ALKB-1-dependent tRNA methylation is required for efficient paternal mitochondrial elimination

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
Maternal mitochondrial inheritance is secured by mechanisms that exclude paternal mitochondrial DNA (mtDNA). While, epigenetic modifications are vital for spermatogenesis and embryo development, their roles in the paternal mitochondrial elimination (PME) remain poorly understood. Here, we identify ALKB-1, a DNA/RNA demethylase, as a pivotal factor for efficient PME in Caenorhabditis elegans (C. elegans), acting through ALKB-1-dependent modulation of tRNA m1A methylation. Mechanistically, ALKB-1 inactivation leads to m1A hypermethylation of tRNA, which subsequently disrupts protein translation, impairs mitochondrial proteostasis, and increases ROS levels. This cascade activates the oxidative stress response factor SKN-1/Nrf2 and initiates the mitochondrial unfolded protein response (UPRmt) through ATFS-1, causing accumulation of mitochondria and mtDNA in sperm, which ultimately impedes efficient paternal mitochondrial removal and negatively impacts male fertility and embryonic development. Our findings describe a mechanism whereby ALKB-1-mediated tRNA m1A epitranscriptomic modifications are necessary for maintaining mitochondrial quality control, thereby influencing PME efficiency, underscoring the importance of this epitranscriptomic stress checkpoint in upholding proper mitochondrial inheritance during reproduction.

Introduction
The coordination of nuclear and mitochondrial genome expression is crucial for maintaining mitochondrial function and homeostasis, despite their unique inheritance patterns. Maternal inheritance of the mitochondrial genome (mtDNA) is predominant in eukaryotes, except for certain fungi and mussels. Paternal mitochondria are selectively eliminated during spermatogenesis and early embryogenesis, a key step for maintaining maternal mtDNA. Accumulation of mutations and heteroplasmy in mtDNA is linked to a spectrum of mitochondrial pathologies, including diabetes and Parkinson’s disease. Maternal transmission of mtDNA hinders the spread of harmful mutations to subsequent generations. Multiple mechanisms involving mitochondrial endonuclease G (EndoG) in Caenorhabditis elegans (C. elegans) and Drosophila, the mitochondrial transcription factor A (TFAM) in human sperm, and autophagy, mitophagy-lysosome, and ubiquitin-proteasome pathways have been implicated in the elimination of paternal mitochondria.

Epigenetic modifications, including DNA or RNA methylation, are crucial for regulating spermatogenesis and embryo development. Changes in RNA methylation are linked to sperm motility, while alterations in DNA methylation patterns are associated with increased sperm mtDNA copy number and abnormalities. Sperm cells are characterized by high levels of DNA methylation, which are essential for their development and function. Nevertheless, the role of methylation-related epigenetic modifications in paternal mitochondrial elimination (PME) and the relevant mechanisms involved are not well understood.

In this study, we investigated the role of ALKBH1, or ALKB-1, in C. elegans PME. ALKBH1 is a dioxygenase with broad demethylation activity that depends on Fe(II) and α-ketoglutarate, and affects both DNA and RNA. Its importance in gene regulation, translation, and mitochondrial function is well-documented. Studies have shown that Alkbh1 null mice exhibit high embryonic lethality, sexual dimorphism, infertility, and developmental issues in the eyes, skeleton, and genitalia. Additionally, ALKBH1 has been shown to modulate gene expression during spermatogenesis and early embryonic stages by changing methylation patterns, underscoring its fundamental biological role in both pre- and post-fertilization processes.

Our study identified ALKB-1 is essential for efficient PME in C. elegans, through its tRNA N1-methyladenosine (m1A) demethylase activity, separate from the DNA N6-methyldeoxyadenosine (6mA) demethylase activity reported previously. Disrupting ALKB-1 function perturbed mitochondrial protein homeostasis, which, in turn, activated transcription factors SKN-1/Nrf2 and the mitochondrial unfolded protein response (UPRmt). Loss of ALKB-1 promotes “clonal expansion” of paternal mitochondria and mtDNA, consequently delaying the PME process and negatively impacting sperm fertility and embryo viability. Our study characterizes the role of epigenetic modification in PME and offers insights into the mechanisms of maternal mitochondrial inheritance. These findings advance our understanding of mitochondrial diseases and may inform future therapeutic strategies.

Results
The demethylase ALKB-1 modulates paternal mitochondrial elimination dynamics
To monitor PME in embryos, we employed the mptDf1 mtDNA allele, with a 179bp deletion, which is smaller than the 3053bp deletion present in the uaDf5 mtDNA allele used in our previous study. We used a nested PCR assay to track the presence of sperm-derived mtDNA during embryogenesis. Crossing mptDf1/+ heteroplasmic males with N2 hermaphrodites revealed that mptDf1 mtDNA is present in early-stage embryos but disappeared before the 64-cell stage, consistent with previous reports of early paternal mtDNA elimination.

