TDP-43 stabilises the processing intermediates of mitochondrial transcripts

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

The trans-activating response region DNA-binding protein 43 (TDP-43) is a member of the family of heterogeneous nuclear ribonucleoproteins and contains two highly conserved RNA recognition motifs (RRMs) and a non-conserved C-terminal region that mediates protein-protein interactions 1 . TDP-43 binds tightly the (UG) n motif 1-4 and helps regulate several aspects of mRNA biogenesis including transcription, pre-mRNA splicing and export, and mRNA stability 2,4,5 . TDP-43 is one of the causative gene products of amyotrophic lateral sclerosis (ALS) that is an adult-onset neurodegenerative disorder characterised by progressive degeneration of upper and lower motor neurons 6,7 . The majority of TDP-43 mutations that cause ALS are found in the C-terminal region, although some are found in the RRMs [8][9][10] . ALS mutant TDP-43 proteins, including those with mutation of the RRMs have an increased half-life in cell models 9,11,12 .
Although TDP-43 shuttles between the nucleus and the cytoplasm, it mainly resides in the nucleus under physiological conditions 13 . TDP-43 is cleared from the nuclear compartment and is incorporated into cytoplasmic ubiquitinated and/or hyperphosphorylated inclusions in patients with familial or sporadic ALS and in patients with frontotemporal lobar degeneration 6,[14][15][16] . Several animal models over-expressing wild type TDP-43 leads to neurodegeneration similar to that observed in ALS [17][18][19][20][21][22][23][24] , accumulate mitochondria in cytoplasmic inclusions in motor neurons 23 , lack mitochondria in motor axon terminals 24 , or have abnormal juxtanuclear aggregates Results TDP-43 binds mitochondrial (mt-) tRNA Asn , mt-tRNA Gln and mt-tRNA Pro directly in vivo. Given that TDP-43 contains RNA recognition motifs (RRMs), we first investigated the association of RNA with exogenously expressed TDP-43 using mass spectrometry (MS) and the Ariadne search engine [30][31][32] . We established human Flp-In TM T-REx TM 293 (T-REx 293) cell line expressing doxycycline-inducible wild-type human TDP-43 carrying a triple affinity-purification tag (DAP-tag: 6 × histidine, biotin, and FLAG; Supplementary Fig. 1a), and showed that the DAP-TDP-43 expression reduced the level of endogenous TDP-43, suggesting that the exogenous DAP-TDP-43 is behaved as native TDP-43 by constitutive negative feedback mechanism ( Supplementary  Fig. 1b) 4 . We pulled down TDP-43-associated RNAs and detected ~70-nt RNAs by denaturing urea-PAGE (Fig. 1a). The MS-based identification method revealed that the mitochondrial (mt) genome regions encoding mt-tRNA Asn , mt-tRNA Gln , and mt-tRNA Pro matched very well with our RNase T1-digested oligonucleotide fragments ( Fig. 1b and Supplementary Table 1). These three mt-tRNAs were also detected by northern blotting (Supplementary Fig. 1c) and are encoded in the L-strand of mtDNA. We confirmed the association of endogenous TDP-43 with these mt-tRNAs using anti-TDP-43 immunoprecipitation (Fig. 1c), and the interactions were determined to occur directly within intact cells as assessed with UV cross-linking in vivo and immunoprecipitation (Fig. 1d). To determine the region of TDP-43 responsible for the binding with the mt-tRNAs, we established T-REx 293 cell lines expressing TDP-43 mutants that had a domain defect (ΔRRM1, ΔRRM2, ΔGR, Δ315), a point mutation at amino acid residue 136, 140 or 145, or double mutations at residues 147 and 149 ( Supplementary Fig. 1d), and showed that residues K136, K145, and F147/149 in the TDP-43 RRM1 are critical for the interaction of TDP-43 with mt-tRNA Asn and mt-tRNA Gln (Supplementary Fig. 1e and Fig. 1e). We also utilised recombinant TDP-43 and synthesised five mt-tRNA Asn mutants in which nucleotide (nt) sequence replacements were made corresponding to a specific region of mt-tRNA Leu [Ac-stem(Leu), D-loop(Leu), pAntiCdn(Leu), Var-R(Leu), or T-loop(Leu)] as well as mt-tRNA Leu as a negative control ( Supplementary Fig. 1f). An electrophoretic mobility shift assay revealed that recombinant TDP-43 binds mt-tRNA Asn but not mt-tRNA Leu ( Supplementary Fig. 1g) and that the region corresponding to UGUUU (nt 44-48) of mt-tRNA Asn is required for interaction with TDP-43 (Supplementary Fig. 1f and Fig. 1f). Competitive interaction analysis using synthetic RNAs (sRNA-1, sRNA-2, sRNA-3, sRNA-4, and sRNA-5, Fig. 1g) suggested that an additional sequence GUGG (nt 49-53) in mt-tRNA Asn is required for inhibiting the binding to TDP-43 (Fig. 1g).

