PRMT5 in mitochondria regulates mtDNA stability through TFAM arginine methylation

Domain: chemistry
Published: 2026-02-23
Editorial outcome: minor_revision

Abstract
Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation and regulates cellular functions such as proliferation, RNA splicing, and nuclear DNA damage response. This study uncovers that a fraction of nuclear-encoded PRMT5 localizes to the mitochondria, which is critical for maintaining mitochondrial DNA (mtDNA) homeostasis. PRMT5 knockout (PRMT5-/-) cells had reduced nucleoid counts, diminished mtDNA copy numbers, disrupted the balance of the mitochondrial fission-fusion cycle, impaired mitochondrial plasticity, and nucleoid trafficking. PRMT5-/- cells are hypersensitive to mtDNA-damaging agents, exhibit reduced mitochondrial transcripts, oxidative phosphorylation, and respiratory capacity that triggers cell death. We identify TFAM as a previously unrecognized interacting partner of PRMT5, which catalyzes symmetric dimethylation of TFAM at R82 residue, which is crucial for mtDNA binding and protection. Defective R82-methylation destabilizes TFAM, which is then degraded by LonP1. Together, we establish that PRMT5 is a mitochondrial enzyme and a key regulator of TFAM in mtDNA maintenance.

Introduction
Arginine methylation is a key post-translational modification (PTM) responsible for the addition of the methyl group to arginine residues. It is increasingly recognized as a pivotal PTM orchestrating a variety of cellular processes, including epigenetic regulation, DNA repair, and nuclear genome maintenance1,2. Protein arginine methylation is an irreversible modification in which the guanidinium group of arginyl residues becomes methylated in a reaction with S-adenosylmethionine as the methyl donor. PRMTs are grouped into three types: type 1 (including PRMT1-4, PRMT6, PRMT8), type II (comprising PRMT5 and PRMT9), and type III (represented by PRMT7) enzymes3. This classification is determined by their ability to catalyze the formation of asymmetric dimethylated arginine (ADMA), symmetric dimethylated arginine (SDMA), and monomethylated arginine (MMA), respectively.

PRMT5 has emerged as a major symmetric dimethylating enzyme involved in the arginine methylation of a myriad of substrates4–7. PRMT5 knockout in mice causes early embryonic death, while its depletion leads to defects in cell proliferation8,9. PRMT5 is primarily known for its role in regulating gene expression and chromatin structure through the methylation of histone proteins8. PRMT5 preserves genomic stability by methylating arginine residues on key DNA damage response (DDR) proteins, including 53BP1, FEN1, RAD9, RUVBL1, and TDP14,6,7,10–13. But how PRMT5-mediated arginine methylation regulates mitochondrial function is still unknown.

The mitochondrial genome lacks the genes essential for mitochondrial DNA (mtDNA) replication, transcription, repair, and maintenance, compelling it to rely only on proteins expressed in the nucleus for mtDNA integrity. Nuclear encoded mitochondrial transcription factor A (TFAM) belongs to the high-mobility group (HMG) of structural proteins; it acts as a transcription factor, replication initiator, and mtDNA packaging factor, thus becoming the master regulator of mitochondrial biogenesis14,15. Human mtDNA, which is circular DNA (~16.5 kb), is a crucial component of eukaryotic cells that encodes a critical subset of mitochondrial proteins required for cellular ATP generation16–18.

TFAM binds mtDNA and plays a crucial role in the formation of mitochondrial nucleoids. TFAM wraps around mtDNA and plays a crucial role in the formation of mitochondrial nucleoids for mtDNA maintenance and ATP production through oxidative phosphorylation (OXPHOS)14,19–21. TFAM binds mtDNA in both a sequence-dependent and sequence-independent manner and contributes to mtDNA compaction and nucleoid formation14,19,21. Sequence-dependent binding to mtDNA promoter regions is required for initiating mitochondrial transcription, which may also serve as the RNA primer for replication initiation21,22. In addition, TFAM binds mtDNA in a sequence-independent manner to compact the genome and protect it from mtDNA damage14,20,23,24.

TFAM also regulates the dynamics of the mitochondrial network, which involves intricate processes of fission and fusion, which are essential for maintaining its structural integrity and functional efficacy25,26. Mitochondrial fusion has been proposed to compensate for mtDNA damage by facilitating the transfer of nucleoids, ensuring the preservation of mtDNA copies27–29. Reductions in mtDNA copy number and the accumulation of mtDNA damage have been linked to various neurological disorders, including Parkinson’s, Alzheimer’s, and Huntington’s diseases, which have been correlated with TFAM; its exact role is elusive23,30. There is limited evidence for the post-translational regulation of TFAM31–33. While studies have investigated the phosphorylation and acetylation of TFAM, which are implicated in its dissociation from mtDNA31,32, other PTMs of TFAM that play a role in its selective binding to mtDNA and its role in fission-fusion dynamics are not known.

This study is the first to demonstrate the localization of catalytically active PRMT5 within mitochondria and its functional coupling with TFAM. In this article, we identify that PRMT5 catalyzes dimethylation of TFAM at R82, which promotes the mitochondrial fission-fusion cycle, stimulates its recruitment to mtDNA, shields the mitochondrial nucleoid, regulates mitochondrial gene transcription and oxygen consumption, providing cellular protection against mitochondria-targeted DNA-damaging agents.

Results
Nuclear-encoded PRMT5 is translocated to the mitochondria
While performing immunofluorescence staining, we observed that PRMT5 was primarily located in the cytoplasm (Fig. 1A), consistent with previous reports8. To determine whether PRMT5 localizes to the mitochondria, we co-stained MCF7 cells with endogenous PRMT5 and the mitochondria-specific marker HSP60 and analyzed them by super-resolution microscopy. Upon immunofluorescence, we detected significant colocalization of PRMT5 (red) with mitochondrial matrix protein HSP60 (green), suggesting the presence of a substantial fraction of PRMT5 in mitochondria (Fig. 1A, B). Under identical conditions, immunofluorescence using the anti-PRMT5 antibody in PRMT5 knockout (PRMT5-/-) cells (Supplementary Fig. 1A) revealed no detectable PRMT5 signal in the mitochondria. Next, we examined the expression of the exogenous FLAG-PRMT5 fusion protein using super-resolution microscopy. Figure 1C, D demonstrate the colocalization of the FLAG-PRMT5 (red) fluorescence pattern with HSP60 (green) upon ectopic expression of PRMT5 in MCF7 cells (Supplementary Fig. 1B). Quantitative image analysis (Fig. 1A, C) demonstrates significant colocalization between PRMT5 and HSP60, as evident by the Pearson correlation coefficient (Fig. 1E). Together, these immunofluorescence data indicate that PRMT5 localizes to mitochondria.

Next, we performed cellular fractionation and Western blotting to confirm the immunofluorescence findings. We detected the presence of PRMT5 as a single band with a similar molecular weight (72.7 kDa) in the mitochondrial (M) fractions together with nuclear (N), cytoplasmic (C) of PRMT5-competent cells (Fig. 1F and Supplementary Fig. 1C). It is unlikely that the mitochondrial PRMT5 signal was the result of nuclear contamination because nuclear Lamin B1, which is an abundant nuclear protein, was undetectable in the mitochondrial fraction (Fig. 1F and Supplementary Fig. 1C). The relative purity of the mitochondrial and nuclear fractions was further assessed by the analysis of TOM20, Cyt C, COX IV and LONP1 which were only detectable in the mitochondrial fractions (Fig. 1F and Supplementary Fig. 1C). The cytoplasmic fraction is checked by GAPDH analysis.

To examine PRMT5 function in the mitochondria, we developed CRISPR-Cas9 to knock out PRMT5 expression and developed the PRMT5-/- MCF7 isogenic cell line (Supplementary Fig. 1D). The knockout efficiency was confirmed by Western blot analysis (Supplementary Fig. 1E). Notably, we failed to detect the PRMT5 signal in any of the subcellular fractions in the PRMT5-/- cell line (Fig. 1F), suggesting PRMT5 is translocated to mitochondria.

To further confirm that PRMT5 is present inside mitochondria rather than adventitiously associated with the outer surface of the organelle, we performed proteinase K (ProK) protection assays. ProK treatment of purified mitochondria has often been used to assess the mitochondrial localization of a protein34. We observed a significant fraction of PRMT5 resistant to ProK-treatment. Furthermore, upon addition of Triton X-100 to disrupt the mitochondrial membrane, PRMT5 in the mitochondrial fraction was ProK-sensitive (Supplementary Fig. 1F, compare lanes 2 and 3), suggesting the presence of PRMT5 within the mitochondria. To precisely determine PRMT5’s mitochondrial localization, we conducted a ProK protection assay under iso-osmotic and hypo-osmotic conditions using isolated mitochondria (Fig. 1G). Under iso-osmotic conditions, ProK degraded the outer membrane (OM) protein TOM20, while PRMT5 remained intact, confirming it is not OM-associated (Fig. 1G, lanes 1 and 2). Hypo-osmotic swelling further disrupted the OM, yet PRMT5 remained resistant to ProK digestion (Fig. 1G, lanes 5 and 6), mirroring the matrix protein LonP1. These results indicate that PRMT5 is localized within the mitoplast, protected by the inner membrane (IM). To further validate PRMT5’s mitochondrial matrix localization, we isolated mitoplasts by treating ultra-purified mitochondria with digitonin, which selectively disrupts the cholesterol-rich outer membrane (OM) while preserving the inner membrane (IM). Following ProK/digitonin treatment, TOM20 was completely degraded, whereas the IM protein COXIV showed partial resistance to ProK but was fully degraded upon digitonin treatment. Notably, PRMT5, like the matrix protein LONP1, remained entirely resistant to ProK/digitonin digestion, reinforcing its localization within the mitochondrial matrix (Fig. 1H).

Finally, we performed an in vitro mitochondrial import assay using recombinant His-PRMT5 and ultra-purified mitochondria in the presence of ProK and the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 1I). The results revealed that His-PRMT5 was transported to a protease-protected compartment, and its import was strictly dependent on membrane potential (Fig. 1I), a defining characteristic of proteins localized to the inner membrane (IM) and mitochondrial matrix35.

Further, the presence of PRMT5 was established in the tissue lysates obtained from mitochondria isolated from the heart, brain, and liver tissue of mice, which have an abundance of mitochondria (Fig. 1J). The presence of PRMT5 in the mitochondria was confirmed by the symmetric dimethylation of arginine residues of mitochondrial proteins detected in PRMT5 proficient mitochondria extract (Fig. 1K), which was markedly abrogated in PRMT5-/- cells. Notably, complementation with the catalytic dead mutant PRMT5R368A in PRMT5-/- (PRMT5-/-/R368A) cells failed to restore SDMA levels, whereas PRMT5 WT effectively rescued the methylation in PRMT5-/-/WT cells (Fig. 1K). Together, these results reveal the first evidence for the presence of a catalytically active PRMT5 enzyme in the mitochondria.

PRMT5 knockout cells are more susceptible to mitochondrial DNA damage
Mitochondrial nucleoids play a crucial role in protecting mtDNA from intrinsic or extrinsic damage. To determine if nucleoid counts were altered by the loss of PRMT5, we used PicoGreen staining and further compared in PRMT5+/+, PRMT5-/-, PRMT5-/-/WT cells using live cell microscopy. We observed a ~4-fold reduction in nucleoid count and enlarged nucleoids in PRMT5-/- cells within elongated mitochondrial networks (Fig. 2A, B; untreated, and Supplementary Fig. 2A). This phenotype was rescued in PRMT5-/-/WT and PRMT5+/+ cells under comparable PRMT5 expression without exogenous DNA damage (Supplementary Fig. 2B). To rule out PicoGreen binding bias due to altered mtDNA supercoiling, we quantified nucleoids in fixed cells using an anti-dsDNA antibody, which yielded similar results (Supplementary Fig. 2C, D).

Given PRMT5’s involvement in nuclear DNA damage response6,7, we explored its role in mitochondrial DNA (mtDNA) damage and repair. MIL-Iri, which induces mtDNA damage by trapping mitochondrial Topoisomerase 1-mtDNA covalent complexes (Top1mtcc)36,37, showed that PRMT5-/- cells had a ~3-fold increase in susceptibility to mtDNA damage compared to PRMT5+/+ cells (Fig. 2A, B; panel MIL-Iri treated). The reduction in nucleoid count prompted us to assess the mtDNA copy number using qPCR in PRMT5+/+, PRMT5-/-, PRMT5-/-/WT and PRMT5-/-/R368A cells. Our results reveal statistically significant rescue of mtDNA copy number in PRMT5-/-/WT cells, whereas no such rescue was observed in PRMT5-/-/R368A cells, demonstrating that PRMT5’s catalytic activity is essential for mtDNA maintenance (Fig. 2C).

MtDNA replication and transcription is a coupled process38,39. As a result, we assessed the mRNA expression of genes transcribed by mtDNA. PRMT5-/- cells showed a significant decrease in mRNA expression of mtDNA-encoded OXPHOS subunits (Supplementary Fig. 2E). Next, we measured the extent of mtDNA damage and repair in the presence of mtDNA-damaging agents (MIL-Iri and H2O2) using a gene-specific long-range quantitative PCR assay, which is an established method34,36,37. We found that PRMT5-/- cells exhibited rapid mtDNA depletion (~2-fold) and slower repair kinetics (~1.5-fold) compared to PRMT5+/+ cells (Fig. 2D, E and Supplementary Fig. 2F, G) upon inducing mtDNA damage. This suggests PRMT5 protects mtDNA from damage and aids in its repair. Cell survival assays revealed increased sensitivity to mtDNA-damaging agents (MIL-Iri, H2O2, and menadione) and respiratory chain inhibitors (rotenone, antimycin, and oligomycin) in PRMT5-/- cells compared to PRMT5+/+ cells (Fig. 2F and Supplementary Fig. 2H–L). This increase in cytotoxicity in PRMT5-/- cells was consistent across different types of mitochondria-targeted damage. We performed PI/Annexin V flow cytometry to more accurately assess cell viability and apoptosis with MIL-Iri, independent of mitochondrial enzyme activity. MIL-Iri treatment induced significantly higher apoptosis in PRMT5-/- cells, which was rescued by PRMT5 WT complementation, consistent with the survival assay (Supplementary Fig. 2M–P).

Finally, we assessed mitochondrial health using the Seahorse XF24 flux analyzer. PRMT5-/- cells showed reduced respiration, further diminished by MIL-Iri treatment compared with PRMT5+/+ cells (Fig. 2G). ATP-linked, basal, maximal, and spare respiratory capacities were significantly reduced in PRMT5-/- cells and further decreased with mtDNA damage compared with PRMT5+/+ cells (Fig. 2H). Mitochondrial respiration assays revealed that only wild-type PRMT5 restored respiration in PRMT5-/-/WT cells, while the catalytically inactive R368A mutant (PRMT5-/-/R368A) failed, underscoring the essential role of PRMT5 enzymatic activity (Fig. 2I, J). These findings demonstrate that PRMT5 plays a crucial role in maintaining mitochondrial nucleoid numbers and protects mtDNA from extrinsic damage.

PRMT5 knockout cells produce hyperfused mitochondria
To investigate the role of PRMT5 in regulating mitochondrial plasticity, we measured mitochondrial length in PRMT5-/- cells and compared them with cells expressing PRMT5 variants (PRMT5+/+ or PRMT5-/-/WT) using MitoTracker Red (Fig. 3A). Mitochondrial length was categorized into three groups: hyperfused mitochondria (>5 µm), moderate length (2–5 µm), and fragmented mitochondria (<2 µm) as done previously25. Notably, in the absence of mtDNA damage, PRMT5-/- cells exhibited a significant increase (~ 2-fold) in hyperfused mitochondrial networks and a concurrent ~1.5-fold decrease in fragmented mitochondria, which could be rescued in PRMT5-/-/WT or wild-type PRMT5+/+ cells (Fig. 3B and Supplementary Fig. 3A). Accordingly, we observed increased expression levels of Mfn1 and Mfn2 in PRMT5-/- cells (Fig. 3C). However, this increase was reversed in PRMT5-/-/WT and in PRMT5+/+ cells (Fig. 3D).

Next, we measured mitochondrial mass by staining cells with MitoTracker Green dye and performing flow cytometry analysis. Indeed, we detected a marked increase in mitochondrial volume (Supplementary Fig. 3B) consistent with hyperfused mitochondria (Fig. 3A, B), suggesting that PRMT5-/- cells employ an alternative mechanism to regulate mitochondrial dynamics. This supports the concept that PRMT5 protects mitochondrial structure and dynamics and is closely linked to proper nucleoid distribution and the removal of intrinsically damaged mtDNA, consistent with previous studies25.

Next, to explore PRMT5’s role in mitochondrial network dynamics, particularly in fission and fusion rates, we utilized live-cell microscopy and the Fluorescence Recovery After Photobleaching (FRAP) technique37. Cells were transfected with mitochondrial matrix-localized Mito-YFP (yellow fluorescent protein) and the fission/fusion rate of mitochondria was analyzed by measuring the rate of redistribution of Mito-YFP, ectopically expressed in PRMT5+/+, PRMT5-/-, or PRMT5-/-/WT cells (Fig. 3E). In the absence of MIL-Iri, PRMT5+/+ cells showed moderate FRAP recovery, reaching a maximum intensity of ~71% at 60 s (Fig. 3E, F), indicating that, under steady-state conditions, there is a balance between the diffusion of Mito-YFP due to the existence of a moderately interconnected mitochondrial network (Fig. 3A, B). Under similar conditions, the PRMT5-/- cells exhibited a 10% increase in FRAP recovery compared to the PRMT5+/+ cells, with a maximum intensity of ~85–90% at 60 s (Fig. 3E, F), indicating elevated levels of the close mitochondrial network due to the presence of hyperfused mitochondria (Fig. 3A, B). Intriguingly, complementation of FLAG-tagged human PRMT5 (PRMT5-/-/WT) restored the fission-fusion cycle to levels similar to PRMT5+/+ cells (maximum intensity of ~75% at 60 s) (Fig. 3F), indicating PRMT5 knockout triggers hyperfused mitochondrial network (Fig. 3B). To further assess PRMT5’s role in mitochondrial fission-fusion dynamics, we performed a FRAP assay in the presence of the DRP1 inhibitor Mdivi-140. In the presence of Mdivi-1, PRMT5+/+ cells showed rapid FRAP recovery, reaching ~95% fluorescence intensity at 60 seconds, consistent with a hyperfused mitochondrial network (Supplementary Fig. 3C, D). Under the same conditions, PRMT5-/- cells exhibited similar recovery (~85–90%), indicating a comparably interconnected mitochondrial network due to impaired fission.

