-RNASET2 (C65R, C118R)-HAHisPC, PQCXIP-RNASET2-GFP, PQCXIP-RNASET2 (C184R)-GFP, PET28A-RNASET2-HisPC, PQCXIP-EndoG-HAHis, PET28A-EndoG-HAHis, PQCXIP-APEX-HisPC, PQCXIP-RNASET2-APEX-HisPC, PQCXIP-MIA40-APEX-HisPC, PQCXIP-PNPASE-HAHis, PQCXIP-MIA40-Avi-HisFlag, PQCXIP-RNASET2-Avi-HisFlag, and PQCXIP-TOM22-BirA-HAHis. See Supplemental Experimental Procedures for construction details.
Isolation of crude mammalian mitochondria and cytosol
Cells were washed once with PBS buffer, resuspended in ice-cold mitoprep buffer (0.225 mol/L manifold, 0.075 mol/L sucrose, and 20 mmol/L HEPES pH 7.4), and broken in a glass-Teflon homogenizer on ice with 30 strokes. Nuclei and unbroken cells were pelleted at 700 ×g for 5 min, and the homogenization repeated once. Supernatants from both times were centrifuged again at 700 ×g. Crude mitochondria were pelleted from second-round supernatants at 11,000 ×g for 5 min, washed once with mitoprep buffer and resuspended in mitoprep buffer of desired volume for further use. Post-mitochondrial supernatant was spun at 21,000 ×g for 10 min and the supernatant was collected as cytosol.
Hypotonic treatment was performed by incubating mitochondria for 20 min on ice by diluting mitoprep buffer with 10 volumes of 20 mmol/L HEPES pH 7.4 with one gentle vortexing at 10 min. Mitoplasts were separated from IMS by centrifugation at 15,000 ×g for 4 min. Salt wash was performed by adding 300 mmol/L of NaCl (from 5 mol/L stock) into mitoprep buffer (in cases of intact mitochondria) or mitoplasting mixture (in cases of mitoplasts) after 20 min on ice for another 5 min. Mitochondrial matrix was isolated by sonicating the salt-washed mitoplast in mitoprep buffer and separating the soluble fraction (matrix) from membrane by 10 min 21,000 ×g centrifugation. Total soluble was isolated by sonicating mitochondria in mitoprep buffer and separating the soluble fraction (total soluble) from membranes by 10 min 21,000 ×g centrifugation. The pellet was washed twice with mitoprep buffer and used as total membranes.
Mitoplasting after in organello mtRNA synthesis was carried out by proteinase K treatment. 500 μg mitochondria in 200 μL in organello synthesis mixture were pelleted by 12,000 ×g 4 min spin, washed with 1 mL mitoprep buffer, and resuspended in 300 μL mitoprep buffer with 2 μg proteinase K. The mixture was incubated on ice for 15 min with one vortexing at 8 min. 1 mL mitoprep buffer was added after the incubation and mitoplasts were pelleted at 15,000 ×g for 4 min, washed once with 1 mL mitoprep buffer, and proceeded to in organello degradation. Control mitochondria samples skipped the last two spins, as spinning and resuspending at this stage ruptures the outer membrane. This new method was used to ensure degradation of some inner membrane bound proteins that might be involved in mtRNA export from the matrix.
In vitro degradation assay
In vitro degradation assay for PNPASE was performed as previously described (Wang et al., 2010). All the other assays were performed in 20 mmol/L HEPES pH 7.4 at 37°C for 10 min if not otherwise specified. Mitochondrial lysates that contain membrane fractions had 0.5% Triton X. For experiments with membrane samples, equal amount of Triton X was added into all samples, normally the final concentration not exceeding 0.1% because of dilution. ~2 μg of IMS, ~2 μg of Matrix or ~10 μg of membrane was used for each 20 μL reaction. For purified protein samples, RNaseI (Thermo) and RNaseA (Thermo), 50 ng was used for each reaction. Reactions with bacterial samples were incubated for 20 min instead of 10 min. Substrates included 1 ng biotinylated UCP2, and 300 ng mtRNA. For effects of metal ions on the degradation, different concentrations of MgCl2, CuSO4, MnCl2, or ZnCl2 were used. Reaction was stopped by adding equal volume of SDS-Urea-EDTA buffer (2× SDS loading buffer with 8 mol/L urea and 15 mmol/L EDTA) and incubating at 90°C for 5 min. Samples were cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane, and detected with nucleic acid detection kit (Thermo). mtRNA samples were run either on SDS-PAGE or agarose gels and stained with EtBr.
In organello RNA synthesis
In organello RNA synthesis was performed in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2 and 1 μL Biotin RNA Labeling Mix (Roche) with 500 μg mitochondria at 37°C. For each time point (0 min, 30 min, and 60 min), 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step. For loading, samples were taken out of −80°C, quickly dissolved in 30 μL SDS-Urea-EDTA buffer (SDS loading buffer with 8 mol/L urea and 15 mmol/L EDTA) preheated to 90°C, and incubated at 90°C for 5 min. Samples were then cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane (400 mA for 1.5 h), and detected with nucleic acid detection kit (Thermo). These synthesis conditions are less inhibitory to mtRNA degradation, and were used for the in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions.