TDP-43 is localised in mitochondria in T-REx 293 cells.
To assess localisation of TDP-43 in mitochondria further, we treated a mitochondrial fraction with proteinase K and detected both endogenous and exogenous TDP-43; TDP-43 was not detected, however, if the mitochondrial membrane was permeabilised with Triton X-100 prior to proteinase K treatment (Fig. 2a). Furthermore, we detected endogenous TDP-43 in mitochondria by immunoelectron microscopy (Fig. 2b). Taken together, these data indicated that TDP-43 has the ability to associate directly with mt-tRNA Asn , mt-tRNA Gln , and mt-tRNA Pro present within the mitochondria of T-REx 293 cells.
TDP-43 stabilises not only the cellular levels of the TDP-43-bound mt-tRNAs but also that of TDP-43-unbound mt-tRNA. In human mitochondria, large polycistronic RNA transcripts are generated from the L-and H-strand promoters of mtDNA 33 . The L-strand encodes one oxidative phosphorylation system subunit (ND6) and eight mt-tRNAs (for Pro, Glu, Ser (UCN), Tyr, Cys, Asn, Ala, and Gln) ( Supplementary  Fig. 2a). Of these, the mt-tRNAs for Asn, Gln, and Pro bound directly to TDP-43. To gain insight into the physiological roles of the binding of TDP-43 to the L-strand-encoded mt-tRNAs, we examined whether TDP-43 is involved in stabilisation of those three mt-tRNAs via direct binding. We used ethidium bromide (EtBr) to inhibit mtDNA transcription and first examined the effect of EtBr on stability of L-strand coded mt-RNAs in T-REx 293 cells. EtBr reduced the levels of all of the L-strand-encoded mt-tRNAs with time but it did not affect a genomic tRNA Met (Supplementary Fig. 2b). It also reduced the levels of H-strand-encoded mt-tRNA Leu(UUR) (Supplementary Fig. 2b) and mRNAs for ND1, ND2 and COXI, whereas it did not affect that of 28S rRNA ( Supplementary Fig. 2c). We then examined the stabilities of TDP-43-bound mt-tRNA Asn and mt-tRNA Gln and TDP-43-unbound mt-tRNA Leu(UUR) and mt-12S rRNA by inhibiting mtDNA transcription with EtBr before and after doxycycline induction of TDP-43. Overexpression of TDP-43 slowed the degradation of mt-tRNA Asn  (Fig. 3a and b). Moreover, TDP-43 overexpression unexpectedly slowed the degradation of mt-tRNA Leu(UUR) , whereas it had only a negligible effect on the degradation of mt-12S rRNA (Fig. 3b). These results suggested that TDP-43 could increase the abundance of particular mt-tRNAs independent of mtDNA transcription. Although the stabilisation of mt-tRNA Asn and mt-tRNA Gln may be attributable, at least in part, to the direct binding of TDP-43, the stabilisation of mt-tRNA Leu(UUR) upon overexpression of TDP-43 cannot be attributed to stabilisation mediated by direct binding of TDP-43.