Next, in the presence of mtDNA damage with MIL-Iri, we detected that the hyperfused mitochondria in PRMT5-/- cells (Fig. 3A) were more prone to fragmentation compared to PRMT5-proficient cells (PRMT5+/+ and PRMT5-/-/WT) (Fig. 3G, H). Accordingly, Mil-Iri treatment markedly delayed (15–10%) the FRAP recovery of YFP-mito in PRMT5-/- cells compared to PRMT5-proficient cells (PRMT5+/+ and PRMT5-/-/WT) (Fig. 3I-J), suggesting the existence of a dispersed mitochondrial network due to increased mitochondrial fission in PRMT5-/- cells. Collectively, these data indicate that PRMT5 plays a crucial role in maintaining the fission-fusion dynamics of mitochondria (Fig. 3K), which is essential for proper nucleoid distribution within cells.

PRMT5 catalyzes TFAM dimethylation at R82
To investigate the significance of PRMT5 localization in mitochondria, we co-immunoprecipitated FLAG-PRMT5 ectopically expressed in HEK293 cells and conducted an unbiased proteomics study using tandem mass spectrometry (MS). MS and PSM (Peptide Spectrum Match) analysis identified TFAM and other mitochondrial matrix-associated proteins (PHB1, TOP1mt, and LONP1) interacting with FLAG-PRMT5 (Fig. 4A). Gene enrichment confirmed a strong mitochondrial association, with TFAM (~19 unique peptides) emerging as a top hit (Supplementary Fig. 4A, B).

To examine the mass spectrometry results, we performed co-immunoprecipitation (co-IP) of ectopic FLAG-PRMT5 in HEK293 cells that pulled down endogenous TFAM, confirming the association of PRMT5 with TFAM (Fig. 4B). Reverse co-IP in cells expressing Clover-TFAM further confirmed the presence of PRMT5 in the TFAM complex (Fig. 4C). We further validated PRMT5 interactions with endogenous mitochondrial matrix proteins—including TOP1mt, LONP1, PHB1, COX4, and TOM20, supporting the mass spectrometry findings (Supplementary Fig. 4C).

Next, we performed immunostaining in MCF7 cells expressing ectopic FLAG-PRMT5 with anti-FLAG antibodies and co-stained with anti-TFAM antibodies to detect its colocalization with endogenous TFAM. Indeed, Fig. 4D, E shows a significant colocalization of PRMT5 with TFAM, which is in keeping with PRMT5-TFAM binding in co-IP experiments (Fig. 4B, C). To determine if the PRMT5-TFAM association occurs within the mitochondria, we extracted mitochondria from HEK293 cells and performed a co-immunoprecipitation (Co-IP) of endogenous TFAM with an anti-TFAM antibody (Fig. 4F). Figure 4G detected PRMT5 in the TFAM-immune complex in the mitochondrial fraction, supporting the plausibility that PRMT5 binds with TFAM in the mitochondria.

Because PRMT5 binds to TFAM, we examined TFAM arginine methylation using MS analysis. MS analysis of the FLAG-PRMT5 immunoprecipitation complex identified TFAM-R82 as a potential dimethylated arginine residue (Supplementary Fig. 4B). The R82 residue of TFAM is highly conserved among species and is located within the conserved DNA-binding HMG Box A domain, adjacent to the mtDNA (Fig. 4H)21.

Prompted by the MS findings, we immunoprecipitated ectopic Clover-TFAM with an anti-GFP antibody from HEK293 cells, and probed with antibodies that recognize symmetrically dimethylated arginine residues (anti-SDMA)6. Fig. 4I shows that Clover-TFAM WT reacts with the SDMA-specific antibody and recognizes bands (*band indicated the precursor form of TFAM) with a similar molecular weight as that of Clover-TFAM, suggesting the possibility of TFAM arginine methylation. To validate R82 as an arginine methylation site on TFAM, we immunoprecipitated the ectopically expressed methylation mutant Clover-TFAMR82K from HEK293 cells, probed with an SDMA-specific antibody. Fig. 4I shows a marked reduction (~80-90%) in the methylation signal in R82K mutant TFAM, confirming that R82 is the primary site of TFAM methylation in cells.

To establish whether PRMT5 is responsible for TFAM arginine methylation, we immunoprecipitated ectopically expressed Clover-TFAM WT in PRMT5+/+ and PRMT5-/- cells with an anti-GFP antibody and probed with an anti-SDMA antibody. Fig. 4J shows that PRMT5 knockout resulted in a marked decrease (80%) in arginine-methylated TFAM, showing that TFAM not only physically interacts (see Fig. 4B–E) but is also arginine-methylated in vivo by PRMT5. To explore TFAM-methylation in mitochondria, we performed PLA assays using SDMA- and TFAM-specific antibodies with MitoTracker Green staining. TFAM-SDMA PLA signals primarily colocalized with mitochondria, confirming that symmetrically dimethylated TFAM is present predominantly within this compartment. (Supplementary Fig. 4E–G).

Finally, to obtain direct evidence for TFAM R82 methylation by PRMT5, we conducted in vitro methylation assays using recombinant His-tagged TFAM variants (His-TFAMWT and His-TFAMR82K) as substrates and immunoprecipitated PRMT5 in the presence of S-adenosylmethionine (SAM). Methylation of TFAMWT by PRMT5 was abrogated in the TFAMR82K mutant (Fig. 4K), demonstrating that R82 is the major residue for PRMT5-mediated TFAM arginine methylation (Fig. 4L). Symmetric arginine dimethylation is primarily catalyzed by PRMT5, with PRMT9 also proposed for catalysis of SDMA41. To test PRMT5’s specificity for TFAM methylation, we performed in vitro methylation assays using immunoprecipitated PRMT9 (Supplementary Fig. 4H). TFAM was methylated by PRMT5, but not by PRMT9, confirming PRMT5 as the specific methyltransferase (Supplementary Fig. 4I). Because TFAM R82 as well as PRMT5 are conserved along the phylogeny, we conclude that R82 of TFAM is the plausible cellular target for PRMT5 across species.

R82-methylation of TFAM shields mtDNA from exogenous damage
To assess the functional impact of R82 methylation of TFAM, we generated a TFAM knockout isogenic clone in the MCF7 cell line (TFAM-/-) using the CRISPR-Cas9 system. The knockout efficiency was confirmed by Western blot analysis (Supplementary Fig. 5A, B). Because TFAM preserves and packages the mitochondrial genome14,20,21, next, we used TFAM-/- cells to complement with human Clover-tagged wild-type TFAM (TFAM-/-/WT) or a methylation mutant R82K-TFAM (TFAM-/-/R82K), which was also confirmed by Western blotting (Supplementary Fig. 5C). Live cell microscopy with DNA-specific PicoGreen dye shows that TFAM-/- cells have a marked reduction in mitochondrial nucleoid count (~7 fold) as compared to TFAM+/+ cells (Fig. 5A; quantification in Fig. 5B), which is in keeping with previous reports25. Under similar conditions, TFAM methylation mutant (TFAM-/-/R82K) cells were defective to rescue the nucleoid count by ~2–3 fold as compared to TFAM+/+ cells or TFAM-/-/WT cells (Fig. 5B), suggesting the role of TFAM-R82 methylation in the maintenance of mitochondrial nucleoids akin to PRMT5-/- cells (Fig. 2A, B). Because TFAM plays a critical role in the replication of mtDNA19. Therefore, we hypothesized that TFAM-/- cells or TFAM-/-/R82K would be more susceptible to the mtDNA damage associated with MIL-Iri-induced Top1mtcc (Fig. 5C). Upon treatment with MIL-Iri, we observed that TFAM-/- cells show a lower mt-nucleoid count (~3 fold) as compared to the wild-type cells which is consistent with TFAM’s protective role (Fig. 5C; quantification in Fig. 5D). However, the mt-nucleoids in TFAM-/-/R82K cells exhibited higher susceptibility (~2 fold) to mtDNA damage as compared to TFAM-/-/WT cells. (Fig. 5C; quantification in Fig. 5D). Accordingly, we detected a marked reduction in mitochondrial copy number in TFAM-/- cells or TFAM-/-/R82K compared to its wild-type counterpart (Fig. 5E).

To further investigate the role of TFAM-R82 methylation in regulating mitochondrial dynamics, we measured the mitochondrial length in TFAM-/- cells complemented with the R82K methylation mutant (TFAM-/-/R82K) and compared it to the wild-type counterpart (TFAM-/-/WT) (Fig. 5A; quantification in Fig. 5F; left panel). TFAM knockout cells showed a significant increase in hyperfused mitochondria (~3 fold) as compared to TFAM proficient cells (TFAM+/+ and TFAM-/-/WT), which could not be rescued by R82K mutant (Fig. 5A; quantification in Fig. 5F; left panel). Accordingly, we detected an increase in mitochondrial volume in R82K methylation mutant (TFAM-/-/R82K), which were reversed in TFAM-proficient (TFAM+/+ and TFAM-/-/WT) cells (Supplementary Fig. 5D). Next, following mtDNA damage with MIL-Iri, we observed that the hyperfused mitochondria in TFAM-/- and TFAM-/-/R82K cells (Fig. 5C) were more prone to fragmentation compared to those in TFAM-proficient cells (TFAM+/+ and TFAM-/-/WT) (Fig. 5C; quantification in Fig. 5F; right panel). This implies that TFAM-R82 methylation is essential for proper nucleoid maintenance and distribution.

TFAM is a transcription factor that activates transcription by binding to the two main promoters of mitochondrial DNA (mtDNA): the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1)21. To investigate the role of R82 methylation, we measured the transcript levels in cells expressing different TFAM variants (TFAM+/+, TFAM-/-, TFAM-/-/WT, and the methylation mutant TFAM-/-/R82K). The TFAM-/-/R82K cells showed a significant decrease in mRNA expression of OXPHOS subunits encoded by polycistronic heavy- and light-chain transcripts of mtDNA, including 12s rRNA, ND1, ND2, ND6, CYTB, COX2 (Fig. 5G). This suggests that PRMT5-mediated TFAM-R82 methylation not only protects mtDNA nucleoids but is also crucial for mtDNA transcription.

R82-methylation of TFAM increases mtDNA binding
TFAM-mtDNA binding is critical for mtDNA compaction and transcriptional activation14,20. In its role in mtDNA compaction, TFAM is believed to interact with mtDNA globally14,20,21. Circular dichroism spectroscopy comparing WT and R82K TFAM showed no structural changes, indicating that the R82 mutation does not alter TFAM function independently of PRMT5-mediated dimethylation (Supplementary Fig. 6A). Next, to assess the binding abilities of TFAM-R82 methylation mutations to mtDNA, we performed both in vivo and in vitro mtDNA binding assays. First, we used chromatin immunoprecipitation (ChIP) with an anti-TFAM antibody, followed by quantitative polymerase chain reaction (qPCR) analysis targeting the D-loop site containing HSP1 and NCR (non-coding regions) and coding regions containing ND4, CYTB and COX1 (Fig. 6A–C). As shown in Fig. 6B, the enrichment of endogenous TFAM on the HSP1 sequence of mtDNA was significantly reduced (~7 fold) in PRMT5-/- cells compared to PRMT5+/+ cells. Accordingly, TFAM defective in R82 methylation (TFAMR82K) exhibited a marked reduction in mtDNA enrichment (~3 fold) to the HSP1 sequence compared to the TFAM WT counterpart (Fig. 6C) in ChIP assays. These findings were further confirmed by measuring the intrinsic fluorescence of TFAM-R82K mutant in the presence of increasing concentrations of a double-stranded Light Strand Promoter (LSP) specific oligo (Supplementary Fig. 6A). The R82-methylated His-TFAM WT exhibited increased DNA binding activity compared to its non-methylated counterpart or methylated His-TFAM R82K (Supplementary Fig. 6B–G). The defective DNA binding ability in the HSP and LSP regions may attribute to the marked reduction in transcriptional activation of OXPHOS subunits encoded by polycistronic heavy- and light-chain promoters of mtDNA in TFAM-/-/R82K cells (Fig. 5G).

Next, we examined the effect of R82 methylation on TFAM’s global DNA binding activity using linearized plasmid DNA and recombinant His-tagged TFAM variants42. We conducted in vitro methylation of recombinant TFAM with immunoprecipitated PRMT5 in the presence of SAM, followed by DNA binding assays (Fig. 6D). Figures 6E and F demonstrate that, with increasing concentrations of recombinant TFAM, the percentage of band retardation is higher (~2.5 fold) for the R82-methylated His-TFAM (Rmet TFAM) compared to the non-methylated His-TFAM. Furthermore, the DNA binding activity of methylated His-TFAM R82K was significantly reduced (~2 fold) compared to the R82-methylated wild-type TFAM (Rmet His-TFAM WT) at higher DNA concentrations (Fig. 6G, H), which may facilitate mtDNA compaction and protection from mtDNA damage. Together, we provide evidence that R82-methylation of TFAM promotes its DNA binding ability (Fig. 6I).

Loss of R82-methylation in TFAM leads to LonP1-mediated degradation and induces mtDNA Damage
The mtDNA-unbound form of TFAM is more sensitive to proteolytic degradation by Lon protease 1 (LONP1)32,33 and arginine methylation of downstream target proteins such as KLF4, 53BP1, and TDP1 protects it from degradation4,6,7. Therefore, we tested endogenous TFAM half-life in cycloheximide (CHX) chase experiments using PRMT5+/+ and PRMT5-/- cells. In the absence of PRMT5, the half-life of endogenous TFAM is significantly decreased (Fig. 7A, B), which is consistent with the increase of mtDNA-unbound TFAM (Fig. 6B). Considering LONP1’s direct involvement in TFAM degradation32, we investigated its role in regulating TFAM half-life in PRMT5 knockout cells using a specific LONP1 protease inhibitor, CDDO-Me (Fig. 7C, D). The relative extent of TFAM accumulation in PRMT5-/- was markedly increased upon CDDO-Me treatment (Fig. 7C, D) similar to PRMT5+/+ wild-type cells (Supplementary Fig. 7A, B) suggesting PRMT5-mediated arginine methylation not only increases mtDNA binding abilities but also protects TFAM from LONP1-mediated degradation.

To further explore the impact of TFAM arginine dimethylation on its stability, we assessed the half-lives of exogenously expressed Clover-tagged TFAM WT and a Clover-tagged arginine methylation mutant TFAM R82K in the CHX chase experiments in the presence and absence of CDDO-Me. Figures 7E and F show that in the absence of CDDO-Me, TFAM WT exhibits an increased half-life compared to TFAM R82K. However, CDDO-Me treatment restored the half-life of TFAM R82K (Fig. 7G, H), similar to the wild-type (Supplementary Fig. 7C, D), suggesting that the R82-methylation not only enhances the binding capacity of TFAM to mtDNA but also protects TFAM from LONP1 degradation.

To investigate the biological significance of TFAM-R82 methylation, we measured the extent of mtDNA damage and repair in cells expressing TFAM variants using gene-specific long-range quantitative PCR assays34,36,37. Interestingly, both the methylation-defective TFAM-/-/R82K cells and the TFAM-/- cells exhibited rapid mtDNA damage (~1.5 fold) and slower recovery (~1.2 fold) as compared to their wild-type counterparts (TFAM+/+ or TFAM-/-/WT) (Fig. 7I, J). Next, to assess endogenous mtDNA breaks in PRMT5-/- or TFAM-/- mitochondria we directly measured in vivo trapping of Top1mtcc in isolated mitochondria using ICE (immunocomplex of enzyme) assays using antibodies that detect Top1mt (Supplementary Fig. 7E–H). Topoisomerase 1 cleavage complex (Top1cc) intermediates are trapped by endogenous DNA lesions, including abasic sites, mismatches, oxidized bases, nicks, and other endogenous DNA adducts37,43,44. We observed increased accumulation of Top1mtcc in mitochondria from PRMT5-/- cells under basal conditions compared to wild-type, indicating elevated endogenous mtDNA breaks in the absence of PRMT5 (Supplementary Fig. 7E, F). Notably, Top1mtcc trapping was further exacerbated by MIL-Iri, a Top1mtcc poison that induces mtDNA damage (Supplementary Fig. 7G–H). Similarly, TFAM-/- and methylation-defective TFAM-/-/R82K cells exhibited heightened Top1mtcc trapping both at baseline and following DNA damage, whereas TFAM+/+ and TFAM-/-/WT cells showed significantly reduced mtDNA damage (Supplementary Fig. 7E–H).