To investigate the function of methylation in the PME process, we conducted an RNA interference (RNAi) screen against potential homologs of the ALKB demethylase family in the C. elegans genome, including B0564.2, F09F7.7, alkb-1, alkb-7, and alkb-8. Notably, only alkb-1 knockdown resulted in delayed elimination of sperm-derived mptDf1 mtDNA, which was detectable up to the 1.5-fold stage of embryonic development. Subsequent microscopic analyses showed a substantial increase in the number of MitoTracker Red (MTR)-labeled paternal mitochondria in the 32- and 64-cell stage embryos, after alkb-1 RNAi, compared to control RNAi. These results collectively suggested that alkb-1 knockdown significantly delayed the PME process in cross-fertilized embryos. High mtDNA heteroplasmy has been shown to promote mtDNA replication, potentially impacting its clearance. To determine whether ALKB-1’s contribution to PME delay involves elevated heteroplasmy, we quantified mptDf1 mtDNA levels in mptDf1/+; him-5(e1467) males treated with control or alkb-1 RNAi. No significant difference was detected, indicating that ALKB-1’s effect on PME occurs independently of changes in heteroplasmy.

ALKBH1/ALKB-1 has been reported to localize to the mitochondria, where it is involved in mitochondrial protein synthesis, respiration, and the maintenance of mutant mtDNA in mammals and C. elegans. The probability of ALKB-1 mitochondrial localization by MitoProt is 0.4168. Western blot analyses of tbb-2p::ALKB-1::3×FLAG transgenic strains confirmed the presence of ALKB-1 in mitochondria, while deletion of mitochondrial targeting peptide sequence (MTS, Δ2-35) abolished this localization. Several attempts to directly visualize ALKB-1 in C. elegans have been unsuccessful, likely due to relatively low protein level. To overcome this limitation, we utilized ectopic expression in HeLa cells and observed that ALKB-1 is localized to both the nucleus and mitochondria, with MTS deletion similarly disrupting mitochondrial targeting. Thus, our analyses, implementing both western blotting and direct visualization techniques, demonstrated that ALKB-1 exhibited localization in the nucleus, cytosol, and mitochondria. This finding deviates from a previous report that suggested ALKB-1 was solely localized to mitochondria. We speculated that this discrepancy might be attributed to the use of different promoters to drive transgene expression in our study, compared to the earlier report. Nevertheless, our results were in agreement with another study that described similar localization patterns. Cumulatively, these findings provided compelling evidence for the localization of ALKB-1 in the mitochondria, although not exclusively. The localization in the nucleus, cytosol, and mitochondria indicates a more complex role for ALKB-1 in cellular processes than previously recognized.

Given that ALKBH1 is a known 6mA demethylase dependent on the ferrous iron-coordinating residue D233, and sequence alignment revealed its conserved counterpart, D247, in ALKB-1, we purified both full-length ALKB-1 protein and a catalytically inactive variant (D247A), and tested for demethylase activity. In vitro demethylation assays showed that recombinant wild-type ALKB-1 proteins could decrease 6mA levels in double-stranded oligonucleotides, while the mutant could not, indicating that C. elegans ALKB-1 possesses 6mA demethylase activity in vitro, similar to ALKBH1, and that D247 is the key catalytic residue.

To delineate the precise role of ALKB-1 in PME, we employed CRISPR/Cas9 to generate a series of alkb-1 mutant C. elegans strains, including an alkb-1 knockout strain (alkb-1(ajz209)/+), an MTS deletion strain (alkb-1 (ajz102)/+), and a demethylase inactive mutant strain, with the D247A substitution (alkb-1(ajz103)). Utilizing an mptDf1 mtDNA-based PCR assay, we observed that strains ajz209/+ and ajz103, but not ajz102/+, showed a significant delay in PME. Microscopic analyses further confirmed these findings. As alkb-1-/- is not viable, we focused on the ajz103 (alkb-1D247A mutant) to elucidate the regulatory mechanisms of ALKB-1 demethylase activity in PME. Re-expression of either wild-type ALKB-1 or its MTS-deleted variant rescued the PME defect in alkb-1D247A mutant, whereas the catalytically inactive D247A variant did not. These findings collectively underscore the pivotal role of ALKB-1’s demethylase activity, independent of its mitochondrial localization, in preventing transmission of paternal mitochondria to offspring.

To further explore the requirement for ALKB-1 in PME across different lineages, we conducted microscopic assays. Paternal alkb-1 mutations resulted in a significant increase in paternal mitochondria in 64-cell stage embryos compared with wild-type controls. Embryos from mothers carrying alkb-1 mutations showed a minor delay in PME, with increased MTR-labeled mitochondrial clusters, visible at the 32-cell stage only. To evaluate the relative contribution of ALKB-1 in the sperm, we conducted rescue experiments in alkb-1D247A mutants. Sperm-specific expression of ALKB-1 significantly alleviated PME defects, whereas germline-specific expression had a minor and statistically insignificant effect. These findings suggest that ALKB-1 catalytic activity contributes to PME in both lineages, with a more pronounced impact in the paternal lineage, particularly in sperm. This observation prompted our hypothesis that ALKB-1 deficiency might interfere with the typical decrease in sperm mitochondria and mtDNA content during spermatogenesis. Indeed, there was a substantial increase in the number of mitochondria and mtDNA in the sperm of alkb-1D247A mutants, compared to wild-type controls. Overall, our results demonstrate the critical role of ALKB-1 demethylase activity in the PME process, during spermatogenesis and early embryogenesis.