TDP-43 stabilises the L-strand-encoded mt-tRNAs and their precursors. We next used northern blotting to address whether overexpression of TDP-43 affects cellular levels of L-strand-encoded mt-tRNAs first other than those bound by TDP-43, using nuclear genome-encoded RNAs (tRNA Met , U1 snRNA, and 5.8S rRNA) as the references. Overexpression of TDP-43 significantly increased the cellular levels of all L-strand-encoded mt-tRNAs (Fig. 4a). Consistent with the result described above (Fig. 3b), it also increased the cellular level of H-strand-encoded mt-tRNA Leu(UUR) (Fig. 4a). In addition, the RNA species detected in the northern blots for mt-tRNA Asn , mt-tRNA Cys , mt-tRNA Tyr , mt-tRNA Ser(UCN) , and mt-tRNA Leu(UUR) appeared slightly larger than the mature transcripts upon overexpression of TDP-43 (Fig. 4b). These data suggested that TDP-43 might stabilise the production of L-strand-encoded mt-tRNAs and possibly H-strand-encoded mt-tRNAs at the level of their precursors or polycistronic RNA transcripts.
To explore this possibility, we next investigated the accumulation of L-strand-encoded polycistronic transcripts in TDP-43-expressing T-REx 293 cells by L-strand-specific reverse-transcription PCR (RT-PCR). The RT-PCR primers were designed to amplify the boundaries among mt-tRNAs or between mt-tRNAs and mt-mRNAs in the region containing mt-tRNA Tyr (Y)-mt-tRNA Ala (A), the region complementary to COXI mRNA (mirrorCOXI)-Y, mirrorND2-mt-tRNA Gln (Q), and mirrorCYB-mt-tRNA Glu (E) to test for cleavage within these boundary regions. All boundaries were found to increase in abundance with time after doxycycline induction compared with GAPDH mRNA (Fig. 4c). In addition, we used a synthetic precursor RNA encompassing a region containing mirrorCOXI and mt-tRNA Tyr -mt-tRNA Cys -mt-tRNA Asn -mt-tRNA Ala (YCNA) and confirmed the direct binding of the precursor RNA to recombinant TDP-43 ( Fig. 4d) and the stabilisation of the precursor RNA by recombinant TDP-43 in mitochondrial extracts (Fig. 4e). These results suggested that increased expression of TDP-43 stabilises the mt-tRNA-containing regions of mitochondrial polycistronic transcripts and increases the abundance of their processed products. Thus, TDP-43 appears to participate in the stabilisation of large polycistronic transcripts from L-strand mtDNA and regulates the cellular levels of the precursor and processed mt-tRNA products.
Two RNase activities are required for proper mt-tRNA and mt-mRNA maturation: mitochondrial RNase P (MRPP3)-mediated cleavage of precursor molecules at the 5′ end, and tRNase Z (ELAC2)-mediated cleavage at the 3′ end 34 . Therefore, accumulation of large precursor transcripts was also investigated by northern blotting after knockdown of MRPP3 or ELAC2 (Supplementary Fig. 3c and d). The analyses revealed that short interfering RNA (siRNA)-mediated knockdown of MRPP3 but not that of ELAC2 resulted in the accumulation of processing intermediates corresponding to 16S-mirrorQ (detected by probes 1-3), ND1-mirrorQ (detected by probes 3 and 4), and W-COXII (detected by probes 6-9) relative to 28S rRNA staining compared with a mock knockdown with a sequence-scrambled RNA (scRNA; Supplementary Fig. 3d). All these processing intermediates accumulated in WB with HRP-conjugated streptavidin or by NB with the probes indicated on the right. 5S rRNA served as a loading control for RNA and was detected using SYBR Gold staining. (f) Recombinant trigger factor-fused TDP-43 (TF-TDP-43) was mixed with synthesised mt-tRNA Asn or its mutant [Ac-stem(Leu), D-loop(Leu), pAntiCdn(Leu), Var-R(Leu) or T-loop(Leu)] shown in Supplementary Fig. 1f, and analysed by electrophoretic mobility shift assay. tRNAs were detected by NB with probes indicated on the left. (g) Top: RNAs were immunoprecipitated from TDP-43-expressing T-REx 293 cells in the absence (NT) or presence of synthetic RNA (sRNA-1, sRNA-2, sRNA-3, sRNA-4, or sRNA-5) and analysed by NB with probes indicated on the right. T-REx 293 cells (TR) served as a negative control. Bottom: Sequences of the synthetic RNAs. Asterisks correspond to the sequence that was replaced in Var-R(Leu).