For comparison of in organello synthesis between different cell lines, more inhibitory synthesis conditions were used to ensure the yield of newly synthesized RNA more representing the real synthesis rate. Therefore, in organello mtRNA synthesis were carried out in buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 0.5 mmol/L CaCl2, 10 mmol/L MgCl2, and 1 mg/mL HEK cytosol.
In organello mtRNA degradation
For in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions, 500 μg mitochondria first underwent mtRNA synthesis in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2, and 1 μL Biotin RNA Labeling Mix (Roche) at 37°C for 45 min. Mitochondria were pelleted at 12,000 ×g for 4 min at 4°C, washed with 1 mL ice cold mitoprep buffer, resuspended in 150 μL mitoprep buffer, and incubated on ice for 15 min with one vortexing at 8 min. 50 μL ice cold cocktail containing 4 mmol/L UTP, 40 mmol/L Ca2+, 1 μL Ribolock RNase inhibitor (Thermo), and 1 μg RNaseI (Thermo) (original buffer exchanged to mitoprep buffer) in mitoprep buffer was added to the sample (RNase inhibitor was used to eliminate cytosolic interference and RNaseI was used to quickly degrade the RNA from broken mitochondria). The samples were shifted to 37°C. For each time point (0 min, 30 min, and 60 min), 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step. For loading, samples were taken out of −80°C, quickly dissolved in 30 μL SDS-Urea-EDTA buffer (SDS loading buffer with 8 M urea and 15 mmol/L EDTA) preheated to 90°C, and incubated at 90°C for 5 min. Samples were then cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane (400 mA for 1.5 h), and detected with nucleic acid detection kit (Thermo).
For comparison of in organello mtRNA degradation between different cell lines, in organello mtRNA synthesis were carried out in buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 0.5 mmol/L CaCl2, and 1 mg/mL HEK cytosol for 45 min at 37°C before the following wash and degradation steps. Mitochondria should be isolated from stable cells lines grown to less than 5 doubling times from the initial frozen stocks.
Biotin-phenol labeling with APEX in live cells
500 μmol/L biotin-phenol (ApexBio) was added to cell medium for 30 min. Then 1 mmol/L of H2O2 was added and the plates were gently agitated for 1 min. The reaction was quickly quenched by replacing the medium with DPBS containing 5 mmol/L Trolox (abcam) and 10 mmol/L sodium ascorbate (Solarbio). Cells were then washed with the same solution three times. For Ni-NTA (Qigen) and HA bead (Thermo Scientific) enrichment of PNPASE, mitochondria were isolated from the biotin-labeled cells and PNPASE was first purified from the lysate under denaturing condition using Ni-NTA beads and then using HA beads under native conditions according to the manufactures’ instructions.
In vivo biotinylation of Avi-tagged protein by spatially localized biotin ligase (BirA)
HEK293 cells co-transfected with the Avi-tagged protein expressing plasmid and BirA fusion protein expressing plasmid were gown in medium containing 25 μmol/L D-Biotin (Amresco) for 12 h. The cells were harvested, washed with PBS time times, lysed in 1× SDS protein loading buffer, and run on SDS-PAGE for biotin detection.
Additional procedures include protein purification, Western blotting, mtRNA isolation, qPCR, Optiprep gradient centrifugation, identification of IMS ribonuclease, fluorescence microscopy and image acquisition, and in vitro transcription. See Supplemental Experimental Procedures for details.
We thank Haiteng Deng for the help on Mass Spec and Zhi Lu and Hongwei Wang for discussion. This research was supported by the Priority Research Program of the Ministry of Science and Technology 2017YFA0504600, the National Natural Science Foundation of China (Grant Nos. 31371439 and 91649103), and Ministry of Education 1000 youth program.
APEX, ascorbate peroxidase; BirA, biotin ligase; IMS, intermembrane space; mtRNAs, mitochondrial RNAs
COMPLIANCE WITH ETHICS GUIDELINES
Peipei Liu, Jinliang Huang, Qian Zheng, Leiming Xie, Xinping Lu, Jie Jin, and Geng Wang declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
- Portnoy V, Palnizky G, Yehudai-Resheff S, Glaser F, Schuster G (2008) Analysis of the human polynucleotide phosphorylase (PNPase) reveals differences in RNA binding and response to phosphate compared to its bacterial and chloroplast counterparts. RNA 14:297–309CrossRefPubMedPubMedCentralGoogle Scholar
- Sato R, Arai-Ichinoi N, Kikuchi A, Matsuhashi T, Numata-Uematsu Y, Uematsu M, Fujii Y, Murayama K, Ohtake A, Abe T et al (2017) Novel biallelic mutations in the PNPT1 gene encoding a mitochondrial-RNA-import protein PNPase cause delayed myelination. Clin Genet. doi:10.1111/cge.13068 PubMedGoogle Scholar
- Vedrenne V, Gowher A, De Lonlay P, Nitschke P, Serre V, Boddaert N, Altuzarra C, Mager-Heckel AM, Chretien F, Entelis N et al (2012) Mutation in PNPT1, which encodes a polyribonucleotide nucleotidyltransferase, impairs RNA import into mitochondria and causes respiratory-chain deficiency. Am J Hum Genet 91:912–918CrossRefPubMedPubMedCentralGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.