ALKB-1 regulates mitochondrial proteostasis through tRNA m1A modification
In humans, ALKBH1 is known to catalyze DNA 6mA demethylation, which we confirmed through in vitro biochemical assays. However, Dot blot and LC-MS analyses showed no significant changes in 6mA levels in genomic DNA (gDNA) or mtDNA from the alkb-1D247A mutants, contradicting a recent report that identified ALKB-1 as a mtDNA 6mA demethylase. This discrepancy could be attributed to the sensitivity limitations of our detection methods, which may potentially be insufficient to detect changes in 6mA levels in the alkb-1D247A mutants. To further ascertain the role of 6mA in PME, we overexpressed the methyltransferase DAMT-1, which is known to increase 6mA levels in both gDNA and mtDNA. Surprisingly, DAMT-1 overexpression did not affect the PME process. Collectively, these data indicate that the ALKB-1-mediated delay in PME is not related to its DNA 6mA demethylase activity.

Considering the role of mammalian ALKB family members in RNA demethylation, we measured N6-methyladenosine (m6A) mRNA levels in the alkb-1D247A mutant and found no significant differences compared to the wild-type. In light of a recent study demonstrating the demethylation of m1A in tRNA by ALKBH1 in mammals, we investigated the potential role of ALKB-1 in tRNA m1A demethylation. LC-MS analyses of tRNA from the alkb-1D247A mutant showed significant elevation of m1A modification. These findings were further corroborated by dot blot assays, using two distinct m1A-specific antibodies from distinct companies, yielding consistent results. Additionally, in vitro demethylation assays demonstrated that recombinant wild-type ALKB-1 protein significantly reduced m¹A modification in stem-loop tRNA probes, whereas the D247A variant showed no such activity. Taken together, these findings demonstrated that ALKB-1 demethylates tRNA m1A in C. elegans.

To further elucidate the role of ALKB-1 in tRNA m1A modification, we performed tRNA m1A sequencing. These analyses revealed significantly elevated m¹A levels in 11 tRNA isodecoders and marked reductions in 4 others. Further analyses of the upregulated isodecoders revealed 9 cytosolic and 2 mitochondrial tRNAs, with 8 of the 9 cytosolic species being tRNA-Gly (TCC/GCC) isoacceptors, indicating ALKB-1 preferentially demethylates m¹A in tRNA-Gly. Collectively, these findings indicated that ALKB-1 demethylates m¹A on both cytosolic and mitochondrial tRNAs, with a primary preference for cytosolic substrates. Concurrently, tRNA sequencing detected no significant change in the abundance of any identified tRNAs, a finding validated by biotin-labeled Northern blot analyses of six selected tRNAs (Gly-TCC, iMet-CAT, Asp-GTC, Gln-TGG, Lys-CTT, and Ser-AGA), which revealed comparable levels between wild-type and alkb-1D247A worms.

The m1A modification in tRNA is crucial for tRNA stability, translation, and protein synthesis. We conducted ribosome nascent-chain complex sequencing (RNC-seq) and proteomics analysis, revealing a substantial impact of ALKB-1 on translation. RNC-seq analysis revealed a significant translation efficiency (TE) alteration in alkb-1D247A worms compared to the wild-type N2, with 471 translated transcripts upregulated and 140 transcripts downregulated. These findings indicate that hypermethylation of tRNA at position m1A might contribute to TE alterations. Gene Ontology (GO) analysis showed that the upregulated genes are primarily involved in cGMP biosynthesis, metabolic processes, and the regulation of cellular and biological processes. Gene Set Enrichment Analysis (GSEA) revealed an enrichment of gene sets associated with oxidative phosphorylation (OXPHOS) and citrate cycle (TCA cycle), in wild-type animals. Proteomics analysis identified 121 upregulated and 47 downregulated proteins in alkb-1D247A worms, versus wild-type N2 worms. Importantly, MRPL-13, a component of the mitochondrial ribosome, was significantly less abundant in alkb-1D247A worms, hinting at a possible impairment of mitochondrial translation. Consistent with this, RNA-seq and qPCR validation showed elevated mRNA levels of most mtDNA-encoded genes, consistent with a compensatory response to translational inhibition, as previously reported. Subcellular localization analyses, using the WoLF PSORT software, identified 15 nuclear-encoded proteins that localize in mitochondria and were significantly upregulated, indicating enhanced mitochondrial protein import. This upregulation may be associated with disrupted proteostasis and potential abnormal protein aggregation. To investigate this, we employed a well-established NS-mtGFP reporter system in C. elegans by generating a transgenic strain with the construct myo-3p::SDHB-1 MTS::gfp(S65C)(no stop codon)::unc-54 3’UTR AAAA n, a method validated in yeast and mammalian models. Imaging analyses revealed punctate mitochondrial aggregates in alkb-1D247A body wall muscle, contrasting with the uniform GFP distribution in controls, providing direct evidence of aberrant protein aggregation. Collectively, these findings indicated that ALKB-1-mediated m¹A modification of tRNA influences both cytoplasmic and mitochondrial translation. ALKB-1 inhibition enhances cytoplasmic translation efficiency while simultaneously impairing mitochondrial translation, thereby disrupting the balance of protein homeostasis between the cytoplasm and mitochondria.