Finally, mitochondrial health was assessed using the Seahorse XF24 flux analyzer. TFAM-/- cells showed reduced respiration, further diminished by MIL-Iri treatment as compared to TFAM+/+ cells (Supplementary Fig. 7I). Both basal and maximal respiratory capacities were significantly lower in TFAM-/- cells, with additional reductions following mtDNA damage (Supplementary Fig. 7J). The methylation mutant TFAM-/-/R82K cells demonstrated a ~2-fold reduction in oxygen consumption rate, both in the absence and presence of MIL-Iri, compared to TFAM-/-/WT cells (Fig. 7K and Supplementary Fig. 7K). Additionally, TFAM-/-/R82K and TFAM-/- cells were more sensitive to MIL-Iri and H2O2 in survival assays, indicating that TFAM-R82 methylation is crucial for cell survival under DNA damage with Top1mtcc and oxidative stress (Fig. 7L and Supplementary Fig. 7L). This sensitivity aligns with the observed decline in oxygen consumption rate in TFAM-/-/R82K cells (Fig. 7K and Supplementary Fig. 7L). Annexin V/PI staining showed that MIL-Iri treatment markedly increased early and late apoptosis in TFAM-/-/R82K cells (Supplementary Fig. 7M–P). Clonogenic assays revealed that TFAM knockout heightened MIL-Iri sensitivity, which was rescued by TFAM WT, wild-type but not by TFAM R82K, indicating that methylation-deficient TFAM fail to restore survival (Supplementary Fig. 7Q, R). Together, our results indicate that deficiencies in TFAM or PRMT5 cause comparable mitochondrial dysfunctions, including reduced mtDNA content, impaired repair kinetics, and compromised respiration in response to mtDNA damage (Fig. 8). This establishes a functional link between PRMT5 and TFAM through R82 methylation.

Methods
Treatment and transfections
Cells were exposed to different concentrations of MIL-Iri for the designated durations as mentioned in the figure legends. For cycloheximide (CHX) experiments, cells at 60–70% confluence were cultured in six-well plates, and 24 h later, treated with CDDO-Me (1 μM) for 24 h immediately followed by CHX at a final concentration of 100 µg/mL. For the FRAP assay, cells were treated with Mdivi-1 at a concentration of 2.5 μm, 4 h. Transfections were carried out using plasmid DNA and either Lipofectamine 2000 (Invitrogen) or Transit Transfection Reagent (Mirus), following the manufacturer’s instructions. All experiments were conducted 48 h post-transfection61–64.

Cell culture, plasmids, and CRISPR/Cas9-mediated gene knockout
Cell cultures were maintained in DMEM medium supplemented with 10% FBS obtained from Life Technologies at 5% CO2 and 37°C temperature. The HEK293 and MCF7 cell lines were procured from ATCC. The cloning procedure for the pCMV-Tag2B-FLAG-PRMT5 construct was previously described6. Clover-tagged-TFAM and pET28A His-tagged-TFAM plasmids were purchased from Addgene. Clover-tagged-TFAMR82K and pET28A His-tagged-TFAMR82K constructs were generated using the ‘QuickChange’ protocol (Stratagene, La Jolla, CA, USA). The His-tagged PRMT5 constructs were generated by polymerase chain reaction amplification using full-length PRMT5 (FLAG-PRMT5 WT) as template and cloned into the bacterial expression vectors pET28A. All constructs were confirmed by DNA sequencing.

The CRISPR-based PRMT5 and TFAM gene knockout was performed in MCF7 cells as described previously7,12. Briefly, the pSpCas9(BB)-2A-GFP (PX458) vector containing the PRMT5 guide RNA (5’ CCTGAATTGCGTCCCCGAAATAG 3’) targeting exon 1 and TFAM guide RNA (5’ AGGTGGCCTCGCTACCGCAAAGA 3’) targeting exon 1 was used to transfect MCF7 using lipofectamine 2000. GFP-expressing cells were sorted using a FACSCell Sorter, and an isogenic clone was generated from a single cell. The loss of PRMT5 and TFAM protein expression in these clones was confirmed by Western blot analysis. The pYFP-Mito construct37, which was used for FRAP analysis, was a kind gift from Dr. Richard J. Youle (NIH). Lipofectamine 2000 (Invitrogen) was used for transfection purposes following the manufacturer’s instructions.

Cell extracts and immunoblotting
Cell lysates were prepared using a previously described method6,7,64. Briefly, the cells were trypsinized, pelleted, and lysed using a standard lysis buffer. The lysis buffer consisted of 10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP40, and 0.5% Na-deoxycholate, supplemented with complete protease inhibitors and phosphatase inhibitors. The lysate was then subjected to intermittent vortexing and incubated at 4°C for 1 h. Subsequently, the lysate was centrifuged at 12,000 g at 4°C for 10 m to collect the supernatant, which was then stored at -80°C for further use. Gel electrophoresis was performed using 10% Tris-glycine gels, followed by standard immunoblotting procedures. For Western blot analysis, following SDS–PAGE it was transferred onto PVDF membranes (Millipore). The membranes were blocked with 3% BSA in PBS containing 0.1% Tween for 1 h, followed by incubation with the primary antibody (1:1000) overnight at 4°C. After washing with 0.1% Tween-PBS, secondary antibodies were applied at a 1:10,000 dilution and incubated for 2 h at room temperature. Detection of immunoreactivity was achieved using an ECL chemiluminescence reaction (Amersham) in the ChemiDoc TM MP System (Bio-Rad, USA), and densitometric analysis of the immunoblots was conducted with ImageJ software.

Immunoprecipitation
For immunoprecipitation, cells were lysed in a buffer containing 50 mM Tris–HCl (pH 7.4), 300 mM NaCl, 0.4% NP40, 10 mM MgCl2, and 0.5 mM dithiothreitol, along with protease and phosphatase inhibitors. The lysate was centrifuged at 15,000 g for 20 m at 4°C, and the supernatant was collected. Protein A/G-PLUS agarose beads (Santa Cruz, CA, USA) were added to approximately 50 μL of the lysate and incubated overnight at 4°C with precleared lysate and the specified antibodies (2–5 mg/ml). Immunocomplexes were isolated by centrifugation, washed three times with the lysis buffer, and analyzed via immunoblotting.

Preparation of mitochondria
The mitochondria were isolated using a previously described protocol34. In summary, cells were scraped and pelleted, and the resulting pellet was resuspended in a buffer containing 10 mM NaCl, 1.5 mM CaCl2, and 10 mM Tris-HCl (pH 7.5) for 5 m to induce osmotic shock. After that, the cells were homogenized for 1 h using a glass Dounce homogenizer, followed by the addition of a stabilizing buffer (2 M sucrose, 35 mM EDTA, and 50 mM Tris-HCl, pH 7.5). Next, the lysate was centrifuged at 750 × g for 5 m to remove nuclei (N) and cellular debris. The resulting supernatant was subjected to a second round of centrifugation at 10,000 × g for 20 m to isolate the mitochondrial pellet, while the supernatant contained the cytoplasm (C). The mitochondrial pellet (M) was then washed three times with mito buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 5 mM DTT, and 20 mM Hepes-KOH, pH 7.4) and eventually resuspended and stored in mito buffer at -80°C for further use.

Ultrapure mitochondria were isolated as previously described65. Briefly, crude mitochondria were washed in mito buffer and further purified by centrifugation (95,000 × g, 30 m, 4°C) on a 30% Percoll density gradient using an SW41 rotor in a Beckman Coulter Optima L-100 XP ultracentrifuge. Ultra-Clear centrifuge tubes (14 × 89 mm; Beckman Instruments) were used for separation. The purified mitochondrial fraction, appearing as a dense band near the tube bottom, was carefully collected using a Pasteur pipette. Mitochondrial pellets were flash-frozen and stored at -80°C until further use.

For the isolation of mitochondria from mouse tissue, mice were euthanized by decapitation according to approved ethical guidelines, and heart, brain, and liver tissue were promptly collected. The tissue was rinsed with ice-cold MSE-A buffer (225 mM Mannitol, 75 mM Sucrose, 1 mM EGTA, 20 mM MOPS, and 0.5 mM DTT, pH 7.5) to remove blood and extraneous tissue as described previously66. Next, the tissue was minced with scissors in a small volume of ice-cold MSE-A buffer for 5 m, using a ratio of 5 ml of MSE-A per 1 gram of tissue. The minced tissue was transferred to a glass Teflon homogenizer and homogenized further in ice-cold MSE-A. The homogenate was centrifuged at 500-600 g for 10 m at 4°C, and the resulting pellet was designated as the whole cell extract (W). The supernatant was collected and centrifuged again at 900–1200 g for 10-15 m at 4°C. The pellet, which is the nuclear fraction (N), was saved, and the supernatant was filtered through a cheesecloth. The filtered supernatant was then centrifuged at 10,000 g for 15-20 m at 4°C. The resulting pellet contained the mitochondrial fraction (M), while the supernatant held the cytoplasmic fraction (C). The mitochondrial pellet was resuspended in MSE-A containing 0.02% digitonin. Following an additional centrifugation at 12,000 × g for 10 min MSE-A, the resulting pellet was re-suspended in MSE-B, composed of 225 mM mannitol, 75 mM sucrose, 20 mM MOPS, 0.1 mM EGTA, 0.5 mM DTT, and ddH2O, adjusted to pH 7.4.

Proteinase K protection assay
Proteinase K protection assays were performed as done previously34. In brief, equal amounts of freshly isolated mitochondria from cells were incubated with 0.5 mg/ml proteinase K (Sigma) for 15 m at 25°C, either with or without 1% Triton-X-100 (Sigma). Proteinase K activity was halted by adding 2 volumes of 20% trichloroacetic acid at 0°C for 20 m. The resulting protein pellets were washed with ice-cold acetone, resuspended in 2× Laemmli sample buffer, and analyzed by Western blotting as described.

Mitoplast analysis
Isolated mitochondria were resuspended in either 250 mM sucrose buffer (250 mM sucrose, 20 mM HEPES-KOH, pH 7.4) or 5 mM sucrose buffer (5 mM sucrose, 20 mM HEPES-KOH, pH 7.4) and incubated on ice for 30 m as done previously67. For proteinase protection assays, aliquots were treated with 25 μg/mL proteinase K for 5 m on ice, followed by PMSF inhibition. Samples were centrifuged at 20,000 × g for 10 m at 4°C, with supernatant proteins recovered by pyrogallol red precipitation. Pellets were washed with 100 μL sucrose buffers, re-pelleted, and solubilized in urea sample buffer containing 50 mM DTT for SDS-PAGE and Western blot analysis.

For digitonin-based mitoplasting, ultrapure mitochondria were treated with 0.2% digitonin (10 m, ice) to disrupt the outer membrane, followed by centrifugation at 15,000 × g for 10 m at 4°C. Mitoplasts were divided for proteinase K treatment (25 μg/mL, 37°C, 30 min) to assess protein localization.

Live cell confocal microscopy and Immunocytochemistry
Live cell imaging was conducted following previously established protocols36,37. Briefly, cells were plated on confocal imaging dishes (Genetix) 24 hours prior to transfection. Live-cell imaging was subsequently performed using either conventional confocal microscopy (Leica SP8 system) or super-resolution imaging (Leica STELLARIS 8 FALCON) operating in Lightning mode with a 63×/1.4 NA oil-immersion objective. The white light laser lines UV (Diode 405 nm), 488 nm (Argon), 561 nm (DPSS 561), or 633 nm (HeNe 633) wavelengths were used to excite DAPI/ Hoechst, MitoTracker red, PicoGreen, and RFP/GFP/YFP fluorophores. The confocal dish stage is set within a chamber that maintains a temperature of 37°C and has a 5% CO2 supply. Fiji ImageJ (NIH) was used to analyze the fluorescence pattern of transfected cells.

Immunofluorescence staining was conducted following previously established protocols6,7,37,68. In summary, cells were fixed with 4% paraformaldehyde for 20 m at room temperature. Primary antibodies targeting PRMT5, COXIV, FLAG, dsDNA, and TFAM were detected using Alexa Fluor 488-labeled anti-mouse, Alexa Fluor 568-labeled anti-mouse, Alexa Fluor 488-labeled anti-rabbit, and Alexa Fluor 568-labeled anti-rabbit IgG secondary antibodies (all from Invitrogen). The cells were mounted in an anti-fade solution containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and examined with a Leica TCS SP8/STELLARIS laser scanning confocal microscope. Images were captured and processed using Leica LASX software and then resized in Adobe Photoshop. Colocalization was assessed by measuring fluorescence intensities for each channel in the LASX software.

Analysis of nucleoid area and mitochondrial length
The nucleoid area and perimeter of the mitochondria were assessed using Fiji ImageJ software. After applying scale information from the LASX (Leica), the images were converted to binary format, and the area of each nucleoid or the perimeter of each mitochondrion was measured using the ‘Analyze Particles’ function in ImageJ. The nucleoid area within each mitochondrion in every cell was analysed and categorized into groups as mentioned in the figure legend according to previous work25. The perimeter of each mitochondrion within every cell was analysed and categorized into hyperfused, intermediate, and fragmented as done previously25. This was done for approximately 50 cells and plotted as a percentage of mitochondria based on different types for each cell.

Quantification of mitochondrial transcripts and mtDNA by real-time PCR
Total RNA was extracted from the specified cells (1 × 106) using the TRIzol reagent (15596018, Invitrogen) following the manufacturer’s instructions, including deoxyribonuclease (AMPD1; Sigma-Aldrich) treatment for each sample. A 1 µg aliquot of RNA was reverse-transcribed using a reverse transcription kit (4368814, Applied Biosystems)37,69. Real-time PCR was then carried out with SYBR Green PCR master mix on the ABI 7500 thermocycler (Applied Biosystems). Each reaction mixture contained 5 µl of 2× SYBR Green PCR master mix and 2 µl of cDNA, generated by reverse transcription and diluted 10-fold, in a final volume of 10 µl with primers at 25 nM. The thermocycling conditions were set at 95°C for 5 m, followed by 40 cycles of 95°C for 50 s, a variable 50°C for 50 s, and 72°C for 60 s. Relative gene expression was calculated as the ratio of the expression level of the gene of interest to that of β-actin RNA, with the expression levels in wild-type cells defined as 100%.

For mtDNA copy number estimation, total DNA was extracted from cell samples using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions, including treatments with proteinase K and RNase. To quantify mtDNA copy number, the ratio of the expression level of the gene of interest was calculated to that of β-actin DNA, with the expression levels in wild-type cells defined as 100%.

Mitochondrial fission-fusion analysis using live cell microscopy and FRAP analysis
A confocal laser-scanning microscope (Leica TCS SP8) was used for the photobleaching experiments64 to assess the mitochondrial network in live cells as described previously37. Briefly, cells were transfected with mitochondrial matrix-targeted protein (pYFP-Mito) in PRMT5 proficient and deficient cells. For FRAP (Fluorescence recovery after photobleaching), photobleaching of YFP-mito at a specific ROI (region of interest) within the cell was done using a 514 nm laser for 30 ms. Thereafter, successive images were taken at intervals of 500 ms for 60 s. The fluorescence intensity of the bleached region was calculated using LasX software, and the background signal was adjusted. The fluorescence signal within the ROI was normalized to the pre-bleach signal, and the FRAP curve was obtained using the equation: ROI=(I0-It)/(I0-Ibg)×100. Here, I0 is the pre-bleaching intensity, It is the intensity at a time point (t), and Ibg is the background signal determined in a region outside the mitochondria.

Measurement of mitochondrial mass
Mitochondrial mass was assessed using MitoTracker Green FM (Molecular Probes) staining. Cells were trypsinized, resuspended in PBS, and incubated with 200 nM MitoTracker Green FM for 30 m at 37°C in the dark. The stained cells were then analyzed by flow cytometry using a BD FACSAria III system.

Cell survival and Apoptosis assays
Cytotoxicity assays were performed as described previously7,37,61. Briefly, PRMT5+/+, PRMT5-/-, TFAM+/+, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K MCF7 were seeded at a density of 1 × 103 cells in 96-well plates (BD Biosciences, USA) on the first day. The cells were treated with the specified drugs (H2O2, MIL-Iri, rotenone, oligomycin, menadione, antimycin) after 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added after an additional 48 hrs and colorimetric detection was done at 570 nm using a Molecular Devices SpectraMax M2 Microplate Reader. The percentage of cell survival was calculated by normalizing with the untreated control. The data presented represent the mean values ± S.D. obtained from three independent experiments. To evaluate cell apoptosis, PRMT5 and TFAM variant cell lines were plated at 5 × 105 cells/mL and were treated with vehicle control (DMSO) or MIL-Iri. Then, cells were washed and stained by Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Pharmingen). Data acquisition was conducted by flow cytometry using a BD FACSAria III system.

Clonogenic assays
TFAM+/+ and TFAM-/- cells were seeded in 6-well plates at 50% confluency. TFAM -/- cells were transfected with Clover-TFAM WT or Clover-TFAM R82K using the TransIT-X2 Dynamic Delivery System (Mirus), following the manufacturer’s protocol. After 48 hours, the cells were treated with MIL-Iri at the indicated concentrations for 3 hours. Cells were then trypsinized and re-seeded at 1 × 10³ cells per well in 6-well plates. Following two weeks of incubation at 37°C, the medium was aspirated, and the cells were washed with PBS. The washed cells were fixed with ice-cold methanol for 10 minutes and stained with 0.5% crystal violet for 1 hour. Excess stain was removed by rinsing with water, and the plates were air-dried. Colony counts were obtained using ImageJ software.

Mass spectrometry analysis of PRMT5
Immunoprecipitation of ectopic FLAG-PRMT5 complexes was performed using an anti-FLAG antibody, as detailed earlier. The immunoprecipitated samples were then subjected to tryptic digestion at 37°C overnight. Subsequently, the digested samples were lyophilized, reconstituted, and fractionated using strong cation exchange (SCX) liquid chromatography (LC). Finally, the fractions obtained were subjected to mass spectrometry analysis, following previously described methods6,61. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE repository with dataset identifier PXD057645. Raw MS files were processed using Proteome Discoverer (Thermo Fisher Scientific), and database searches were performed with MS Amanda (v2.5.0.16129) against a merged database containing the human UniProt reference proteome and MitoCarta 3.0 mitochondrial proteins70.