the TDP-43-overexpressing cells (Fig. 5a-c). Thus, the phenotype observed with mitochondrial RNase P deficiency is similar to that observed upon overexpression of TDP-43.
Overexpression of TDP-43 causes mitochondrial dysfunction. Concurrent with the accumulation of processing intermediates, overexpression of TDP-43 in T-REx 293 cells caused irregular staining of mitochondria (Fig. 6a) and led to an increase in amount of reactive oxygen species in cells over time after doxycycline induction (Fig. 6b). Accordingly, TDP-43 overexpression inhibited cell proliferation, whereas the TDP-43 mutants having reduced ability to bind mt-tRNA Asn and mt-tRNA Gln (K136A, K145A, and F147/149L) did not inhibit cell proliferation (Fig. 6c). Notably, we also found that inhibition of mitochondrial transcription with EtBr abolished the inhibitory effect of excess TDP-43 expression on cell proliferation (Fig. 6d). A previous report demonstrated that the defects in the processing of mt-tRNA precursors impair the translation of mtRNAs 35,36 . To examine whether the cellular level of TDP-43 affects this translation, we estimated the levels of mitochondrial proteins in TDP-43-overexpressing cells and found apparently reduced levels of a number of proteins including ND2, ND5, CYB, and ATP8 at 24-48 h after doxycycline induction (Fig. 6e,f). These results suggested that TDP-43 overexpression deregulates the normal processing of mitochondrial polycistronic transcripts for the production of both tRNAs and mitochondrial proteins. Thus, TDP-43-induced cytotoxicity correlated with mitochondrial functions in human cells as reported for yeast cells expressing human TDP-43 26 .
TDP-43 is required for maintaining mitochondrial function. Given the results that TDP-43 affected the processing of mitochondrial polycistronic transcripts and the expression of their processing products, we used several approaches to determine the function of TDP-43 in mitochondria further. We first examined the effects of siRNA-mediated knockdown of TDP-43 on mitochondrial function. TDP-43 knockdown reduced mitochondrial membrane potential and the cellular levels of ATP and the enzyme activity of electron transfer complex I relative to levels in scRNA-treated cells (Fig. 7a-c). In addition, TDP-43 knockdown caused significant reductions on 16S-mirrorQ and mirrorN-COXI, as revealed by northern blotting (Fig. 7d). Thus, TDP-43 appears to be  mt-12S rRNA was detected by SYBR Gold staining and MS-based analysis (b). The graphs present data for the average ± SD (three independent experiments) of staining intensities at each time point relative to 0 h. Each band intensity was normalised to that of 5S rRNA detected using SYBR Gold staining. required for maintaining appropriate levels of at least certain mt-tRNAs, mt-mRNAs, and mt-mRNA precursors and for maintaining mitochondrial function.