ALKB-1 impairment delays PME through the induction of UPRmt
Imbalances in mitochondrial protein homeostasis commonly trigger UPRmt. To explore the effects of ALKB-1 on UPRmt, we employed the hsp-6p::gfp strain reporter in C. elegans and found that alkb-1D247A mutants exhibited robust UPRmt induction in both males and hermaphrodites. This effect was recapitulated by alkb-1 RNAi. Silencing atfs-1, a central UPRmt regulator, effectively abolished the UPRmt triggered by alkb-1 loss, with no effect observed in wild-type backgrounds. Thus, we deduced that UPRmt activation contributed to the delayed PME phenotype observed in alkb-1D247A mutants. Consistent with this hypothesis, deletion of atfs-1 rescued the PME defect in alkb-1D247A embryos, while atfs-1 mutants displayed normal PME.

In C. elegans, UPRmt activation typically involves the cooperative function of the transcription factor ATFS-1, together with DVE-1, UBL-5, and LIN-65, to facilitate retrograde signaling from the mitochondria to the nucleus, during mitochondrial stress. Consistent with this role of ATFS-1, loss of dve-1, ubl-5, or lin-65 restored normal PME in alkb-1D247A embryos, and mutants of these factors displayed PME comparable to wild-type worms. Repression of dve-1, ubl-5, or lin-65 by RNAi also counteracted UPRmt activation, induced by alkb-1 lesion. Additionally, alkb-1D247A mutants showed increased protein levels and nuclear translocation of DVE-1::GFP and UBL-5::GFP. Collectively, these results demonstrate that ALKB-1 regulates the PME processes through its impact on UPRmt activation and retrograde signaling to the nucleus.

ATFS-1 possesses both mitochondrial and nuclear localization signals. Nuclear ATFS-1 activates UPRmt, while mitochondrial ATFS-1 promotes mtDNA replication, to facilitate mtDNA “clonal expansion” in dysfunctional mitochondria. The protein level of ATFS-1 is primarily regulated by the ATP-dependent protease LONP-1. Upon mitochondrial damage, ATFS-1 accumulates in the mitochondria and enhances mtDNA replication by facilitating the binding of POLG to mtDNA. We observed that the knockdown of alkb-1 significantly increased both the mitochondrially localized form (the lower band produced after MTS cleavage) and the unprocessed form of ATFS-1. Remarkably, the alkb-1D247A mutation resulted in a significant elevation of mtDNA copy number in males, early embryos, and sperm, which was dependent on ATFS-1, as evidenced by the mitigation of this effect upon atfs-1 RNAi. Additionally, absence of paternal ATFS-1 was sufficient to rescue the PME defect caused by ALKB-1 inactivation, while maternal ATFS-1 had no effect, highlighting the key role of ATFS-1 in pre-fertilization mtDNA amplification and PME suppression, in alkb-1D247A mutants. Taken together, these findings indicate that the delay in PME, resulting from ALKB-1 inactivation, depends on UPRmt induction and ATFS-1-mediated mtDNA replication.

ALKB-1 impairment delays PME, dependent on transcription factor SKN-1
Results from translatomics and proteomics, in conjunction with UPRmt activation upon mitochondrial proteostasis imbalance, indicated that ALKB-1 inactivation led to mitochondrial dysfunction. It is well established that compromised mitochondrial function can increase reactive oxygen species (ROS) production in cells. GO analysis of RNC-seq data indicated that alkb-1 inactivation affected ETC function and the oxidative stress response, hinting at a potential link between ALKB-1-mediated PME and increased ROS production. Utilizing the ROS sensor H2DCF-DA, we observe a significant increase in ROS levels in both alkb-1D247A mutant hermaphrodites and males, compared to wild-type controls. Additionally, mitochondrial ROS (mtROS) levels were higher in the alkb-1D247A mutant, as indicated by mtSOX Red staining. Treatment with the antioxidants N-Acetyl-L-cysteine (NAC) or Pyrroloquinoline quinone (PQQ) effectively reversed the PME delay and reduced mtROS levels in the alkb-1D247A mutant. Notably, induction of oxidative stress by paraquat treatment delayed the PME process. Collectively, these findings suggest an association between the ALKB-1-mediated PME process and ROS production.

Elevated ROS levels typically activate the oxidative stress response transcription factor SKN-1/Nrf2, which subsequently upregulates genes involved in antioxidant and phase II detoxification pathways. A significant upregulation of the SKN-1 target gene gst-4 was observed in the alkb-1D247A mutant, indicating that impairment of ALKB-1 leads to SKN-1 activation. Microscopic assays confirmed that skn-1 knockdown in alkb-1D247A embryos abrogated the PME delay. Notably, unlike atfs-1, skn-1 knockdown in either the paternal or maternal lineage only moderately alleviated the PME defect induced by ALKB-1 inactivation, suggesting that both paternal and maternal SKN-1 play roles in the PME delay following ALKB-1 inactivation. These findings support a model in which SKN-1 offers broad antioxidant and mitochondrial buffering capabilities, contributing to PME regulation in both maternal and paternal lineages, whereas ATFS-1, although induced in both gametes, likely acts as a mitochondria-centric, sperm-biased surveillance mechanism, predominantly regulating PME in the paternal lineage.

SKN-1 also facilitates mitochondrial biogenesis through interaction with outer mitochondrial membrane proteins, a process that may require coordination with ATFS-1. We investigated whether mitochondrial biogenesis mediated by SKN-1 contributes to the delay in PME, induced by ALKB-1 inactivation. By using the ges-1p::mitoGFP reporter strain, we detected a significant increase in mitochondrial content, in alkb-1D247A mutants of both sexes compared to wild-type controls, with a corresponding increase observed in male sperm by MTR staining. This phenotype was reversed by silencing skn-1 or atfs-1. Together, these findings suggest that ALKB-1 inactivation initiates a mitochondrial retrograde signaling pathway that regulates mitochondrial biogenesis, pre- and post-fertilization, mainly through the coordination of the transcription factors SKN-1 and ATFS-1.