Quantification of mtDNA damage
MtDNA damage and repair were quantified using a PCR-based assay known as Long-range PCR, as described previously34,36,37. Briefly, indicated cell variants were seeded in a 12-well plate and treated with mtDNA-damaging agents (MIL-Iri and Hydrogen peroxide) for specific durations as described in the figure legend. Alternatively, after drug treatment, cells were grown in drug-free media for an indicated time to study the repair kinetics. Total DNA was extracted from cell samples using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions, including treatments with proteinase K and RNase. MtDNA-specific Long-range primers were used to amplify a mtDNA fragment of 9 kbp and COX-2 specific primer was used to amplify a smaller fragment of 110 base pairs for normalization. The PCR cycle was restricted to 18 cycles so that the amplification occurs within an exponential phase. The mtDNA damage was quantified by the ratio of the long-range PCR product (LR) to the short-range PCR product (SR) (LR/SR).

ICE bioassay
The isolation of Top1mt-DNA cleavage complexes was performed using an in vivo complex of enzyme (ICE) bioassay as previously described37. Briefly, cells (5 × 10⁶) expressing PRMT5 and TFAM variants were either left untreated or exposed to MIL-Iri (2.5 μm, 3 h). Mitochondria were isolated using the aforementioned protocol and lysed in 6 M guanidinium thiocyanate buffer [10 mM Tris-HCl (pH 6.5), 20 mM EDTA, 4% Triton X-100, 1% N-lauroylsarcosine, 1% DTT]. Lysates were precipitated with 0.4 mL ice-cold 100% ethanol (-20°C, 5 min), followed by centrifugation (12,000 × g, 10 min). Pellets were washed twice with ethanol, then resuspended in freshly prepared 8 mM NaOH and briefly sonicated (20% amplitude, 10–20 sec). For detection, mtDNA samples were slot-blotted onto nitrocellulose membranes (Millipore) using a Bio-Rad vacuum manifold. Top1mt-DNA complexes were immunodetected with anti-Top1mt antibodies, while anti-dsDNA antibodies served as loading controls. Protein-DNA adducts were visualized by enhanced chemiluminescence (ChemiDoc MP Imaging System, Bio-Rad).

Proximity ligation assay (PLA)
Proximity ligation assays were performed using a commercial PLA kit (Olink Bioscience) following the manufacturer’s instructions. For each experimental condition, a minimum of fifty cells were analyzed to ensure statistical reliability. Cells were counterstained with Mitotracker Green.

Circular dichroism analysis
All circular dichroism data were collected using a Jasco Circular Dichroism Spectrophotometer J-1500 with a 1-mm path length cell. The protein sample of WT or R82K mutant was 10 μM in 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 1 mM DTT. Far-UV circular dichroism spectra were collected at 25°C, with a data averaging time of 5 s. The data were collected every 1 nm from 195 to 260 nm.

Chromatin immunoprecipitation and RT-qPCR
Chromatin immunoprecipitation (ChIP) was performed following previously described protocol71. Briefly, PRMT5+/+, PRMT5-/- and TFAM-/- cells (1 × 107) expressing TFAM variants; TFAM WT and TFAM R82K were fixed in formaldehyde (1.1%) at room temperature for 10 m, followed by quenching for 5 m by addition of glycine (125 mM). The cells were then washed with PBS twice, scraped, and lysed in 0.5 ml of ChIP lysis buffer (50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8), 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitor) for 30 m in ice. Sonication was done ten times for 30 s at 30% power to fragment the DNA corresponding to 200 to 300 base pairs size, followed by centrifugation at 13000 rpm for 30 m at 4°C. The remaining supernatant was diluted in a buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA (pH 8), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitors. It was then pre-cleared by incubation with protein-G-sepharose beads/salmon sperm DNA at 4°C for 1 h. For each immunoprecipitation, 500 μl of the pre-cleared chromatin was incubated with 5 µg of GFP and TFAM-specific antibodies on a revolving platform at 4°C for 1 h. A control reaction containing an equivalent amount of Goat/Rabbit IgG was included to assess background levels. Additionally, 10% of the precleared chromatin was reserved as an input control. Following antibody incubation, 150 μl of protein-G-sepharose beads/salmon sperm DNA was added and incubated overnight under the same conditions. The next day, the beads were washed sequentially with a high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl; pH 8.1, 500 mM NaCl), a LiCl wash buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl; pH 8.0), and finally TE buffer (10 mM Tris-HCl; pH 8.0, 1 mM EDTA). DNA was then eluted using an elution buffer containing 1% SDS and 100 mM NaHCO₃. The elutes were incubated overnight at 65°C in the presence of 5 M NaCl, reaching a final concentration of 200 mM, to reverse formaldehyde cross-linking. Following this, samples were digested with proteinase K (50 mg/ml) at 55°C for 3 h and further treated with RNase (20 mg/ml) at 37°C before undergoing DNA isolation using the phenol-chloroform method. For qPCR analysis, the input sample was diluted 50-fold. PCR reactions were performed in triplicate using an ABI 7500 thermocycler with the following cycling conditions: initial denaturation at 95°C for 10 m, followed by 40 cycles of amplification at 95°C for 15 s, variable 50°C for 15 s, and 72°C for 30 s, with signal acquisition at the end of each cycle. TFAM enrichment on mtDNA was quantified using the percentage input method.

In vitro methylation assays
The in vitro methylation assays were performed as previously described6. In brief, PRMT5 and PRMT9 were immunoprecipitated and incubated with either recombinant His-TFAM WT or His-TFAM R82K proteins (1 μg). The methylation reactions were conducted in a buffer (50 mM Tris–HCl, pH 8.5, 5 mM MgCl2, 4 mM DTT) containing 100 μM unlabeled S-(5’-adenosyl)-L-methionine chloride dihydrochloride (SAM) (A7007) (Sigma) for 2 h at 30°C. The reactions were stopped by adding 2× SDS loading buffer (Invitrogen) and boiling the samples for 5 m. The resulting methylation products were separated by SDS–PAGE, transferred onto a PVDF membrane, and analyzed by Western blotting using an anti-SDMA antibody.

Recombinant protein purification
TFAM purification was done using pET28a-TFAM expression vector (Addgene), which encompasses residues 43–246, representing the full-length TFAM protein as described previously21. PRMT5 purification was done using the pET28a-PRMT5 expression vector. BL21 (DE3) E. coli was transformed with either pET28a-TFAM or pET28a-PRMT5 plasmids, and a single colony was inoculated in 20 ml of LB media containing 50 μg/ml Kanamycin and incubated for 12 h at 37°C. The next day, the culture was scaled up to 1 litre until the OD600 reached 1.0. Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was grown overnight. Thereafter, the cells were harvested and lysed in a 20 ml resuspension/lysis buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) by subsequently vortexing at an interval of 30 m for 4 h. The lysate was then sonicated using a probe sonicator on ice for an additional 30 m with 30% amplitude pulse for 30 s (10 s on and 20 s off) and centrifuged at 13000 rpm for 30 m at 4°C. The supernatant was then subjected to Ni-NTA-based affinity chromatography to isolate the His-tagged TFAM and His-tagged PRMT5. The protein was eluted in an elution buffer (20 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, pH 7.5) and then concentrated using an Amicon Ultra-15 10 kDA-cut-off concentrator (Millipore). The protein was aliquoted and stored in -80°C.

Mitochondrial Protein Import Assay
Protein import assays were performed as previously described35,72 with modifications. Purified mitochondria were resuspended in import buffer (250 mM sucrose, 10 mM MOPS-KOH [pH 7.2], 80 mM KCl, 2 mM KH₂PO₄, 5 mM MgCl₂, 5 mM methionine, 3% fatty acid-free BSA) supplemented with 2 mM ATP, 2 mM NADH, 5 mM creatine phosphate, and 100 μg/mL creatine kinase. Recombinant His-tagged PRMT5 (His-PRMT5) was incubated with mitochondria at 25°C to initiate import. Reactions were terminated by transferring samples to ice and dissipating the mitochondrial membrane potential using 0.2 mM CCCP. Non-imported precursors were removed by proteinase K treatment (25 μg/mL, on ice), followed by protease inhibition with 1 mM PMSF. Mitochondria were re-isolated by centrifugation, washed with isolation buffer, and lysed under denaturing conditions. Imported His-PRMT5 was detected by using anti-His antibodies.

EMSA
Electrophoretic mobility shift assay (EMSA) was performed as described previously42. The pBluescript SK(+) plasmid, approximately 3 kb in size, was subjected to Xho1 digestion at a single site to linearize the DNA. Linearized plasmids (125.5 ng) were then incubated with varying amounts of hTFAM (500, 1000, 1250, 1500, 1750, and 2000 ng) in a reaction mixture, which corresponds to a ratio of 1 hTFAM per 11.3, 5.6, 4.5, 3.8, 3.2, and 2.8 base pairs of plasmid DNA, respectively42. This mixture was incubated in an EMSA buffer (35 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM DTT, 5 mM spermidine, 0.1 mg/ml BSA, and 10 mM EDTA) at 37°C for 30 m. After incubation, the protein-DNA complex was subjected to electrophoresis in agarose gel (1%) in the absence of ethidium bromide (EtBr). The TFAM-mtDNA binding was visualized in a ChemiDoc instrument (BioRad) after staining with EtBr. The percentage retardation was calculated as indicated in the figure legend by measuring the migration, which is equal to the distance between the lane and the DNA-protein band in each lane, and normalizing with the only plasmid DNA lane.

DNA binding measurements
The intrinsic tryptophan fluorescence of TFAM was utilized as the basis for a binding assay to study its interaction with DNA. Among the four tryptophan residues present in TFAM, three of them are located within 3.6 Å from the mitochondrial DNA (mtDNA). The intrinsic fluorescence of only TFAM protein (25 nM) in a quenching buffer (20 mM Tris–HCl (pH 7.5), 300 mM NaCl, and 1 mM DTT) was initially measured. Subsequently, a double-stranded oligo corresponding to the LSP (light strand promoter) region (5’ GTGTTAGTTGGGGGGTGACTGTTAAAAGTG 3’) was added in increasing concentrations. This measurement was done in a FluoroMax-3 spectrofluorimeter (Jobin–Yvon–Horiba). Excitation wavelength of 280 nm was used and emission spectra were taken in a range of 320–400 nm. The slit width of the excitation and emission of the instrument was set at 4 nm and 10 nm, respectively. Each binding measurement was carried out in triplicate to ensure accuracy and reproducibility. To assess the binding affinity, the percent reduction in fluorescence (% quenching) was determined and plotted against the total concentration of DNA used in the assay.

Mitochondrial bioenergetic profiling
Cellular respiration was analyzed using the Seahorse XF24 analyzer (Seahorse Biosciences, Agilent Technologies) following the manufacturer’s protocols. In brief, 30,000 cells were seeded into assay microplates the night before the experiment. On the following day, the cells were incubated in 500 μl of assay media at 37°C in a CO2-free incubator for 1 h before the assay. The oxygen consumption rate (OCR) was measured over time using the XF24 analyzer after the sequential addition of various respiratory inhibitors. The respiratory inhibitors used were oligomycin (0.5 μM), FCCP (1 μM), and rotenone (0.5 μM), all obtained from Seahorse mito stress kit (Agilent). Following the assay, the media was removed, and the cells were lysed for further analysis. The data were normalized based on protein concentration, which was determined using the Bradford assay (Bio-Rad).