Discussion
In human mtDNA, the genes are located in three different transcription units (one in the L-strand and two in the H-strand) that are transcribed to different extents. The one H-strand transcription unit that includes only the mt-rDNA region is transcribed most frequently and is responsible for producing the majority of the mt-rRNAs 37 . The product of the other H-strand transcription unit is a polycistronic RNA molecule corresponding to nearly the entire length of the H-strand that is a precursor for the majority of mt-tRNAs and mt-mRNAs 37 . This H-strand unit is transcribed at ~4% of the frequency of the H-strand mt-rDNA transcription unit 37 . On the other hand, the L-strand unit is transcribed 10-16 times more frequently than the nearly entire H-strand unit 33,37 . Our present data suggest that TDP-43 overexpression stabilises at least two regions (corresponding to mt-tRNA Pro and mt-tRNA Asn /mt-tRNA Gln ) of this L-strand transcription unit, although we cannot exclude the possibility that the entire L-strand transcript is stabilised by the binding of TDP-43 because the region containing mt-tRNA Pro is located in the first part of the L-strand mtDNA that is transcribed, and the region containing mt-tRNA Asn / mt-tRNA Gln is located at the 3′ end. It is also likely that TDP-43 binds to regions of mt-transcripts other than the regions corresponding to the three mt-tRNAs because of its general RNA-binding properties 1,2 . We also propose that the increase in the L-strand transcript and/or its processing products induced by TDP-43 overexpression may stabilise all or part of the H-strand transcript and/or its processing products through complementary association, resulting in increased levels of these processing products (Fig. 8). Rackham et al. 36 recently reported that mitochondrial MRPP3 is required for the biogenesis of mitochondrial ribosomal subunits that are produced co-transcriptionally on an unprocessed RNA containing the 16S rRNA. Our present data indicate that increased expression of TDP-43 causes accumulation of unprocessed RNA containing the 16S rRNA similar to that caused by conditional knockout of MRPP3 36 . In addition, the results that increased expression of TDP-43 upregulated the levels of mt-transcripts and their products are consistent with those of Rackham et al. 36 in that the conditional knockdown of MRPP3 enhances the rate of transcription and impairs protein synthesis. Moreover, it is also possible that TDP-43 stabilises mt-transcripts and increases the ratio of these to MRPP3, resulting in the accumulation of processing intermediates. Finally, we should note that the unprocessed RNA containing 16S rRNA (indicated as 16S-mirrorQ) seems to be corresponded to RNA 19 that is increased in diseases associated with mt-tRNA Leu(UUR) gene mutations 38,39 .
Because TDP-43 strictly controls its own expression through a negative feedback loop in which TDP-43 binds to the 3′ untranslated region in its own mRNA 4 , our findings provide important insight into the physiological roles of this negative feedback control and indicate that the strict control of TDP-43 expression is critical for maintaining mitochondrial homeostasis. In this context, mutations in TDP-43 that affect its stability or expression are expected to reduce mitochondrial function. Our present data provides a new mechanism underlying disruption of mitochondrial function by TDP-43 that differs from the mechanisms that involve perturbation of Mfn2 27 , the VAPB-PTPIP51 interaction 29 , or translation of ND3 and ND6 28 .

Immunoprecipitation of DAP-tagged protein associated complex. Confluent cells were washed
with PBS and suspended in five packed cell volumes of lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% (w/v) IGEPAL CA-630) containing 2 mM ribonucleoside-vanadyl complex, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin for 30 min on ice. After removal of any insoluble residue by centrifugation at 20,000 × g for 30 min at 4 °C, the supernatants were used as cell lysate for the following immunoprecipitation. Cell lysate (5 mg) prepared from 2 × 10 7 cells were incubated with 15 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) for 2 h at 4 °C. The beads were washed five times with 1 ml of lysis buffer, and proteins and RNA were eluted with 150 µl of Protein-RNA extraction buffer (7 M urea, 350 mM NaCl, 1% SDS, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 2% 2-mercaptoethanol) for 5 min. The eluted proteins and RNA were subjected to SDS-PAGE or denaturing (8 M) urea PAGE, respectively, as described previously 40-42 . In-gel RNA digestion and LC-MS for RNA analysis. In-gel RNA digestion was performed as described previously 31 . LC-MS analysis of RNA, and database search and interpretation of MS/MS spectra of RNA were performed as described 30,31,40,41 .