SKN-1 acts upstream of ATFS-1 during ALKB-1-mediated PME
To elucidate how ATFS-1 and SKN-1 interact in the PME delay caused by ALKB-1 inactivation, we conducted epistasis analyses. Overexpression of ATFS-1 using the gain-of-function mutant atfs-1 (et17), known for its constitutive UPRmt activation, partially restored the delayed PME phenotype of the alkb-1D247A; skn-1 RNAi mutant, suggesting that ATFS-1 functions downstream of SKN-1 to mediate PME delay upon ALKB-1 inactivation. Notably, atfs-1 (et17) harbors a disrupted mitochondrial targeting sequence, resulting in exclusive nuclear localization. Our study unveiled that PME delay, induced by ALKB-1 impairment, depends on the subcellular localization of ATFS-1 in both mitochondria and the nucleus. Due to a lack of mitochondrial localization, the atfs-1 (et17) mutant was incapable of fully restoring the phenotype of alkb-1D247A; skn-1 RNAi, to levels comparable to the alkb-1D247A mutant. Furthermore, treatment with NAC, PQQ, and skn-1 RNAi significantly inhibited the induction of UPRmt and the mitochondrial content increase, induced by the alkb-1D247A mutant, indicating that ROS modulates the effect of ALKB-1 on the PME process. We also found that skn-1 RNAi, atfs-1 RNAi, or their combination suppressed the increase in mitochondrial biogenesis and mtDNA copy number caused by the alkb-1D247A mutation, with atfs-1 RNAi exerting a stronger inhibitory effect. This further supports the involvement of both ATFS-1 and SKN-1 in mtDNA amplification and mitochondrial biogenesis, induced by the alkb-1 mutant, with ATFS-1 acting downstream of SKN-1 in this process.

ALKB-1 facilitates PME to maintain sperm fertility and embryo viability
Previous studies have shown that an abnormally high number of sperm mitochondria and PME defects can negatively impact sperm and early embryonic development. We assessed sperm development using GFP::NKB-2 reporter and quantified sperm numbers via DAPI staining. Neither alkb-1 RNAi nor alkb-1D247A mutation affected sperm differentiation at the L4 or adult stages, nor altered sperm counts, indicating that ALKB-1 is dispensable for male sperm development. However, we observed reduced fertility of alkb-1D247A sperm, as evidenced by a significant decrease in the number of progeny from N2 hermaphrodites, mated with alkb-1D247A males. Additionally, we observed significantly increased embryonic lethality in embryos with delayed PME, caused by the paternal alkb-1 mutation, compared to wild-type controls. These findings suggest that ALKB-1 inactivation leads to reduced male fertility and hinders embryo development, possibly due to delayed PME.

Methods
C. elegans strains and cultures
The wild-type strain Bristol N2, and the mutant strains CB1467, him-5(e1467)V; SJ4100, zcIs13 [hsp-6p::GFP+lin-15(+)]; SJ4143, zcIs17 [ges-1::GFP (mit)]; SJZ328, foxSi75 [eft-3p::tomm-20::mKate2::HA::tbb-2 3’ UTR (oxti185)I]; CL2166, dvIs19 [(pAF15) gst-4p::GFP::NLS]III; OP675, wgIs675 [atfs-1::TY1::EGFP::3×FLAG + unc-119 (+)]; TM4919, atfs-1 (tm4919)V; QC117, atfs-1 (et17)V; TM4803, dve-1 (tm4803)X; MT13232, lin-65 (n3441)I; VC2654, ubl-5 (ok3389)I; and DA2123, adIs2122 [lgg-1p::GFP::lgg-1 + rol-6B (su1006)], were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA) and the National BioResource Project (Tokyo, Japan). The strains SJ4197, zcIs39II [dve-1p::dve-1::GFP] and SJ4151, zcIs19 [ubl-5p::ubl-5::GFP], were the gifts from Dr. Ye Tian of the Genetics and Developmental Biology, Chinese Academy of Sciences. The strain LMW467, ibp96 [gfp::nkb-2] was the gifts from Dr Long Miao of the Key Laboratory of RNA Biology, Chinese Academy of Sciences. The strains JNU102, alkb-1(ajz102)/+II; JNU103, alkb-1(ajz103)II; JNU105, ajzIs105 [pie-1p::ALKB-1::3xFlag::tbb-2 3’UTR; unc119 (ed3)]; JNU111, ajzIs111 [trp-3p:: ALKB-1::3xFlag::trp-3 3’UTR; unc-119 (ed3)]; JNU209, alkb-1 (ajz209)/+II; JNU213, ajzIs213 [pMD18-T::damt-1p::DAMT-1::3xHA::3×Flag::tbb-2 3’UTR, myo-2p::mCherry] and JNU214, ajzIs214 [tbb-2p::ALKB-1::3×Flag::tbb-2 3’UTR] were generated by SunyBiotech (Fuzhou, Fujian, China). The double mutants were constructed using standard genetic techniques.
All worms were cultured at 20°C on standard nematode growth medium (NGM) plates seeded with Escherichia coli (E. coli) strain OP50 (unless otherwise stated).