Statistics and reproducibility
All experiments were conducted independently at least two-three times. Data are expressed as mean ± standard error of the mean (S.E.M.). The number of experiments and the quantified cells per field are detailed in the figure legends. Each experiment (n) was performed using different cell variants. Statistical significance (P values) was determined using Sidak’s or Tukey’s multiple comparisons two-way ANOVA and t-test, as indicated in the figure legends. Data analysis and graphing were performed using Prism 8 (GraphPad Software) and OriginLab. No specific method was applied to pre-determine sample size, and no data were excluded from the analysis. Images were randomly selected, and data analysis was carried out in a blinded manner.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "TFAM is a master regulator of mitochondrial genome organisation and expression. Bhattacharjee et al convincingly demonstrate that TFAM can be methylated at R82, and this novel modification impacts TFAM binding to mtDNA and its proteolytic degradation by LONP. This is an important finding that improves our basic understanding of TFAM post-translational regulation. The authors argue that the arginine methyltransferase, PRMT5, catalyses the methylation of TFAM R82. The authors argue that TFAM methylation occurs within the mitochondrial matrix and identify mitochondrial-localised PRMT5 for the first time.\n\nMajor comments\n1. The mito localisation of PRMT5 is novel and crucial for the mechanism but I believe the data are too premature to conclude that PRMT5 localises to the mitochondrial matrix. The main issues with the current experiments are highlighted below:\na) PRMT5 is absent from MitoCarta3.0 and is also absent from recent high-confidence proteomic analysis of mitochondrial-localised proteins (PMID: 34800366). Can the authors comment on why this might be?\nb) Unfortunately, I find the confocal microscopy to be unconvincing – a linescan from one cell with fragmented mitochondria at this resolution is insufficient to determine if PRMT5 is indeed within mitochondrial matrix. Super-resolution would be required to show matrix localisation by IF where quantified overlap with mitochondrial matrix marker could be performed. If this is not possible then further biochemical assays should be performed (e.g import assay as mentioned below). Also, please validate PRMT5 antibody for IF using PRMT5-/- cells.\nc) The authors performed a proteinase K protection assay (Fig. 1F) to argue that PRMT5 is localised within mitochondrial matrix. Only COXIV (IM marker) is used as a control and this behaves the same as PRMT5. From this single experiment, one could interpret PRMT5 to be in the IMS instead. In the discussion, the authors state that PRMT5 does not have a mitochondrial targeting sequence (MTS) to guide it to the mitochondrial matrix. Can the authors demonstrate PRMT5 import using in vitro import assays? Is PRMT5 import dependent on mitochondrial membrane potential? Is it possible that PRMT5-dependent TFAM methylation occurs outside mitochondria prior to TFAM being imported?\nd) In general authors need to use outer mito membrane and matrix markers to help with data interpretation in Figs 1E, F . Ideally, a pure mitochondrial fraction should be tested as well (e.g percoll density ultracentrifugation) since differential centrifugation provides crude mitochondrial fractions mixed with many heavy membranes e.g ER.\ne) To conclude that Fig. 1H proves “presence of catalytic active PRMT5 enzyme inside mitochondria”, authors should perform rescue experiment with PRMT5 WT and catalytic dead mutant.\n2. The reduction of mtDNA copy number in PRMT5 KO cells (Fig. 2C) is not rescued significantly by re-expression of WT PRMT5. I’m confused how the authors can conclude “2-fold reduction in PRMT5-/- cells compared to PRMT5+/+ and PRMT5-/-/WT cells?” This raises questions over the phenotype data that follows. The authors should use complemented PRMT5-/-/WT cells to check that respiration defect in PRMT5 KO cells is indeed PRMT5 dependent (ideally along with a catalytic mutant).\n3. The methylation of TFAM by PRMT5 in vitro appears convincing (Fig. 4J) but why is there some methylation of TFAM R82K? Furthermore, can the authors demonstrate this methylation is specific to PRMT5 and not also catalysed by another PRMT?\n4. Authors state that “MS analysis revealed that approximately a subset of mitochondrial-targeted proteins binds with FLAG-PRMT5…” But they only show TFAM as a hit. Supp Fig4A does not include any information on how many other interaction partners were detected and their localisation etc. These data should be provided in a table at least.\n\nOther comments\n1. Why do PRMT5-/- cells have picogreen in nucleus but WT and complemented cells do not (Fig.2A)?\n2. Does ectopically expressed Clover-TFAM localise purely to mitochondria or is it possible that methylation is occurring outside the mitochondria?", "reviewer_2": "The manuscript by Bhattacharjee and colleagues presents an interesting new role of PRMT5 in mitochondrial DNA regulation. Two main points should be addressed before the publication:\n1. In several cases the exogenous FLAG-PRMT5 (-/-WT) does not rescue PRMT5 KO (-/-). This is for example in Fig. 2B, 2C, 3B, 5B. How do the authors explain this? Is the exogenous FLAG-PRMT5 functional? Is there another secondary mechanism that contributes to the phenotype?\n2. Authors strongly suggest that PRMT5 protects against mtDNA damage. To support this statement authors should provide evidence that endogenous mtDNA damage is induced in PRMT5 KO or PRMT5 R82K cells. In the current data authors only show exogenously induced damage and its physiological relevance is elusive.\n\nOther comments\nIntroduction:\nLine 57 – please remove “covalently closed” as mtDNA may exist in many topological conformations\nLines 60-63 – Suggested changes to improve clarity: “Unlike nuclear chromosomes, mtDNA lacks histones. Instead, TFAM binds mtDNA and plays a crucial role in the formation of mitochondrial nucleoids.”\nLine 64 – replace “counts” with “formation”\nLines 71-72 – remove “Due to the close association of OXPHOS, mtDNA is more vulnerable to oxidative DNA damage”. There are publications showing no evidence of oxidative damage in mtDNA. Authors may consider writing that mtDNA mutation rates are higher than nuclear DNA mutation rates instead.\nLine 72 - and mitochondrial fusion has been proposed to compensate for mtDNA damage. Please add “has been proposed to”\nLine 76 – please add “which have been correlated with TFAM, nevertheless its exact role is elusive”\nResults:\nLines 90-108 and Fig.1E – How is PRMT5 imported into mitochondria? Does it have MTS?\nLines 119-125 – Please add mitoplast analysis with digitonin treatment to show if PRMT5 is localized to the mitochondrial matrix\nLine 117 – Please remove “suggesting nuclear-encoded PRMT5 is translocated to mitochondria.” This is confusing\nLine 180 – based on which criteria did the authors grouped hyperfused mitochondria (>5 μm), moderate length (2–5 μm), and fragmented mitochondria (<2 μm)? How the sizes were selected?\nLines 188-194 – did the authors consider alternative hypothesis. Does PRMT5 KO influences MitoTracker import or mitochondrial membrane potential?\nLines 195-210 – Please use orthogonal approach to support this data. Authors are advised to use chemical and genetic fission/fussion inhibitors.\nLine 225: What are the other proteins identified by proteomics? Please include a table with all identified proteins as well as western blot confirmation of selected hits.\nLine 345- please change “promotes” to “increases”\nDiscussion:\nLine 397 – how do the authors explain increased sensitivity of PRMT5 KO cells to ETC inhibitors (anti/rot/oligo)?\nLine 403 – please change “critical” to “new”\nLine 406 – authors write that PRMT5 is localized mainly in the cytoplasm, but they write that its main function is in histone methylation. Is PRMT5 translocated to the nucleus? Are there any conditions that stimulate PRMT5 localization to nucleus or mitochondria from the cytoplasm?\nLine 462 – please change to TFAM's role in neuroprotection was suggested”\nFigures:\nFig2A – why is PicoGreen nuclear signal present only in PRMT5 KO cells?\nFig2 – please add additional control of nuclear PRMT5 (non-mito localized) to ensure direct effects in the mitochondria\nFig5B – why is WT and R82K more similar than +/+ and -/-WT? This argues against the role of R82K in the nucleoid protection\nFig7B-D – please include comparison of PRMT5 +/+ to PRMT5 -/- + CDDO\nFig7F-H – please include comparison of WT to R82K + CDDO"}, "author_response": "POINT BY POINT RESPONSE TO REVIEWER’S\nReviewers' comments:\nReviewer #1: (Remarks to the Author)\nOverall assessment:\nTFAM is a master regulator of mitochondrial genome organisation and expression.\nBhattacharjee et al convincingly demonstrate that TFAM can be methylated at R82, and this\nnovel modification impacts TFAM binding to mtDNA and its proteolytic degradation by\nLONP. This is an important finding that improves our basic understanding of TFAM post-\ntranslational regulation. The authors argue that the arginine methyltransferase, PRMT5,\ncatalyses the methylation of TFAM R82. The authors argue that TFAM methylation occurs\nwithin the mitochondrial matrix and identify mitochondrial-localised PRMT5 for the first\ntime.\nAnswer: Thank you for acknowledging, “This is an important finding that improves our basic\nunderstanding of TFAM post-translational regulation.” We have carefully addressed all the\npoints raised in our manuscript by conducting several new experiments.\nMajor comments\nQuestion 1: The mito localisation of PRMT5 is novel and crucial for the mechanism but I\nbelieve the data are too premature to conclude that PRMT5 localises to the mitochondrial\nmatrix. The main issues with the current experiments are highlighted below:\nAnswer: We have conducted additional experiments to strengthen our manuscript and\naddress your concern: “the data are too premature to conclude that PRMT5 localizes to the\nmitochondrial matrix.” These new findings have been incorporated into the results and\ndiscussion sections of the revised manuscript. We have also thoroughly addressed each of\nyour comments.\na) PRMT5 is absent from MitoCarta3.0 and is also absent from recent high-confidence\nproteomic analysis of mitochondrial-localised proteins (PMID: 34800366). Can the authors\ncomment on why this might be?\nAnswer: MitoCarta3.0 (PMID: 33174596) provides a curated reference inventory of 1,136\nhuman mitochondrial proteins, compiled through literature review and evidence of\nmitochondrial localization from Gene Ontology (GO) and UniProt annotations. While bottom-\nup proteomics approaches (PMID: 34800366) rely on the generation of proteolytic peptides\nsuitable for mass spectrometry (MS)-based sequencing and are constrained by the sensitivity\nand accuracy of MS-based peptide and protein quantification. Consequently, these methods\nmay fail to detect certain proteins, including newly discovered mitochondrial proteins like\nMOF, KANSL3 (Chatterjee et al., 2016, Cell, PMID: 27768893), NOTCH1 (Wang et al., 2024,\nNature Communications, PMID: 39550366), TDP1 (Das et al. 2010, PNAS, PMID: 21041670)\nand p53 (Marchenko et al., 2000, JBC, PMID: 10821866) despite published experimental\n1\nevidence supporting their mitochondrial localization. The absence of PRMT5 from these\ndatasets, alongside these proteins, may be attributed to its lack of prior identification or\nannotation in existing databases, as well as its potential abundance falling below the detection\nthreshold of the proteomic techniques employed.\nTo address this, we conducted extensive validation experiments, including super-\nresolution microscopy with appropriate mitochondrial matrix marker (Figure 1A-D),\nmitochondrial import assays (Figure 1I), and mitoplast analysis (Figure 1G), and Digitonin\nexperiments to confirm the mitochondrial localization of PRMT5 under the specific conditions\nof our study (Figure 1H). Additionally, we used protein markers to demonstrate that PRMT5 is\nlocalized in the mitochondrial matrix (Figure 1F-H). Furthermore, we extensively validated\nmitochondrial matrix-associated proteins interacting with PRMT5 by coimmunoprecipitation\nand western blot analysis (Supplementary Figure 4C), as identified through mass spectrometry\nanalysis (Figure 4A). These findings provide compelling evidence that PRMT5 is a\nmitochondrial matrix protein. These results have been incorporated into the revised manuscript,\nwhere we have also elaborated on the potential reasons for PRMT5's absence in these datasets\nwithin the discussion section.\nb) Unfortunately, I find the confocal microscopy to be unconvincing – a linescan from one cell\nwith fragmented mitochondria at this resolution is insufficient to determine if PRMT5 is indeed\nwithin mitochondrial matrix. Super-resolution would be required to show matrix localisation\nby IF where. If this is not possible then further biochemical assays should be performed (e.g\nimport assay as mentioned below). Also, please validate PRMT5 antibody for IF using PRMT5-\n/- cells.\nAnswer: Thank you for bringing up the important point “a linescan from one cell with\nfragmented mitochondria at this resolution is insufficient to determine if PRMT5 is indeed\nwithin mitochondrial matrix” and providing us the opportunity to strengthen our conclusions.\nWe have carefully performed several new experiments to address the reviewer’s concern.\nExperiment #1: We performed super-resolution microscopy to detect both endogenous PRMT5\n(Figure 1A-B) and exogenously expressed FLAG-PRMT5 (Figure 1C-D), co-stained with the\nmitochondrial matrix marker HSP60. Our analysis confirms the localization of PRMT5 within\nthe mitochondrial matrix. To further validate this, we quantified the colocalization of PRMT5\nwith HSP60 and provided the Pearson correlation coefficient between PRMT5 and the\nmitochondrial marker (Figure 1E), reinforcing its matrix-specific localization.\nExperiment #2: To confirm the specificity of the PRMT5 antibody, we performed\nimmunofluorescence in PRMT5 knockout (PRMT5-/-) cells using the anti-PRMT5 antibody\n(Supplementary Figure 1A), which showed no detectable PRMT5 signal in the mitochondria.\nExperiment #3: As suggested by one of the reviewers, we conducted an in vitro mitochondrial\nimport assay using recombinant His-PRMT5, with and without CCCP (Figure 1I), to confirm\nthe membrane potential-dependent import of PRMT5 into mitochondria.\nExperiment #4: Furthermore, as suggested, we performed mitoplast analysis by incubating\nisolated mitochondria in iso-osmotic and hypo-osmotic buffers, with or without proteinase K,\n2\nto confirm PRMT5 localization within the mitochondrial matrix. These new experiments were\nconducted alongside the evaluation of specific markers and are presented in Figure 1G.\nExperiment #5: Additionally, to confirm the mitochondrial matrix localization of PRMT5, we\nperformed percoll density gradient fractionation experiments of crude mitochondria to achieve\na high degree of purification, effectively separating mitochondria from cellular contaminants.\nThis approach yielded a cleaner and functionally intact mitochondrial fraction, allowing us to\nconfirm the presence of PRMT5 in the mitochondrial matrix. The purity of the mitochondrial\nfraction was validated by TOM20, which was degraded in the presence of proteinase K (Figure\n1H).\nExperiment#6: Furthermore, we validated PRMT5's mitochondrial matrix localization by\nidentifying its interacting proteins through co-immunoprecipitation and western blot analysis\n(Supplementary Figure 4C), supported by mass spectrometry (Figure 4A). These findings\nstrongly establish PRMT5 as a mitochondrial matrix protein.\nc) The authors performed a proteinase K protection assay (Fig. 1F) to argue that PRMT5 is\nlocalised within mitochondrial matrix. Only COXIV (IM marker) is used as a control and this\nbehaves the same as PRMT5. From this single experiment, one could interpret PRMT5 to be in\nthe IMS instead. In the discussion, the authors state that PRMT5 does not have a mitochondrial\ntargeting sequence (MTS) to guide it to the mitochondrial matrix. Can the authors demonstrate\nPRMT5 import using in vitro import assays? Is PRMT5 import dependent on mitochondrial\nmembrane potential? Is it possible that PRMT5-dependent TFAM methylation occurs outside\nmitochondria prior to TFAM being imported?\nAnswer: We sincerely appreciate the reviewer’s insightful comments, which allowed us to\nclarify and strengthen the evidence supporting PRMT5's localization within the mitochondrial\nmatrix. In response, we conducted a series of rigorous experiments using specific\ncompartmental markers for the mitochondrial outer membrane (OM), inner membrane (IM),\nintermembrane space (IMS), and matrix (Mc).\nTo determine PRMT5's precise localization, we first performed a Proteinase K (ProK)\nprotection assay under iso-osmotic and hypo-osmotic conditions using isolated HEK293\nmitochondria (Figure 1G, revised manuscript). Under iso-osmotic conditions, ProK degraded\nthe OM protein TOM20, while PRMT5 remained intact, indicating it is not an OM-associated\nprotein (Figure 1G, lanes 1 and 2). Hypo-osmotic swelling further disrupted the OM, yet\nPRMT5 remained protected from ProK digestion (Figure 1G, lanes 5 and 6), similar to the\nmatrix protein LonP1, suggesting PRMT5 resides within the mitoplast, protected by the IM.\nTo further confirm PRMT5’s matrix localization, we isolated mitoplasts by treating\nultra-purified mitochondria with digitonin, which selectively disrupts the cholesterol-rich OM\nwhile preserving the IM. Following ProK/digitonin treatment, TOM20 was completely\ndegraded, while COXIV, an IM protein, exhibited partial ProK resistance but was fully\ndegraded after digitonin treatment. PRMT5, like LonP1, remained completely resistant to\nProK/digitonin digestion, supporting its matrix localization (Figure 1H, revised manuscript).\nHis-PRMT5 and ultra-purified mitochondria in the presence of ProK and the uncoupler CCCP\n(Figure 1I). This assay demonstrated that His-PRMT5 was transported to a protease-protected\ncompartment and that its import was membrane potential-dependent—hallmarks of IM and\nmatrix-localized proteins (Jain et al., 2023, EMBO Reports; Ryan et al., 2001, Methods Cell\nBiol).\nTogether, all these new experiments provide compelling and conclusive evidence that\nPRMT5 localizes within the mitochondrial matrix in a membrane potential-dependent manner.\nWe sincerely thank the reviewer for prompting this detailed clarification.\nd) In general authors need to use outer mito membrane and matrix markers to help with data\ninterpretation in Figs 1E, F. Ideally, a pure mitochondrial fraction should be tested as well (e.g\npercoll density ultracentrifugation) since differential centrifugation provides crude\nmitochondrial fractions mixed with many heavy membranes e.g ER.\nAnswer: We thank the reviewer for this important point, which has allowed us to refine our\nmethodology and strengthen our data interpretation. In response, we conducted additional\nexperiments to incorporate specific mitochondrial compartment markers and ensure the purity\nof our mitochondrial preparations.\nTo accurately interpret the data in Figures 1F and Supplementary Figure 1F, we\nincluded well-established markers for distinct mitochondrial compartments: Tom20 (outer\nmembrane), cytochrome c (intermembrane space), CoxIV (inner membrane), and LonP1\n(matrix). These controls confirm PRMT5’s precise localization within the mitochondrial matrix\nand enhance the reliability of our findings.