Northern blot analysis. Total RNA was isolated using the RNAgents Total RNA Isolation System (Promega). For analysis of mt-mRNAs, 5 µg of total RNA was subjected to 2% agarose/formaldehyde gel electrophoresis in 3-(N-morpholino)propanesulfonic acid running buffer. For analysis of mt-tRNAs, 1 µg of total RNA was subjected to denaturing (8 M) urea PAGE in Tris-borate-EDTA running buffer, stained with SYBR Gold (Invitrogen) for 10 min, and visualised using the LAS4000 Luminescent Image Analyzer System (Fujifilm). The separated total RNAs were transferred to a Hybond N + membrane (GE Healthcare). The membrane was dried and subsequently cross-linked using an ultraviolet cross linker (Funakoshi, Tokyo) at 120 mJ/cm 2 . For mt-mRNAs analysis, the membranes were stained with methylene blue. Hybridizatioin with biotin-labeled DNA probes and detection of RNA signals were performed as described previously 40,41 . The oligonucleotides used as probes are shown in Supplementary Table 2. UV-CLIP. T-REx 293 cells expressing DAP-TDP-43 were cross-linked by ultraviolet irradiation at 400 mJ/cm 2 , and DAP-TDP-43-associated complexes were immunoprecipitated with anti-FLAG M2 magnetic beads. After heating for 5 min at 65 °C in SDS sample buffer, DAP-TDP-43-associated complexes were released from the anti-FLAG M2 magnetic beads, and separated by SDS-PAGE using a Bis-Tris gel (Running gel: 360 mM Bis-Tis-HCl (pH 6.8), 8% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate; Stacking gel: 360 mM Bis-Tis-HCl (pH6.8), 5% acrylamide, 0.1% tetramethylethylenediamine, 1% ammonium persulfate) and Bis-Tris running buffer (50 mM Tris, 50 mM 3-(N-morpholino)propanesulfonic acid, 5 mM EDTA, 0.5% SDS). The separated proteins were electrophoretically transferred to a Protran BA 85 Nitrocellulose membrane (Whatman, Maidstone, UK). The membrane was blocked with 5% non-fat dried skim milk in TBST for 30 min, washed with TBST, and then incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific, San Jose, and pulled down with anti-FLAG beads. mirrorCOXI-YCNA was detected by northern blotting, and TF-FL or TF-TDP43-FL by western blotting with anti-FLAG. (e) mirrorCOXI-YCNA was incubated with mitochondrial extract from 293 T cells for the indicated times in the presence of TF-FL or TF-TDP43-FL. TFAM (transcription factor A mitochondrial protein) served as a loading control. Data in the graph represent the mean ± SEM of three independent experiments.