Plasmid construction
The coding sequences of ALKB-1, ALKB-1D247A, ALKB-1(Δ2-35), SDHB-1 MTS, and DAMT-1 was amplified by PCR from wild-type N2 or alkb-1D247A mutant cDNAs. The sequences of tbb-2/damt-1/myo-3 promoter and tbb-2/unc-54 3’UTR were amplified by PCR from N2 genome. The sequence of GFP (S65C) was cloned from plasmid pPD95_75 (Addgene: 1494). The sequence of mCherry was cloned from plasmid Pmyo-2::mCherry::unc- and C.e ALKB-1(Δ2-35)::mCherry fusion proteins in HeLa cells. The C. e ALKB-1::mCherry and C. e ALKB-1(Δ2-35)::mCherry constructs were cloned using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) into the eukaryotic expression plasmid pcDNA3.1(+). To create the plasmids expressing ALKB-1::3×Flag, ALKB-1D247A::3×Flag, ALKB-1(Δ2-35)::3×Flag, DAMT-1::3xHA::3×Flag, and SDHB-1 MTS::gfp(S65C)(no stop codon)::unc-54 3’UTR AAAA n fusion protein in C. elegans, the tbb-2p::ALKB-1::3×Flag::tbb-2 3’UTR, tbb-2p::ALKB-1D247A::3×Flag::tbb-2 3’UTR and tbb-2p::ALKB-1(Δ2-35)::3×Flag::tbb-2 3’UTR cassette were constructed and cloned into the pCFJ356-MCS vector (Addgene: 34871), the damt-1p::DAMT-1::3xHA::3×Flag and myo-3p::SDHB-1 MTS::gfp (S65C) (no stop codon)::unc-54 3’UTR AAAA n cassette was constructed and cloned into the pMD18-T vector (Takara, 6011), using the ClonExpress II One Step Cloning Kit. All plasmids were verified by Sanger sequencing before use.

Microinjection and transgenic strains
Transgenic C. elegans strains were generated by standard microinjection into the syncytial gonads of young adult hermaphrodites. The DNA injection mixture contained an expression plasmid at 50 ng/μL and a fluorescent co-injection marker at 5 ng/μL. Specifically, we used plasmids to express a catalytically inactive ALKB-1 (tbb-2p::ALKB-1D247A::3×Flag::tbb-2 3’UTR), an ALKB-1 variant lacking its N-terminal mitochondrial targeting sequence (tbb-2p::ALKB-1(Δ2-35)::3×Flag::tbb-2 3’UTR), or an NS-mtGFP reporter (myo-3p::SDHB-1 MTS::gfp(S65C)(no stop codon)::unc-54 3’UTR AAAA n). These were co-injected with either Pmyo-2::GFP::H2B or Pmyo-3::mCherry as selection markers into either wild-type N2 or alkb-1D247A mutant backgrounds. F1 progeny exhibiting marker fluorescence were subsequently selected, which maintained the respective transgenes as heritable, extrachromosomal arrays.

Cell culture and transfection
HeLa cells were maintained in DMEM medium (Gibco, C11995500BT) with 10% fetal bovine serum (Gibco, A5256701) and 1% penicillin and streptomycin (Gibco, SH30030.02). Cells were incubated at 37°C in 5% CO2. For the expression of C.e ALKB-1::mCherry or C. e ALKB-1(Δ2-35)::mCherry fusion proteins, HeLa cells were transfected with plasmids using Lipofectamine LTX reagent (Invitrogen, 11668-019) according to the manufacturer’s protocol.

Immunofluorescence analysis in HeLa cells
After transfection of 300 ng of ALKB-1-mCherry or ALKB-1(Δ2-35)-mCherry plasmids into HeLa cells cultured on 10 cm dishes coated with poly-lysine, the cells were subjected to two cold PBS washes 48 h post-transfection. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, followed by three additional PBS washes. The cells were then treated with 0.1% Triton X-100 for 10 min. Following three more PBS washes, the cells were blocked with 5% bovine serum albumin (BSA) for 1.5 h at room temperature. Antibodies against TOM20 (1:200, Proteintech, 11802-1-AP) were then added at a 1:400 dilution and allowed to incubate for 4 h at room temperature. Following this, the cells underwent four PBS washes before being incubated with 488-AffiniPure Goat Anti-Rabbit (diluted 1:1000 in 5% BSA) for 1 h. After six additional PBS washes, the slides were mounted with DAPI-containing mounting medium and imaged using a Leica confocal microscope to capture single-slice images. The co-localization of mCherry with GFP or DAPI was quantified using ImageJ software.

RNA interference (RNAi)
All RNAi strains, with the exception of the alkb-1 RNAi strain, were obtained from the Ahringer library. To generate the RNAi strain targeting alkb-1, the alkb-1 cDNA sequence was amplified from C. elegans N2 cDNA and inserted into a HindIII-digested L4440 vector. Subsequently, the L4440-alkb-1 (cDNA) plasmids were transformed into HT115 (DE3) chemically competent cells through a CaCl2 transformation protocol. The bacteria expressing dsRNA were cultured overnight in LB medium supplemented with 100 mg/mL of ampicillin, after which the bacteria were spread onto NGM plates containing 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). L1 larvae were grown on plates containing gene-specific RNAi bacteria until they reached the L4 or young adult stage for subsequent experiments.