\nTo rule out contamination from heavy membranes, we performed Percoll density\ngradient fractionation (225 mM mannitol, 25 mM HEPES [pH 7.4], 1 mM EGTA, and 30%\nPercoll [vol/vol]) followed by ultracentrifugation (95,000 × g, 30 min, 4 °C) to obtain highly\npurified mitochondria, effectively eliminating mitochondria-associated membranes (MAMs)\nand ER contaminants. Using this ultra-purified mitochondrial fraction, we further validated\nPRMT5’s matrix localization (Figure 1H).\nThese additional controls significantly strengthen our conclusions, and we appreciate\nthe reviewer’s feedback in enhancing the rigor of our study.\ne) To conclude that Fig. 1H proves “presence of catalytic active PRMT5 enzyme inside\nmitochondria”, authors should perform rescue experiment with PRMT5 WT and catalytic dead\nmutant.\nAnswer: We sincerely thank the reviewer for their insightful comments, which have allowed\nus to further strengthen our conclusions. In response, we performed rescue experiments using\nboth wild-type (WT) PRMT5 and a catalytically inactive mutant PRMT5 (R368A) to confirm\nthe presence of catalytically active PRMT5 within mitochondria.\nWe observed a significant reduction in symmetric dimethylarginine (SDMA) levels in\nmitochondrial extracts from PRMT5 knockout cells, highlighting PRMT5’s role in\nmitochondrial protein arginine methylation. Notably, complementation with the R368A mutant\n4\nfailed to restore SDMA levels, whereas WT PRMT5 successfully rescued the methylation\n(Figure 1K, revised manuscript).\nThese results demonstrate that PRMT5’s catalytic activity is essential for symmetric\ndimethylation of mitochondrial proteins, confirming the presence of enzymatically active\nPRMT5 within mitochondria.\nQuestion 2: The reduction of mtDNA copy number in PRMT5 KO cells (Fig. 2C) is not\nrescued significantly by re-expression of WT PRMT5. I’m confused how the authors can\nconclude “2-fold reduction in PRMT5-/- cells compared to PRMT5+/+ and PRMT5-/-/WT\ncells?” This raises questions over the phenotype data that follows. The authors should use\ncomplemented PRMT5-/-/WT cells to check that respiration defect in PRMT5 KO cells is\nindeed PRMT5 dependent (ideally along with a catalytic mutant).\nAnswer: Thank you for raising this point and allowing us to clarify the issue. To address this,\nwe performed new experiments to assess mtDNA copy number in PRMT5+/+, PRMT5-/-, and\ncomplemented PRMT5-/-/WT cells, as well as in PRMT5-/- cells expressing the catalytically\ninactive PRMT5 R368A mutant.\nOur new results reveal a modest and statistically significant rescue of mtDNA copy\nnumber in PRMT5-/-/WT cells, which was absent in PRMT5-/-/R368A cells, indicating that\nPRMT5’s catalytic activity is essential for mtDNA maintenance (Figure 2C, revised\nmanuscript).\nAdditionally, to ensure the specificity of the observed phenotypes, we evaluated\nmitochondrial respiration using Seahorse XF24 flux analysis. Our findings (Figure 2I-J, revised\nmanuscript) confirm that PRMT5’s catalytic activity is crucial for maintaining mitochondrial\nrespiration.\nQuestion 3: The methylation of TFAM by PRMT5 in vitro appears convincing (Fig. 4J) but\nwhy is there some methylation of TFAM R82K? Furthermore, can the authors demonstrate this\nmethylation is specific to PRMT5 and not also catalysed by another PRMT?\nAnswer: Symmetric dimethylation of arginine residues is primarily catalyzed by PRMT5\ninside cells, with PRMT9 also suggested to catalyze this modification. To determine whether\nPRMT5 is exclusively responsible for TFAM methylation, we conducted additional in vitro\nmethylation assays.\nTo test its involvement, we performed in vitro methylation assays using recombinant\nHis-tagged wild-type TFAM (His-TFAMWT) as a substrate, in the presence of S-\nadenosylmethionine (SAM), with immunoprecipitated PRMT5 and PRMT9. Our results\ndemonstrate that TFAM is methylated by PRMT5 but not by PRMT9 (Supplementary Figure\n4H-I, revised manuscript), confirming that PRMT5 specifically catalyzes this modification.\nWe thank the reviewer for your observation that “The methylation of TFAM by PRMT5\nin vitro appears convincing (Fig. 4J).” The significantly reduced SDMA signal in the TFAM\nR82K mutant confirms that R82 is the primary site for arginine methylation. However, this\ndoes not exclude the possibility of additional methylation sites.\nmitochondrial-targeted proteins binds with FLAG-PRMT5…” But they only show TFAM as a\nhit. Supp Fig4A does not include any information on how many other interaction partners were\ndetected and their localisation etc. These data should be provided in a table at least.\nAnswer: We appreciate the opportunity to clarify this point. To address the concern regarding\nmitochondrial-targeted proteins interacting with FLAG-PRMT5, we conducted a detailed\nanalysis of the mass spectrometry (MS) data (Figure 4A and Supplementary Figure 4B, revised\nmanuscript), which includes a plot of potential mitochondrial interaction partners of PRMT5.\nGene enrichment analysis based on biological processes and cellular components\nfurther confirmed the enrichment of mitochondrial-associated proteins, with TFAM emerging\nas a top hit (Supplementary Figure 4B, revised manuscript).\nTo validate these findings, we performed co-immunoprecipitation (co-IP) experiments\nusing ectopically expressed FLAG-PRMT5 in HEK293 cells. These experiments confirmed\nPRMT5 interactions with endogenous mitochondrial proteins, including TOP1mt, LonP1,\nPHB1, COX4, and TOM20, corroborating the MS results (Supplementary Figure 4C). These\nadditional analyses strengthen our findings and provide a comprehensive understanding of\nPRMT5’s mitochondrial interaction network.\nMinor comments\nQuestion 1: Why do PRMT5-/- cells have picogreen in nucleus but WT and complemented\ncells do not (Fig.2A)?\nAnswer: Thank you for bringing up the issue. As suggested, we have conducted new\nexperiments and indeed failed to detect picogreen in the nucleus. The new data are incorporated\nin revised manuscript.\nQuestion 2: Does ectopically expressed Clover-TFAM localise purely to mitochondria or is it\npossible that methylation is occurring outside the mitochondria?\nAnswer: Yes, Clover-TFAM exclusively colocalizes with mitochondria, as shown in Figures\n5A and 5C (right panel). Immunofluorescence analysis (Figure 4D, E) confirms PRMT5-\nTFAM colocalization, while immunoprecipitation of mitochondria-imported PRMT5 with\nTFAM (Figure 4G) further supports their interaction within mitochondria.\nTo validate TFAM methylation in mitochondria, we performed additional new proximity\nligation assays (PLA) using SDMA-specific and TFAM antibodies, combined with\nMitoTracker Green staining. We detected TFAM-SDMA PLA signals colocalizing with\nmitochondria, confirming that symmetric dimethylation of TFAM occurs specifically in this\ncompartment. However, we also observed a few TFAM-SDMA signals outside mitochondria,\nwhich may result from TFAM’s dynamic shuttling between the cytoplasm and mitochondria.\nWhile extramitochondrial methylation cannot be entirely ruled out, our findings strongly\nsupport mitochondrial TFAM methylation. We have updated the discussion and incorporated\nthese new data in the revised manuscript (Supplementary Figure 4E-G).\nOverall assessment:\nThe manuscript by Bhattacharjee and colleagues presents an interesting new role of PRMT5\nin mitochondrial DNA regulation. Two main points should be addressed before the\npublication:\nAnswer: We earnestly thank the reviewer for your constructive comment that “The manuscript\nby Bhattacharjee and colleagues presents an interesting new role of PRMT5 in mitochondrial\nDNA regulation”. We have carefully performed several new experiments to address the\nreviewer’s concern.\nMajor comments\nQuestion 1: In several cases the exogenous FLAG-PRMT5 (-/-WT) does not rescue PRMT5\nKO (-/-). This is for example in Fig. 2B, 2C, 3B, 5B. How do the authors explain this? Is the\nexogenous FLAG-PRMT5 functional? Is there another secondary mechanism that contributes\nto the phenotype?\nAnswer: We sincerely thank the reviewer for their insightful comments, which have\nstrengthened our conclusions. To directly assess the role of catalytically active PRMT5 in\nmitochondria, we performed new rescue experiments by complementing PRMT5-/- cells with\neither wild-type PRMT5 (PRMT5 WT) or the catalytically inactive R368A mutant (PRMT5\nR368A) (Figure 1K). Additionally, we conducted multiple validation experiments, as reported in\nprevious studies.\nFirst, we confirmed that PRMT5 knockout resulted in a marked reduction of symmetric\ndimethylarginine (SDMA) levels in mitochondrial extracts, reinforcing PRMT5’s role in\nmitochondrial protein arginine methylation. Notably, complementation with PRMT5 R368A\nfailed to restore SDMA levels, whereas PRMT5 WT fully rescued the methylation (Figure 1K,\nrevised manuscript).\nTo further validate the functional significance of PRMT5’s catalytic activity in\nmitochondrial nucleoid maintenance, we assessed mtDNA copy number in PRMT5+/+,\nPRMT5-/-, and PRMT5-/- cells complemented with PRMT5 WT or PRMT5 R368A. Our results\nreveal a modest but statistically significant rescue of mtDNA copy number in PRMT5-/-/WT\ncells, whereas no such rescue was observed in PRMT5-/-/R368A cells, demonstrating that\nPRMT5’s catalytic activity is essential for mtDNA maintenance (Figure 2C, revised\nmanuscript). Additionally, our re-quantification of Figure 2B confirms this statistically\nsignificant, rescue effect in complemented PRMT5-/-/WT cells.\nTo further ensure the specificity of these phenotypic effects, we evaluated\nmitochondrial respiration using Seahorse XF24 flux analysis in PRMT5+/+, PRMT5-/-, and\nPRMT5-/- cells complemented with PRMT5 WT or PRMT5 R368A. Our findings (Figure 2G-J,\nrevised manuscript) confirm that PRMT5’s catalytic activity is critical for maintaining\nmitochondrial respiration, though the extent of rescue remains modest.\nRegarding Figures 3B and 5B, our re-quantified analysis supports a statistically\nsignificant, rescue effect. However, the impact of TFAM arginine methylation, particularly at\n6G, H), emphasizing the crucial role of TFAM R82 methylation in mtDNA maintenance.\nTaken together, our data suggest that, beyond TFAM, PRMT5 may have additional\nmitochondrial targets that contribute to mitochondrial function. Moreover, the potential\ninterplay between arginine methylation and other post-translational modifications, as\npreviously reported (Bhattacharjee et al., 2022, Cell Reports), could further influence this\nphenotype. These interconnected regulatory mechanisms likely account for the variability\nobserved in rescue experiments and highlight the complexity of mtDNA regulation.\nQuestion 2: Authors strongly suggest that PRMT5 protects against mtDNA damage. To\nsupport this statement authors should provide evidence that endogenous mtDNA damage is\ninduced in PRMT5 KO or PRMT5 R82K cells. In the current data authors only show\nexogenously induced damage and its physiological relevance is elusive.\nAnswer: Thank you for your constructive comment. We have conducted additional\nexperiments to directly assess endogenous mtDNA damage/breaks in PRMT5-/- or TFAM-/- /R82\ncells.\nTopoisomerase 1 cleavage complex (Top1cc) intermediates are also trapped by endogenous\nDNA lesions, including abasic sites, mismatches, oxidized bases, nicks, and other endogenous\nDNA adducts (Pourquier and Pommier, 2001; Pommier et al., 2006a, Ghosh et al., 2019,\nScience Advances). To assess this, we detected endogenous mtDNA breaks in PRMT5-/- or\nTFAM-/- mitochondria by directly measuring in vivo trapping of Top1mtcc in isolated\nmitochondria using ICE (immunocomplex of enzyme) assays using antibodies that detects\nTop1mt (Supplementary Figure 7E-H, revised manuscript).\nOur results detected accumulations of Top1mtcc in mitochondria isolated from PRMT5 KO\ncells compared to wild-type controls, suggesting that there are endogenous DNA breaks in the\nabsence of PRMT5. This trapping was further enhanced upon treatment with the mtDNA-\ndamaging agent MIL-Iri. Similarly, both methylation-defective TFAM-/-/R82K cells and TFAM-\n/- cells exhibited increased Top1mtcc trapping, reflecting rapid mtDNA damage under both\nbasal conditions and following exogenous DNA damage induction. In contrast, wild-type\n(TFAM+/+) and TFAM-/-/WT-complemented cells showed markedly lower levels of Top1mtcc\ntrapping (Supplementary Figure 7F and 7H, revised manuscript).\nThese findings provide compelling evidence that PRMT5 plays a critical role in protecting\nagainst endogenous mtDNA damage. The increased trapping of Top1mtcc in PRMT5-deficient\nand R82K mutant cells underscores the importance of PRMT5 in mitigating cytotoxic DNA\nlesions in mitochondria, consistent with its proposed role in maintaining mitochondrial genome\nintegrity. These data strengthen our conclusion that PRMT5 is essential for safeguarding\nmtDNA against endogenous damage.\nMinor comments\nIntroduction:\nQuestion 1: Line 57 – please remove “covalently closed” as mtDNA may exist in many\ntopological conformations\nQuestion 2: Lines 60-63 – Suggested changes to improve clarity: “Unlike nuclear\nchromosomes, mtDNA lacks histones. Instead, TFAM binds mtDNA and plays a crucial role in\nthe formation of mitochondrial nucleoids.”\nAnswer: Thank you. We have replaced “Unlike nuclear chromosomes, mtDNA lacks histones”\nwith “TFAM binds mtDNA and plays a crucial role in the formation of mitochondrial\nnucleoids”.\nQuestion 3: Line 64 – replace “counts” with “formation”\nAnswer: Thank you. We have replaced “counts” with “formation”.\nQuestion 4: Lines 71-72 – remove “Due to the close association of OXPHOS, mtDNA is more\nvulnerable to oxidative DNA damage”. There are publications showing no evidence of\noxidative damage in mtDNA. Authors may consider writing that mtDNA mutation rates are\nhigher than nuclear DNA mutation rates instead.\nAnswer: Thank you. We have removed “Due to the close association of OXPHOS, mtDNA is\nmore vulnerable to oxidative DNA damage”.\nQuestion 5: Line 72 - and mitochondrial fusion has been proposed to compensate for mtDNA\ndamage. Please add “has been proposed to”\nAnswer: Thank you. We have changed accordingly.\nQuestion 6: Line 76 – please add “which have been correlated with TFAM, nevertheless its\nexact role is elusive”\nAnswer: Thank you. We have incorporated the suggested line “which have been correlated\nwith TFAM, nevertheless its exact role is elusive” in the revised manuscript.\nResults:\nQuestion 1: Lines 90-108 and Fig.1E – How is PRMT5 imported into mitochondria? Does it\nhave MTS?\nAnswer:\nWe did not identify a canonical mitochondrial targeting signal (MTS) within the PRMT5\nprotein sequence. This suggests that PRMT5 relies on non-canonical targeting mechanisms,\nsimilar to other proteins such as CREB, p53, yeast BCS1, HSP10, TDP1, and ISD11 (Leigh-\nBrown et al., 2010; Vögtle et al., 2009; Das et al., 2010, PNAS; Marchenko et al., 2000, JBC).\nTo investigate this, we conducted an in vitro mitochondrial import assay using recombinant\nHis-PRMT5 and ultra-purified mitochondria in the presence of Proteinase K (ProK) and the\n9\nmitochondrial uncoupler CCCP (Figure 1I). Our results demonstrated that His-PRMT5 was\ntransported to a protease-protected compartment, and its import was strictly membrane\npotential-dependent—a hallmark of proteins localized to the inner membrane (IM) and\nmitochondrial matrix (Jain et al., 2023, EMBO Reports; Ryan et al., 2001, Methods Cell Biol).\nThese findings provide strong evidence that PRMT5 is imported into the mitochondrial matrix\nvia a membrane potential-dependent mechanism. We sincerely thank the reviewer for\nprompting this detailed validation, which has significantly strengthened our conclusions.\nQuestion 2: Lines 119-125 – Please add mitoplast analysis with digitonin treatment to show\nif PRMT5 is localized to the mitochondrial matrix\nAnswer: To address the reviewer's concern, we performed a mitoplast isolation assay by\ntreating ultra-purified mitochondria with digitonin, which selectively solubilizes the\ncholesterol-rich outer membrane (OM) while preserving the integrity of the inner membrane\n(IM) (revised Figure 1H). These treatment exposes intermembrane space (IMS) proteins and\nIM-facing IMS proteins to proteolysis.\nFollowing ProK/digitonin digestion, the OM protein Tom20 was completely degraded,\nconfirming the successful removal of the OM. In contrast, the IM protein CoxIV exhibited\npartial resistance to ProK but was fully degraded after digitonin treatment, indicating its IM\nlocalization. Notably, PRMT5 remained completely resistant to ProK/digitonin digestion,\nsimilar to the mitochondrial matrix protein LonP1, providing strong evidence that PRMT5\nresides within the mitochondrial matrix (Figure 1H, revised manuscript).\nQuestion 3: Line 117 – Please remove “suggesting nuclear-encoded PRMT5 is translocated\nto mitochondria.” This is confusing\nAnswer: Thank you. We have removed “suggesting nuclear-encoded PRMT5 is translocated\nto mitochondria.”.\nQuestion 4: Line 180 – based on which criteria did the authors grouped hyperfused\nmitochondria (>5 μm), moderate length (2–5 μm), and fragmented mitochondria (<2 μm)?\nHow the sizes were selected?\nAnswer:\nWe have classified mitochondria into hyperfused (>5 μm), moderate length (2–5 μm), and\nfragmented (<2 μm) categories was based on the work by West et al. (Nature, 2015) conducted\nby the Gerald Shadel group, which provides a well-defined framework for categorizing\nmitochondrial morphology based on perimeter measurements. We quantified the perimeter of\nindividual mitochondria using the widely recognized image analysis software Fiji ImageJ,\nemploying the \"Analyze Particles\" plugin for precise measurements. By adopting these criteria,\nwe ensured that our analysis aligns with standardized methods, enabling meaningful\ncomparisons with prior studies and facilitating the interpretation of mitochondrial\nmorphological changes in our experimental system.\nKO influences MitoTracker import or mitochondrial membrane potential?\nAnswer: Thank you for raising this point and giving us the opportunity to clarify. To address\nthe reviewer’s concern, we conducted additional experiments to evaluate mitochondrial\nmembrane potential in PRMT5 KO cells.\nWe assessed changes in mitochondrial membrane potential using tetra-methylrhodamine\nmethyl ester (TMRM) staining followed by fluorescence-activated cell sorting (FACS)\nanalysis. Our results demonstrate that PRMT5 KO cells indeed exhibit a change in membrane\npotential as compared to wild-type controls as shown below.\nQuestion 6: Lines 195-210 – Please use orthogonal approach to support this data. Authors\nare advised to use chemical and genetic fission/fussion inhibitors.\nAnswer: Thank you for raising this point and giving us the opportunity to clarify. We have\nperformed additional FRAP assays in PRMT5+/+ and PRMT5-/- cells in the presence of Drp1\ninhibitor (Mdivi-1) to support the role of PRMT5 in regulating the fission-fusion dynamics of\nmitochondria. Cells were transfected with mitochondrial matrix-localized Mito-YFP (yellow\nfluorescent protein), treated with Mdivi-1 (2.5M, 4 h) and the fission/fusion rate of\nmitochondria was analyzed by measuring the rate of redistribution of Mito-YFP, ectopically\nexpressed in PRMT5+/+ and PRMT5-/- (Supplementary Fig.3C-D)\nOur new results demonstrate that in the presence of Mdivi-1, PRMT5+/+ cells exhibited a high\nFRAP recovery, achieving a maximal fluorescence intensity of approximately 95% at 60\nseconds (Supplementary Figure. 