SCientifiC REPORts | 7: 7709 | DOI:10.1038/s41598-017-06953-y CA) for 30 min. After washing three times with TBST for 10 min, the Chemiluminescent Nucleic Acid Detection Module kit (Thermo Fisher Scientific) was used for detecting biotin of DAP-TDP-43. Stained parts of the membrane were excised and incubated in 250 µl of Proteinase K buffer (10 mM Tris-HCl pH 8.0, 30 mM NaCl, 10 mM EDTA, 2 mg/ml Proteinase K) at 37 °C for 30 min. After addition of 250 µl of Protein-RNA extraction buffer, RNA was isolated by phenol-chloroform extraction and isopropanol precipitation and subjected to northern blot analysis.  the manufacturer's instructions. Transcripts were collected by isopropanol precipitation, and purified using reverse-phase liquid chromatography. To synthesize biotinylated mirrorCOXI-YCNA RNA, template DNA for in vitro transcription was prepared. We amplified DNA fragments corresponding to mtDNA between mirror mt-tRNA Asn to COX I with the primer set (mirrorANCY-COXI-for/ mirrorANCY-COXI-for) using KOD neo plus (TOYOBO), and inserted them into the BamHI/PstI site of the pSPT19 vector (Roche) (mirrorANCY-COXI pSPT19). mirrorANCY-COXI pSPT19 linearlized by digesting with SmaI was used as template DNA for in vitro transcription. Biotin-labeled mirrorCOXI-YCNA was in vitro transcribed from the DNA template (0.06 pmol) in the presence of 7.5 mM of ATP, 7.5 mM of CTP, 7.5 mM of UTP, 7.5 mM GTP, 0.5 mM of biotin-UTP (Roche) using CUGA ® 7 in vitro transcription kit (NIPPON GENE, Japan), and purified by 1% agarose gel electrophoresis and the subsequent gel extraction using Zymoclean Gel RNA Recovery Kit (ZYMO RESEARCH).
EMSA. Each binding reaction was performed at room temperature for 20 min. Each reaction was performed in 10 µl binding buffer (40 mM Tris-HCl (pH 7.4), 30 mM KCl, 1 mM MgCl 2 , 0.01% IGEPAL CA-630, 1 mM DTT, 10 µg of yeast tRNA (Ambion), 10 µg BSA, 10 ng of synthetic RNA, 200 ng of recombinant protein), and electrophoresed on a 6% non-denaturing polyacrylamide gel at 100 V for 50 min in 0.5× Tris borate/EDTA buffer (44.5 mM Tris-borate and 1 mM EDTA). The separated RNA-protein complexes were transferred to a Hybond N+ membrane that was dried and UV-crosslinked using the FS-1500 crosslinker (Funakoshi) at 120 mJ/cm 2 . The synthetic RNA was detected by northern blot analysis using the indicated probes. DAP-TDP-43 was used for this assay. Synthetic RNA (sRNA) (1 pmol) was added to the total cell lysate and subjected to immunoprecipitation using anti-FLAG magnetic beads as described above. RNA isolated from DAP-TDP-43 complex was subjected to denaturing urea-PAGE and northern blot analysis.
Mitochondria Isolation. Mitochondria were prepared from T-REx 293 cells or 293 T cells grown in 5 × 15 cm 2 dishes. Cells washed twice with PBS were sedimented (800 × g for 5 min at 4 °C), and resuspended in 9 ml of ice-cold Solution A (20 mM Hepes-KOH pH7.6, 220 mM Mannitol, 70 mM Sucrose, 1 mM EDTA, 1 mM PMSF, 2 mg/ml fatty acid free BSA). Cells were homogenized by passing ten times with a 25 G needle. The nuclei from this suspension were sedimented (800 × g for 10 min at 4 °C), and the centrifugation step was repeated for the supernatant once more. Mitochondria were sedimented from the supernatant by centrifugation (15,000 × g for 20 min at 4 °C), and washed once with 1 ml of Solution A. Protein concentration was measured using NanoDrop. For Proteinase K protection assay, the isolated mitochonaria was diluted into 1 mg/ml with Solution B (10 mM Hepes-KOH pH7.6, 500 mM Sucrose). 100 µg of mitochondria was treated with the indicated concentration of Proteinase K (Takara Bio, Osaka, Japan) for 20 min on ice. Mitochondria were treated with 0.5% (w/v) of Triton X-100 along with the treatment of Proteinase K for a control of Proteinase K protection assay. Proteinase K reaction was stopped with the addition of 1 mM PMSF, and the mitochondria were centrifuged (12,000 × g for 5 min at 4 °C), resuspended with SDS sample buffer, and subjected to western blot analysis.