RNAi Screen
To identify methylation-related genes involved in PME, we performed an RNAi screen to pinpoint genes predicted to encode dealkylating enzymes in C. elegans. We used the nested PCR method to detect the sperm-derived mptDf1 mtDNA in the 1.5-fold stage cross-progeny embryos. Briefly, mptDf1/+; him-5(e1467) and N2 worms were grown on IPTG-added NGM plates containing HT115 (DE3) bacteria harboring specific-gene dsRNA expression constructs for two generations. Afterward, F1 adults mptDf1/+; him-5(e1467) males and N2 hermaphrodites were transferred to fresh RNAi plates for mating experiments. Following mating, 1.5-fold stage cross-progeny embryos dissected from the gravid hermaphrodites and then subjected to the nested PCR to ascertain the presence of mptDf1 deletion.

Detection of mptDf1 mtDNA
To determine the heteroplasmy level of the mptDf1 mtDNA in males and to detect sperm-derived mptDf1 mtDNA in cross-progeny samples, nested PCR assays were performed. For heteroplasmy analyses in males, approximately 20 L4-stage worms were collected and lysed in 20 μL of lysis buffer at 65°C for 60 min. For the detection in cross-progeny, total lysates were prepared from different developmental stages of embryos or larvae. Wild-type mtDNA and mptDf1 mtDNA were distinguished using the following primers: mptDf1 Outer F, 5′-GGTATAATTGGGGCCATCCGT-3′ and mptDf1 Outer R, 5′-TCAAAAGTGGAGCGGTGCTAC-3′; mptDf1 Inner F, 5′-GAACTTAACCGGGCGCCATT-3′ and mptDf1 Inner R, 5′-ACGCTACAGCAGCAAAACAA-3′. The heteroplasmy level in males was subsequently quantified by assessing the relative band intensity (grayscale value) of the wild-type and mptDf1-specific PCR products.

Microscopy and quantification of paternal mitochondria
Young adult males were stained on OP50 NGM plates containing 50 μM MitoTracker® Red CMXRos (Invitrogen, M7512) for 12 h and then transferred to a fresh OP50 NGM plates for 30 min to eliminate residual MTR. The MTR-stained males were subsequently mated with unstained young adult hermaphrodites for 8–10 h. Following mating, the hermaphrodites were dissected to release the cross-fertilized embryos onto an agarose pad. A small volume of M9 buffer was added to the agar pad, and a coverslip was placed on top. The embryos were systematically imaged using a Zeiss Axio Imager Z2 with Apotome.2 microscope at 1000× magnification, capturing images from top to bottom in Z-stacks. Deconvolution was performed on the Z-stacks, and the images were projected onto a single plane to provide a comprehensive visualization of all MTR-labeled mitochondria in the embryo. The number of MTR-stained mitochondrial clusters in cross-fertilized embryos was counted using ImageJ software. At least 10 embryos were scored in each group. Statistical significance was determined using the unpaired student’s t-test or one-way ANOVA to calculate the p-value.

RNA extraction and quantitative RT-PCR
Young adult worms were collected using M9 buffer and subjected to multiple washes. The worm samples were then resuspended in AG RNAex PRO reagent (Accurate Biology, Changsha, China) for RNA extraction. Total RNA was isolated using chloroform extraction followed by isopropanol precipitation. Subsequently, 500 ng of RNA was utilized for cDNA synthesis utilizing a high-capacity cDNA transcription kit (RK20400, ABclonal, Wuhan, China). Quantitative RT-PCR was conducted utilizing SYBR Green Select Master Mix (RK21203, ABclonal, Wuhan, China) on a LightCycler480 real-time system (Roche, USA), with each assay being replicated three times. The mRNA expression levels of the genes were evaluated after normalization to the reference gene cdc-42, and statistical significance was assessed using the unpaired student’s t-test. Details of the primer sequences used in this study were provided in Supplementary Table 1.

mRNA isolation
The mRNA was isolated from the total RNA using the Dynabeads mRNA purification kit (Cat No. 61006, Invitrogen) according to the manufacturer’s instructions. In brief, 75 μg of total RNA was heated at 65°C for 2 min to disrupt the secondary structure. The superparamagnetic Dynabeads™ coupled with oligonucleotide-(dT)25 in the binding buffer, were equilibrated and mixed with the total RNA. The beads were then washed to remove RNA contaminants, and the mRNA was eluted using 5 µL of 10 mM Tris-HCl. The isolated mRNA samples were subsequently utilized for dot blot analyses.

tRNA isolation
Young adult hermaphrodite worms were collected using M9 buffer and underwent several washes. The small RNA fraction (<200 nucleotides, primarily consisting of mature tRNA) was extracted from the worm samples using the mirVana™ miRNA Isolation Kit (AM1561, Life Technologies) according to the manufacturer’s instructions. The isolated tRNA samples were subsequently used for dot blot analyses.