3D), consistent with a hyperfused mitochondrial network in\nthe absence of fission. Under identical conditions, PRMT5-/- cells displayed a comparable\nincrease in FRAP recovery, reaching a maximal intensity of approximately 85–90% at 60\nseconds (Supplementary Figure 3D), suggesting a similarly interconnected mitochondrial\nnetwork attributable to hyperfused mitochondria (Figure 3). These observations imply that\nPRMT5 likely functions in association with DRP1 within the same pathway. Further studies\nare required to elucidate the precise mechanistic relationship between PRMT5 and DRP1 in\nregulating mitochondrial dynamics.\ntable with all identified proteins as well as western blot confirmation of selected hits.\nAnswer: We appreciate the opportunity to clarify this point. To address the concern regarding\nmitochondrial-targeted proteins interacting with FLAG-PRMT5, we conducted a detailed\nanalysis of the mass spectrometry (MS) data (Figure 4A and Supplementary Figure 4B, revised\nmanuscript), which includes a plot of potential mitochondrial interaction partners of PRMT5.\nGene enrichment analysis based on biological processes and cellular components\nfurther confirmed the enrichment of mitochondrial-associated proteins, with TFAM emerging\nas a top hit (Supplementary Figure 4B, revised manuscript).\nTo validate these findings, we performed co-immunoprecipitation (co-IP) experiments\nusing ectopically expressed FLAG-PRMT5 in HEK293 cells. These experiments confirmed\nPRMT5 interactions with endogenous mitochondrial proteins, including Top1mt, LonP1, Phb1,\nCox4, and Tom20, corroborating the MS results (Supplementary Figure 4C). These additional\nanalyses strengthen our findings and provide a comprehensive understanding of PRMT5’s\nmitochondrial interaction network.\nWe believe these additional analyses and experimental validations address the concern and\nprovide a more comprehensive understanding of the interaction partners of PRMT5.\nQuestion 8: Line 345- please change “promotes” to “increases”\nAnswer: Thank you. We have changed accordingly.\nDiscussion:\nQuestion 1: Line 397 – how do the authors explain increased sensitivity of PRMT5 KO cells\nto ETC inhibitors (anti/rot/oligo)?\nAnswer: Thank you for your inquiry and giving us the opportunity to clarify this point. We\nhave checked the expression of OXPHOS subunit in PRMT5+/+, PRMT5-/- , PRMT5-/-WT cells\nand PRMT5-/-R368 as shown below.\nsubunit (nuclear encoded) in the absence of catalytic active PRMT5 as compared to their\nwildtype counterpart. Therefore, enhanced sensitivity of PRMT5 knockout cells to electron\ntransport chain (ETC) inhibitors—such as antimycin, rotenone, and oligomycin is due to\ndefects in the assembly of the ETC. Consequently, the inhibition of mitochondrial complexes\nby ETC inhibitors induces ROS production at levels sufficient to cause mitochondrial DNA\n(mtDNA) damage and trigger cell death pathways.\nQuestion 2: Line 403 – please change “critical” to “new”\nAnswer: Thank you. We have changed accordingly.\nQuestion 3: Line 406 – authors write that PRMT5 is localized mainly in the cytoplasm, but\nthey write that its main function is in histone methylation. Is PRMT5 translocated to the\nnucleus? Are there any conditions that stimulate PRMT5 localization to nucleus or\nmitochondria from the cytoplasm?\nAnswer: Thank you for raising this point. While PRMT5 is primarily cytoplasmic, evidence\nsuggests that it translocates to the nucleus under specific conditions, underscoring its functional\nsignificance.\nArginine symmetric methylation is increasingly recognized as a pivotal post-\ntranslational modification orchestrating key cellular processes, including epigenetic regulation,\nDNA repair, and genome maintenance. Cytoplasmic PRMT5 is predominantly associated with\nhighly proliferative, undifferentiated cells, whereas its nuclear localization correlates with cell\ncycle arrest and differentiation (Tee et al., 2010). For instance, tamoxifen promotes PRMT5\nnuclear translocation to suppress ERα transcriptional activity (Poulard et al., 2023, EMBO\nMolecular Medicine). In mouse primordial germ cells, PRMT5 shuttles between the nucleus\nand cytoplasm in association with the transcriptional repressor BLIMP-1 (Ancelin et al., 2006).\nSimilarly, in osteosarcoma cells, PRMT5 is recruited to the nucleus by SNAIL and its\ncorepressor AJUBA (Hou et al., 2008). More recently, Akt kinase activity has been implicated\nin PRMT5 nuclear export in lung cancer cells, though the precise mechanism remains unclear\n(Liu et al., 2021).\nPRMT5 is frequently overactivated in cancers, driving cellular proliferation through\nsymmetric dimethylation (SDMA) of multiple targets. It represses tumor suppressor genes RB1\nand CUL4A by methylating histones H3 and H4, while also activating key non-histone proteins\nsuch as p53, E2F1, and DNA repair factors (TDP1, FEN1, and RAD9) involved in DNA\nreplication (Rehman et al., 2018; Bhattacharya et al., 2022; Blanc et al., 2017; Guccione et al.,\n2019).\nThe predominantly cytoplasmic localization of PRMT5 is largely attributed to the\npresence of three nuclear export signals (NES) and the absence of a nuclear localization signal\n(NLS) (Gu et al., 2012).\nUnderstanding the molecular partners that mediate PRMT5's localization to different\norganelles remains a critical challenge, as its subcellular distribution is tightly linked to its\ndiverse functional roles.\nQuestion 4: Line 462 – please change to TFAM's role in neuroprotection was suggested”\nFigures:\nQuestion 1: Fig2A – why is PicoGreen nuclear signal present only in PRMT5 KO cells?\nAnswer: Thank you for bringing up the issue. As suggested, we have conducted new\nexperiments and indeed failed to detect picogreen in the nucleus. The new data are incorporated\nin revised manuscript.\nQuestion 2: Fig2 – please add additional control of nuclear PRMT5 (non-mito localized) to\nensure direct effects in the mitochondria.\nAnswer: Our study uncovers a novel role for PRMT5 in mitochondrial function, supported by\nmultiple lines of evidence. Comprehensive mitochondrial analyses (Figure 2) consistently\ndemonstrate PRMT5’s involvement in mitochondrial processes. Cellular fractionation data\n(Figure 1) reveal PRMT5 localization in nuclear, cytoplasmic, and mitochondrial fractions,\nindicating its import into multiple cellular compartments to fulfill distinct functional roles.\nImmunolocalization further confirms PRMT5’s presence across the cytoplasm, mitochondria,\nand nucleus. Given these findings, we believe that additional experiments focusing on non-\nmitochondrial PRMT5 fall outside the scope of this study.\nQuestion 3: Fig5B – why is WT and R82K more similar than +/+ and -/-WT? This argues\nagainst the role of R82K in the nucleoid protection.\nAnswer: Thank you for bringing up the issue. We have performed re-quantification of cells\nand have incorporated the new result in our revised manuscript.\nQuestion 4: Fig7B-D – please include comparison of PRMT5 +/+ to PRMT5 -/- + CDDO\nAnswer: Thank you for your suggestion. We performed a new cycloheximide chase assay to\ncompare the half-life of PRMT5+/+ and PRMT5-/- cells treated with CDDO. The results from\nthis experiment have been incorporated into Supplementary Figure 7A-B of the revised\nmanuscript.\nQuestion 5: Fig7F-H – please include comparison of WT to R82K + CDDO\nAnswer: Thank you for your suggestion. We conducted new cycloheximide chase assay to\ncompare the half-life of as well as WT and the R82K mutant treated with CDDO\n(Supplementary Figure 7C-D), as suggested by the reviewer and incorporated in the revised\nmanuscript.\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all my comments with a number of new experiments. I believe these high-quality data, such as the characterisation of PERMT5 mitochondrial localisation (Fig. 1) strengthen the manuscript. I'd like to congratulate the authors on their exciting discovery.", "reviewer_3": "Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation and controls various pathways, including gene expression, chromatin structure, and preserving genomic stability (line 45). No previous studies reported the localization of PRMT5 in the mitochondria (MT). The authors here analyzed the functionality of PRMT5 in the MT. They demonstrated the presence of PRMT5 in the mitochondria (Fig. 1). They previously developed MT-specific DNA topoisomerase I poison (MIL-Iri) (Ref. 36, 37) and here uncovered the role of PRMT5 in the stability of mitochondria DNA (mtDNA) following exposure of cells to MIL-Iri (Fig. 2). They also showed the role of PRMT5 in controlling balance of the mitochondrial fission-fusion cycle with and without a MIL-Iri treatment (Fig. 3) and in TFAM dimethylation at R82 (Fig. 4). Also, PRMT5-mediated TFAM dimethylation stabilizes mtDNA, maintains the transcription of genes encoded by mtDNA (Fig. 5), increases the binding of TFAM to mtDNA (Fig. 6), and stabilizes TFAM, ensuring efficient mtDNA repair of MIL-Iri-induced DNA damage (Fig. 7). These studies demonstrated the role of PRMT5 in the functionality of MT.\n\nTheir study addressed the timely clinically significant research question. Seven PRMT5 inhibitors, such as AZD3470 and AMG193, are under clinical trials for developing anti-cancer therapy, though previous studies have poorly studied the molecular mechanisms underlying the anti-cancer effect of PRMT5. Although it is widely believed that DNA-damaging anti-cancer therapies, such as DNA topoisomerase I poisons and cisplatin, kill malignant cells by damaging genomic DNA but not mtDNA, the current study will open up a new research field, the anti-cancer effect of mtDNA damage. Future studies will investigate the anti-cancer effect of PRMT5 inhibitors comparing between WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K MCF-7 cells. In conclusion, this manuscript should be published soon if an appropriate revision is done.\n\nSpecific comments.\n1. Line 184: ‘We found that PRMT5-/- cells exhibited rapid mtDNA depletion (~2-fold) and slower repair kinetics (~2-fold) compared to PRMT5+/+ cells (Fig. 2D-E and Supplementary Fig. 2F-G) upon inducing mtDNA damage.’ However, actual Fig. 2E data shows that PRMT5-/- and PRMT5+/+ cells exhibited similar repair kinetics (about 30% increases in the number of long-PCR products during 6 h repair time). Please clarify this point.\n2. Line 187: Cell survival assays revealed increased sensitivity to mtDNA-damaging agents (MIL-Iri, H2O2, and menadione) (Fig. 2F). It is widely accepted that topoisomerase I poison (Iri) kills cancer cells by inducing lethal double-strand breaks (DSBs) in genomic DNA during DNA replication, but not DNA damage to mtDNA, leading to activation of the damage checkpoint and triggering apoptosis. Thus, the cytotoxic effect of a 24 h exposure of cells to MIL-Iri is surprising and also medically crucial if it is true. Therefore, the authors need to completely exclude the possibility that a small fraction of MIL-Iri induces DSBs in genomic DNA by showing no increased gammaH2AX signals after the 24 h exposure of cells to 5 μM MIL-Iri.\n3. In cell survival assays (Line 721) for Figs. 2F and 7L, the authors treated cells with MIL-Iri for 24 h and subjected cells to the MTT assay, which assesses the viability of cells by measuring the catalytic activity of mitochondrial dehydrogenases. Since the PRMT5-/- and TFAM-/-/R82K mutations can reduce their catalytic activity without killing cells, the authors may need to analyze cellular viability by flow cytometric analysis of PI/Annexin-V staining. Another interesting experiment is to stain SA-β-gal signals after incubation with MIL-Iri for 24 h, because it is clinically important if PRMT5 inhibitors block the proliferation of cancer cells by inducing their senescence.\n4. Related to the second comment, if possible, this manuscript would be further improved if the authors could analyze the sensitivity of WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K cells to Irinotecan, which damages both genomic DNA and mtDNA, using the colony survival assay. If TFAM-/-/WT cells are more resistant to Irinotecan than TFAM-/-/R82K cells, this data would reveal an anti-cancer effect of mtDNA damage in addition to the anti-cancer effect of lethal DSBs in genomic DNA.\n5. Line 208: After the authors indicated ‘PRMT5-/- cells exhibited a significant increase (~ 2-fold) in hyperfused mitochondrial networks’, the authors showed that ‘Accordingly, we observed increased expression levels of Mfn1 and Mfn2 in PRMT5-/- cells (Fig. 3C).’ (line 211). Do the authors want to say the causal relationship between ‘increased expression levels of Mfn1 and Mfn2’ and ‘a significant increase (~ 2-fold) in hyperfused mitochondrial networks’? Please clarify this point.\n6. Related the above comment, if ‘increased expression levels of Mfn1 and Mfn2 in PRMT5-/- cells’ causes ‘hyperfused mitochondrial networks’, an interesting experiment is to test whether the ectopic expression of Mfn1 and Mfn2 suppresses hyperfused mitochondrial networks and restores the stability of mtDNA following exposure of PRMT5-/- cells to MIL-Iri. If the ectopic expression of Mfn1 and Mfn2 does not suppress hyperfused mitochondrial networks, the authors should remove Fig. 3C because this data is confusing to general readers.\n7. To suggest that PRMT5 stabilizes mtDNA by demethylating TFAM at R82, the authors showed the data of the four genotypes, WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K MCF-7 cells (Figure 5B, 5D, 5E, and 5G). However, the authors need to exclude the possibility that the R82K affects the functioning of TFAM by changing its conformation. To this end, the authors need to show the phenotypic similarity between TFAM-/-/R82K and PRMT5-/- cells by adding the data of PRMT5-/- MCF-7 cells to Figure 5B, 5D, 5E, and 5G. If possible, this manuscript would be further improved if the authors could show the phenotypic similarity between PRMT5-/- cells, TFAM-/-/R82K cells, and cells with both PRMT5-/- and TFAM-/-/R82K mutations.\n8. This manuscript would be further improved if the authors discuss the potential anti-cancer effect of PRMT5 inhibitors. The authors should shorten the discussion on the localization of PRMT5 in the MT (the third and fourth paragraphs) because the data of Fig. 1 is solid.\n9. The authors presented several exciting phenotypes seen in the PRMT5-/- and TFAM-/-/R82K mutant cells and discuss them in parallel, including abnormal nucleoid counts, mtDNA copy numbers, the increased sensitivity of mtDNA to MIL-Iri, altered fission-fusion cycle of MT, reduced transcription of mtDNA-encoded genes, and compromised MT respiration. However, it is difficult for general readers to understand the relative importance of individual phenotypes and their potential causal relationships. The reviewer feels that PRMT5-/- and TFAM-/-/R82K mutations reduced transcription (Fig. 5G) more significantly than decreases in mtDNA copy number (Fig. 5E), the altered fission-fusion cycle (Fig. 5F), and higher sensitivity of mtDNA to MIL-Iri (Fig. 7J). Also, the reduced transcription could explain the anti-cancer effect of PRMT5 inhibitors. This manuscript would be further improved if the authors discuss the relationship of the several phenotypes.\n\nMinor comments:\nFigs. 2E and 7J legend: The authors need to write the definition of ‘1.0’ on the Y-axis."}, "author_response": "Point-by-point address to REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors addressed all my comments with a number of new experiments. I believe these high-quality\ndata, such as the characterisation of PRMT5 mitochondrial localisation (Fig. 1) strengthen the\nmanuscript. I'd like to congratulate the authors on their exciting discovery.\nAnswer: We sincerely thank the reviewer for recognizing the extensive revisions and newly added\nexperiments. We are glad that the characterization of PRMT5’s mitochondrial localization (Fig. 1) and\nother supporting data addressed the all concerns raised. We appreciate the reviewer’s kind words and\nare encouraged by the positive evaluation of our study. We are grateful for the constructive feedback,\nwhich has significantly improved the quality and clarity of the manuscript.\nReviewer #3 (Remarks to the Author):\nProtein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation and controls various\npathways, including gene expression, chromatin structure, and preserving genomic stability (line 45).\nNo previous studies reported the localization of PRMT5 in the mitochondria (MT). The authors here\nanalyzed the functionality of PRMT5 in the MT. They demonstrated the presence of PRMT5 in the\nmitochondria (Fig. 1). They previously developed MT-specific DNA topoisomerase I poison (MIL-Iri)\n(Ref. 36, 37) and here uncovered the role of PRMT5 in the stability of mitochondria DNA (mtDNA)\nfollowing exposure of cells to MIL-Iri (Fig. 2). They also showed the role of PRMT5 in controlling\nbalance of the mitochondrial fission-fusion cycle with and without a MIL-Iri treatment (Fig. 3). In\nTFAM dimethylation at R82 (Fig. 4). Also, PRMT5-mediated TFAM dimethylation stabilizes mtDNA,\nmaintains the transcription of genes encoded by mtDNA (Fig. 5), increases the binding of TFAM to\nmtDNA (Fig. 6), and stabilizes TFAM, ensuring efficient mtDNA repair of MIL-Iri-induced DNA\ndamage (Fig. 7). These studies demonstrated the role of PRMT5 in the functionality of MT.\nTheir study addressed a timely clinically significant research question. Seven PRMT5 inhibitors, such\nas AZD3470 and AMG193, are under clinical trials for developing anti-cancer therapy, though previous\nstudies have poorly studied the molecular mechanisms underlying the anti-cancer effect of PRMT5.\nAlthough it is widely believed that DNA-damaging anti-cancer therapies, such as DNA topoisomerase\nI poisons and cisplatin, kill malignant cells by damaging genomic DNA but not mtDNA, the current\nstudy will open up a new research field, the anti-cancer effect of mtDNA damage. Future studies will\ninvestigate the anti-cancer effect of PRMT5 inhibitors, comparing between WT, TFAM-/-, TFAM-/-/WT,\nand TFAM-/-/R82K MCF-7 cells. In conclusion, this manuscript should be published soon if an\nappropriate revision is done.\nAnswer: We thank the reviewer for summarizing our findings. As noted, our study is the first to\ndemonstrate mitochondrial localization of PRMT5 and its functional relevance. We show that PRMT5\nregulates mtDNA stability following mitochondria targeted MIL-Iri treatment, H O , Menadione,\n2 2\nmodulates mitochondrial dynamics, and methylates TFAM at R82. This methylation enhances TFAM’s\nbinding to mtDNA, maintains mitochondrial gene transcription, and promotes mtDNA repair.\nCollectively, these results uncover a novel role for PRMT5 in maintaining mitochondrial genome\nintegrity and function.\nThough we appreciate Reviewer #3's comments, the emphasis on PRMT5's anti-cancer\npotential and its broader implications in mtDNA damage falls outside the scope of our current study.\nOur manuscript specifically investigates the discovery of mitochondrial localization of PRMT5 and its\nfunctional interaction with TFAM through arginine methylation at R82 in regulating mtDNA, as pointed\nout by reviewer#1 and by reviewer#2 comments. We respectfully note that several points raised\nintroduce new and unrelated questions not supported by the presented data. While the role of PRMT5\nin cancer may be important, it is not the focus of this work, which is centred on mitochondrial biology,\nas it is well summarised in the 1st paragraph by reviewer#3 comments above. However, we have\nperformed several new experiments to address the reviewer’s concerns, as listed below.\nSpecific comments.\n1. Line 184: 'We found that PRMT5-/- cells exhibited rapid mtDNA depletion (~2-fold) and slower\nrepair kinetics (~2-fold) compared to PRMT5+/+ cells (Fig. 2D-E and Supplementary Fig. 2F-G) upon\ninducing mtDNA damage.' However, actual Fig. 2E data show that PRMT5-/- and PRMT5+/+ cells\nexhibited similar repair kinetics (about 30% increases in the number of long-PCR products during 6 h\nrepair time). Please clarify this point.\nAnswer: Thank you for the opportunity to clarify this point. We observed a ~4-fold reduction in\nnucleoid number and enlarged nucleoids within elongated mitochondrial networks in PRMT5-/- cells\n(Fig. 2A-B; untreated, and Supplementary Fig. 2A). This phenotype was rescued in PRMT5-/- cells\ncomplemented with wild-type PRMT5 (PRMT5⁻/⁻/WT), but not with the catalytic mutant PRMT5⁻/⁻/R368A,\nunder comparable expression levels and without exogenous DNA damage (Supplementary Fig. 2B).\nCorrespondingly, mtDNA copy number was significantly restored in PRMT5-/-/WT but not in PRMT5-/-\n/R368A cells, indicating that PRMT5’s catalytic activity is essential for mtDNA maintenance (Fig. 2C).\nUnder MIL-Iri treatment that exclusively traps mitochondrial Top1cc-mtDNA complexes,\nPRMT5-/- cells showed a ~3-fold increase in mtDNA damage compared to PRMT5⁺/⁺ cells (Fig. 2A-B,\nright panels). This heightened susceptibility was consistent with rapid mtDNA depletion upon exposure\nto MIL-Iri or H₂O₂ (Fig. 2D-E; Supp. Fig. 2F-G). Increased mtDNA damage also correlated with\nreduced cell viability in PRMT5⁻/⁻ cells treated with mtDNA-damaging agents (MIL-Iri, H₂O₂,\nmenadione). While mitochondrial DNA repair is critical as they are exposed to the electron transport\nchain to intrinsic DNA damage and lack protective histones, they therefore have complex backup\npathways for repair.\nAs suggested by the reviewer we have re-quantified the long-range quantitative PCR assay gels\nwhich reflect a statistically consistent ~1.5-fold delay in mtDNA repair in PRMT5⁻/⁻ cells. Together,\nthese findings demonstrate that PRMT5 is critical for maintaining mitochondrial nucleoid structure and\nprotecting mtDNA from exogenous damage. Thank you.\n2. Line 187: Cell survival assays revealed increased sensitivity to mtDNA-damaging agents (MIL-Iri,\nH2O2, and menadione) (Fig. 2F). It is widely accepted that topoisomerase I poison (Iri) kills cancer\ncells by inducing lethal double-strand breaks (DSBs) in genomic DNA during DNA replication, but not\nDNA damage to mtDNA, leading to activation of the damage checkpoint and triggering apoptosis. Thus,\nthe cytotoxic effect of a 24 h exposure of cells to MIL-Iri is surprising and also medically crucial if it is\ntrue. Therefore, the authors need to completely exclude the possibility that a small fraction of MIL-Iri\ninduces DSBs in genomic DNA by showing no increased gammaH2AX signals after the 24 h exposure\nof cells to 5 μM MIL-Iri.\nAnswer: We would like to respectfully clarify that our study focuses on the carrier-mediated,\nmitochondria-targeted delivery of irinotecan using MIL-101(Fe) nanoparticles (MIL-Iri), specifically\ndesigned to selectively trap mitochondrial Top1 covalent complexes (Top1mtcc) without affecting the\nnuclear Top1 isoform (Ghosh et al., 2024; Dalton Transactions; PMID: 38265230). We demonstrate\nthat MIL-Iri induces Top1mtcc formation in a dose-dependent manner at low nanomolar concentrations,\nresulting in mitochondrial DNA (mtDNA) damage in human colon and breast cancer cells. This\nmitochondria-specific genotoxicity contributes to cellular toxicity and underpins a novel therapeutic\nstrategy for targeting cancer.\nAs MIL-Iri-induced Top1mtcc triggers apoptosis in cancer cells (Ghosh et al, 2024; Dalton\nTransaction; PMID# 38265230), the proposed γH2AX signal likely reflects secondary nuclear DNA\nfragmentation during apoptotic progression. Mitochondrial-nuclear crosstalk is well known in\napoptosis, and thus, detection of γH2AX under these conditions is not unexpected. We believe that\nadditional experiments to assess γH2AX levels following 24-hour exposure to 5 μM MIL-Iri are not\ndirectly relevant to the scope of our mitochondrial-targeted approach and would not yield\nmechanistically meaningful insights into primary genomic DNA damage.\n3. In cell survival assays (Line 721) for Figs. 2F and 7L, the authors treated cells with MIL-Iri for 24 h\nand subjected cells to the MTT assay, which assesses the viability of cells by measuring the catalytic\nactivity of mitochondrial dehydrogenases. Since the PRMT5-/- and TFAM-/-/R82K mutations can\nreduce their catalytic activity without killing cells, the authors may need to analyze cellular viability by\nflow cytometric analysis of PI/Annexin-V staining. Another interesting experiment is to stain SA-β-gal\nsignals after incubation with MIL-Iri for 24 h, because it is clinically important if PRMT5 inhibitors\nblock the proliferation of cancer cells by inducing their senescence.\nAnswer: As suggested by the reviewer, MTT assay results may be influenced by changes in\nmitochondrial dehydrogenase activity, particularly in PRMT5-/- and TFAM-/-/R82K cells. To address this\nconcern, we have performed new flow cytometric analysis using PI/Annexin-V staining to more\naccurately assess cell viability and apoptosis, independent of mitochondrial enzyme activity. This\nanalysis confirmed MIL-Iri induced elevated levels of cell death in PRMT5-/- and TFAM-/-/R82K as\nrevealed by a significant increase in early and late apoptotic cell population after MIL-Iri treatment\n(Supplementary Figure 2M-P and Supplementary Figure 7M-P). MIL-Iri -induced apoptotic cellular\npopulation could be protected by complementation of PRMT5WT and TFAMWT in the PRMT5 -/- and\nTFAM-/- cells respectively, consistent with the survival assays.\nWe believe that investigating the senescence mechanism falls beyond the scope of the present\nstudy. However, we are confident that the current manuscript will open several new avenues for future\nresearch in this direction.\n4. Related to the second comment, if possible, this manuscript would be further improved if the authors\ncould analyze the sensitivity of WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K cells to Irinotecan,\nwhich damages both genomic DNA and mtDNA, using the colony survival assay. If TFAM-/-/WT cells\nare more resistant to Irinotecan than TFAM-/-/R82K cells, this data would reveal an anti-cancer effect\nof mtDNA damage in addition to the anti-cancer effect of lethal DSBs in genomic DNA.\nAnswer: Irinotecan is a well-established anticancer drug that exerts its cytotoxic effect by stabilizing\nnuclear Topoisomerase I-DNA covalent complexes (Top1cc), thereby preventing the re-ligation of DNA\nduring replication and transcription. This leads to the accumulation of DNA breaks, replication stress,\nand ultimately cell death, particularly in rapidly dividing cancer cells. Notably, irinotecan primarily\ntargets the nuclear Top1 isoform and does not effectively trap mitochondrial Top1 (Top1mt) due to its\nlimited access to the mitochondrial compartment. Please note MIL-Iri in our study focuses on the\ncarrier-mediated, mitochondria-targeted delivery of irinotecan via MIL-101(Fe) nanoparticles, designed\nto selectively trap Top1mt covalent complexes (Top1mtcc) without affecting the nuclear Top1 isoform\n(Ghosh et al, 2024; Dalton Transaction; PMID# 38265230). Therefore, as suggested by the reviewer,\nwe have performed new colony survival assay using WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K cells\nin response to MIL-Irinotecan (MIL-Iri)—which offers insights into the interplay between\nmitochondrial DNA damage and nuclear DNA perturbation. Clonogenic assays revealed that TFAM\nknockout increased cellular sensitivity to MIL-Iri. This hypersensitivity was rescued by\ncomplementation with wild-type TFAM but not with the R82K mutant, indicating that arginine\nmethylation–deficient TFAM fails to rescue TFAM-deficient cells (Supplementary Figure 7Q-R). All\nthese new data are incorporated in the revised manuscript.\n5. Line 208: After the authors indicated ‘PRMT5-/- cells exhibited a significant increase (~ 2-fold) in\nhyperfused mitochondrial networks’, the authors showed that ‘Accordingly, we observed increased\nexpression levels of Mfn1 and Mfn2 in PRMT5-/- cells (Fig. 3C).’ (line 211). Do the authors want to say\nthe causal relationship between ‘increased expression levels of Mfn1 and Mfn2’ and ‘a significant\nincrease (~ 2-fold) in hyperfused mitochondrial networks’? Please clarify this point.\nAnswer: Mitochondrial dynamics are governed by the opposing processes of fusion and fission, which\nare essential for maintaining mitochondrial integrity, distribution, and function. Fusion helps mitigate\nstress by mixing the contents of partially damaged mitochondria, whereas fission facilitates the removal\nof dysfunctional mitochondria (Ghosh et al., 2019 Sci Adv; PMID 31723605; Chen et al., J. Cell. Biol,\nPMID: 12527753). Mfn1 and Mfn2 are key outer mitochondrial membrane GTPases that regulate\nmitochondrial fusion (Chen et al., J. Cell. Biol, PMID: 12527753). Their increased expression promotes\nthe merging of adjacent mitochondria, resulting in elongated or hyperfused mitochondrial networks.\nThis fusion process counterbalances mitochondrial fission and is essential for maintaining\nmitochondrial morphology, bioenergetics, and cellular homeostasis. Conversely, loss of Mfn1 or Mfn2\ndisrupts fusion, leading to fragmented mitochondria and impaired mitochondrial function (Chen et al.,\nJ. Cell. Biol, PMID: 12527753; Tábara et al., Nature Reviews Molecular Cell Biology (2025): PMID:\n39420231). Disruption in the balance of fusion and fission, as observed through altered expression or\nfunction of MFNs, can lead to mitochondrial dysfunction, genomic instability, and disease. In our study,\nwe demonstrate that PRMT5 plays a crucial role in maintaining mitochondrial nucleoid numbers and\nprotects mtDNA from extrinsic damage. Further, we show that loss of PRMT5 results in mitochondrial\nhyperfusion, accompanied by increased Mfn1/Mfn2 levels, highlighting a critical role of PRMT5 in\nmaintaining mitochondrial dynamics.\n6. Related the above comment, if ‘increased expression levels of Mfn1 and Mfn2 in PRMT5-/- cells’\ncauses ‘hyperfused mitochondrial networks’, an interesting experiment is to test whether the ectopic\nexpression of Mfn1 and Mfn2 suppresses hyperfused mitochondrial networks and restores the stability\nof mtDNA following exposure of PRMT5-/- cells to MIL-Iri. If the ectopic expression of Mfn1 and Mfn2\ndoes not suppress hyperfused mitochondrial networks, the authors should remove Fig. 3C because this\ndata is confusing to general readers.\nAnswer: Thank you for raising this important point and giving us the opportunity to clarify. We would\nlike to bring to the reviewer’s attention that ectopic overexpression of Mfn1 and Mfn2 in PRMT5-/- cells\nwould, in fact, be expected to exacerbate mitochondrial hyperfusion, leading to further elongation of\nthe mitochondrial network rather than suppressing the hyperfused phenotype.\nIn contrast, we have performed orthogonal experiments using FRAP assays in PRMT5+/+ and\nPRMT5-/- cells in the presence of Drp1 inhibitor (Mdivi-1) to support the role of PRMT5 in regulating\nthe fission-fusion dynamics of mitochondria. Based on the suggestion of reviewer# 2, cells were\ntransfected with mitochondrial matrix-localized Mito-YFP (yellow fluorescent protein), treated with\nMdivi-1 (2.5M, 4 h) and the fission/fusion rate of mitochondria was analysed by measuring the rate\nof redistribution of Mito-YFP, ectopically expressed in PRMT5+/+ and PRMT5-/- (Supplementary Fig.\n3C-D). Our new results demonstrate that in the presence of Mdivi-1, PRMT5+/+ cells exhibited a high\nFRAP recovery, achieving a maximal fluorescence intensity of approximately 95% at 60 seconds\n(Supplementary Figure 3D), consistent with a hyperfused mitochondrial network in the absence of\nfission. Under identical conditions, PRMT5-/- cells displayed a comparable increase in FRAP recovery,\nreaching a maximal intensity of approximately 85–90% at 60 seconds (Supplementary Figure 3D),\nsuggesting a similarly interconnected mitochondrial network attributable to hyperfused mitochondria\n(Figure 3). These observations imply that PRMT5 likely functions in association with DRP1 within the\nsame pathway. Therefore, we respectfully disagree with the suggestion to remove Fig. 3C, as we have\nprovided sufficient evidence—such as live-cell microscopy quantifying mitochondrial length and FRAP\nanalysis (Figure 3)—to support that the absence of PRMT5 leads to mitochondrial hyperfusion.\nMoreover, there is strong precedence in the literature demonstrating that increased levels of MFN1 and\nMFN2 are causally linked to mitochondrial hyperfusion (e.g., PMID: 35977476), supporting our\ninterpretation. Thank you for raising this point and giving us the opportunity to clarify.\n7. To suggest that PRMT5 stabilizes mtDNA by demethylating TFAM at R82, the authors showed the\ndata of the four genotypes, WT, TFAM-/-, TFAM-/-/WT, and TFAM-/-/R82K MCF-7 cells (Figure 5B,\n5D, 5E, and 5G). However, the authors need to exclude the possibility that the R82K affects the\nfunctioning of TFAM by changing its conformation. To this end, the authors need to show the phenotypic\nsimilarity between TFAM-/-/R82K and PRMT5-/- cells by adding the data of PRMT5-/- MCF-7 cells to\nFigure 5B, 5D, 5E, and 5G. If possible, this manuscript would be further improved if the authors could\nshow the phenotypic similarity between PRMT5-/- cells, TFAM-/-/R82K cells, and cells with both\nPRMT5-/- and TFAM-/-/R82K mutations.\nAnswer: To address the possibility that the R82K mutation alters TFAM function by affecting its\nconformation, we have performed circular dichroism (CD) spectroscopy comparing wild-type\n(WT) and the R82K mutant TFAM (Supplementary Fig. 6A). The CD analysis confirms that the\nR82 mutation does not induce any significant structural changes that could impact TFAM’s function\nindependently of PRMT5-mediated di-methylation. The new data has been incorporated in the revised\nmanuscript (Supplementary Fig. 6A).\nRegarding the phenotypic comparison with PRMT5 knockout (PRMT5-/-) cells, we would like\nto point out that data related to nucleoid count in the presence or absence of MIL-IRI, mtDNA copy\nnumber, and mitochondrial length in PRMT5-/- cells have already been presented in Figure 2. These\ndata provide relevant insights into the role of PRMT5 in mitochondrial regulation. Accordingly, we\ndetected a marked reduction in mitochondrial copy number in TFAM-/- cells or TFAM-/-/R82K compared\nto its wild-type counterpart (Fig. 5E). This highlights the phenotypic similarity between PRMT5-/- cells\nand TFAM-/-/R82K cells. Like PRMT5 KO cells (Fig 2A &B), TFAM knockout cells showed a significant\nincrease in hyperfused mitochondria (~3 fold) as compared to TFAM and PRMT5 proficient cells,\nwhich could not be rescued by R82K mutant (Fig. 5A; quantification in Fig. 5F; left panel). Accordingly,\nwe detected an increase in mitochondrial volume in R82K methylation mutant (TFAM-/-/R82K), which\nwere reversed in PRMT5+/+; TFAM+/+ proficient wild-type cells (Supplementary Fig.5D). Next,\nfollowing mtDNA damage with MIL-Iri, we observed that the hyperfused mitochondria in TFAM-/- and\nTFAM-/-/R82K cells (Fig. 5C) were more prone to fragmentation compared to those in PRMT5+/+;\nTFAM+/+-proficient cells (Fig. 5C; quantification in Fig. 5F; right panel). This implies that TFAM-R82\nmethylation is essential for proper nucleoid maintenance and distribution. Therefore, we have\nintentionally not included the PRMT5-/- data in Figure 5 to avoid overloading the figure and potentially\nconfusing readers with overlapping datasets. Taken together, both PRMT5-/- cells and TFAM-/-/R82K cells\nshow similar mitochondrial phenotypes, further confirming the critical role of PRMT5-mediated\nTFAM-R82 methylation in the protection of mtDNA nucleoids. We have included a paragraph in the\ndiscussion section of our revised manuscript.\n8. This manuscript would be further improved if the authors discuss the potential anti-cancer effect of\nPRMT5 inhibitors. The authors should shorten the discussion on the localization of PRMT5 in the MT\n(the third and fourth paragraphs) because the data of Fig. 1 is solid.\nAnswer: Thank you for finding the data presented for the PRMT5 in the mitochondria (Fig. 1) is\nappreciated as solid data. The discussion of “the discussion on the localization of PRMT5 in the MT\n(the third and fourth paragraphs)” was expanded based on the request of the 1st and 2nd revisers.\nHowever, based on your recommendation we have reduced paragraph III and omitted the paragraph IV\nconnecting PRMT5 localization in mitochondria.\nWe appreciate the suggestion and we have included a discussion on the potential anti-cancer\neffects of PRMT5 inhibitors in the revised manuscript. Given the emerging therapeutic interest in\ntargeting PRMT5 in various cancers.\n9. The authors presented several exciting phenotypes seen in the PRMT5-/- and TFAM-/-/R82K mutant\ncells and discussed them in parallel, including abnormal nucleoid counts, mtDNA copy numbers, the\nincreased sensitivity of mtDNA to MIL-Iri, altered fission-fusion cycle of MT, reduced transcription of\nmtDNA-encoded genes, and compromised MT respiration. However, it is difficult for general readers\nto understand the relative importance of individual phenotypes and their potential causal relationships.\nThe reviewer feels that PRMT5-/- and TFAM-/-/R82K mutations reduced transcription (Fig. 5G) more\nsignificantly than decreases in mtDNA copy number (Fig. 5E), the altered fission-fusion cycle (Fig. 5F),\nand higher sensitivity of mtDNA to MIL-Iri (Fig. 7J). Also, the reduced transcription could explain the\nanti-cancer effect of PRMT5 inhibitors. This manuscript would be further improved if the authors\ndiscussed the relationship of the several phenotypes.\nAnswer: We thank the reviewer for this thoughtful and insightful comment. We believe that PRMT5-/-\nand TFAM-/-/R82K mutations shows reduced transcription (Fig. 5G) more significantly than decreases in\nmtDNA copy number (Fig. 5E), that altered fission-fusion cycle (Fig. 5F), and higher sensitivity of\nmtDNA to MIL-Iri (Fig. 7J) are all interconnected mitochondrial phenotypes that result in mitochondrial\ndysfunction. We agree that discussing the relative importance and potential causal relationships among\nthe observed phenotypes will improve the clarity and impact of the revised manuscript.\nIn the revised version, we have expanded our discussion to address how these phenotypes—\nparticularly reduced transcription of mtDNA-encoded genes, decreased mtDNA copy number, altered\nmitochondrial dynamics, and increased sensitivity to Mil-IRI—may be functionally interconnected.\nWe have highlighted that the reduction in mitochondrial transcription appears to be one of the more\npronounced and potentially central defects, which could mechanistically link PRMT5 loss or TFAM-\nR82K mutation to mitochondrial dysfunction and, ultimately, the anti-cancer effects of PRMT5\ninhibition.\nMinor comments:\nFigs. 2E and 7J legend: The authors need to write the definition of '1.0' on the Y-axis.\nAnswer: We thank the reviewer for pointing this out. We have revised the figure legends incorporating\n1.0 on the Y axis as fold change (arbitrary unit) for Figures 2E and 7J accordingly."}]

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