Protein purification
The fragments sequences of ALKB-1 and ALKB-1D247A were cloned into the pET-30a(+) vector and then introduced into BL21 (DE3) chemically competent cells using the CaCl2 transformation protocols. Subsequently, these proteins with N-terminal His-tag were expressed in the BL21 (DE3) cells following overnight induction with 0.2 mM IPTG. The cells were collected by centrifugation and resuspended in lysis buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Afterward, the cells were homogenized using an Emulsiflex C3 (Avestin) homogenizer, and the centrifugation-cleared supernatant was loaded onto a HisTrap nickel column (GE Healthcare). The protein bound to the nickel column was eluted using a linear imidazole gradient ranging from 20 to 500 mM. The eluted protein underwent His-tag removal by PreScission protease and was subsequently subjected to ion-exchange chromatography using HiTrap Q HP and HiTrap Heparin HP columns (GE Healthcare). Finally, the protein was purified to homogeneity via size exclusion chromatography on a Superdex 200 increase 10/300 column (GE Healthcare) and concentrated to 15 mg/mL in a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl for subsequent experiments.

Demethylation assays in vitro
The modified 6mA oligo used in this study was obtained from The Beijing Genomics Institute BGI (Shenzhen, Guangdong, China). The sequence of the oligo was as follow: 6mA, 5′-GGGAATTTCCCGGCGATTTGA(N6-Me-dA)TCAAATCGCCGGGAAATTCCC-3′, as previously described. The stem-loop structured tRNA probe modified with m1A was obtained from Sangon Biotech (Shanghai, China), and the sequence of the probe was as follow: 5′-CACGGUUCG(m1A)UUCAAAG-3′, as previously described. To assess the demethylation activity of ALKB-1 in vitro, each substrate was annealed under distinct conditions. The 6mA oligos were denatured at a concentration of 0.1 nmol/μL in a denaturation buffer (10 mM Tris-HCl, 100 mM NaCl, 10 μM EDTA, pH 8.0) by heating at 95°C for 2 min followed by a gradual cooling to 25°C over 1 h. For the m1A-tRNA probe, annealing was carried out at 0.1 μM in RNA annealing buffer (100 mM Tris-HCl, 1 M NaCl, 50 mM MgCl₂, 0.1 mM EDTA, pH 7.5) using DEPC-treated water, with a protocol involving heating to 95°C for 2 min, rapid cooling to 45°C, a 30 min hold, and gradual cooling to 4°C at 0.1°C/s. The demethylation reaction was then conducted in a 20 μL volume containing 0.1 nM of the respective annealed substrate and 1 μg of either ALKB-1 or the mutant ALKB-1D247A protein in reaction buffer (50 μM HEPES pH 7.0, 50 mM KCl, 2 mM MgCl₂, 2 mM ascorbic acid, 1 mM α-ketoglutarate, 200 μM (NH₄)₂Fe(SO₄)₂). Notably, reactions involving the m1A-tRNA substrate were supplemented with 0.2 U/μL RNase Inhibitor. The reactions were then incubated at 25°C for 6 h and terminated by adding EDTA to a final concentration of 5 mM, followed by incubation at 95°C for 10 min. The reaction products were subsequently utilized for dot blot analyses.

Purification of worm genomic DNA
Young adult worms were harvested using M9 and then lysed in a worm gDNA lysis buffer containing 200 mM NaCl, 100 mM Tris-HCl (pH 8.5), 50 mM EDTA (pH 8.0), 0.5% SDS, and proteinase K (0.1 mg/mL). The lysate was incubated at 65°C for 1 h with intermittent vortexing. Proteinase K was then inactivated by heating at 95°C for 20 min. Afterward, RNase A was added and incubated at 37°C for 1 h. The sample was then mixed with an equal volume of phenol: chloroform: isopentanol (25:24:1) and centrifuged at 13,000 rpm for 5 min at room temperature. The aqueous phase was transferred to a new tube and mixed with sodium acetate and 100% ethanol before being incubated at −20°C overnight. After centrifugation at 16,000×g for 10 min at 4°C, the DNA pellet was washed with 70% EtOH, air-dried, and resuspended in TE buffer. Following this, the purified gDNA underwent treatment with RNase A/T1 (Thermo Fisher Scientific) and RNase H (New England Biolabs) at 37°C for 1 h. The process concluded with another purification step, starting with the phenol: chloroform: isoamyl alcohol extraction. The isolated gDNA samples were then prepared for dot blot analyses.

Dot blot
Oligos, gDNA, mRNA, and tRNA samples were denatured at 95°C for 10 min and snap-cooled on ice. A total of 2 μL of each sample, containing Oligos (all reaction products), 300 ng of DNA, 120 ng of mRNA, and 100 ng of tRNA, was spotted onto a membrane (Amersham, Hybond-N+, GE), air-dried, and UV cross-linked for 1.5 h. The membranes were then incubated in a blocking buffer (5% milk, PBS-T) for 2 h at room temperature, followed by an overnight incubation with antibodies against 6mA (1:1000, Synaptic Systems, 202-003) or m1A (1:1000, MBJ Life Science, D345-3 or 1:1000, abcam, ab208196) at 4°C. After three subsequent washes, the membrane was incubated with a secondary antibody at room temperature for 1 h, and the signals were visualized utilizing a Bio-Rad ChemiDoc MP imaging system.

LC-MS based tRNA modification Detection
The LC-MS based tRNA modification detection was conducted by Aksomics (Shanghai, China). Total RNA from each sample was assessed using a NanoDrop ND-1000 instrument, and approximately 20 μg of total RNA was utilized for tRNA isolation using the Urea-PAGE method. The purified tRNA was enzymatically digested into single dephosphorylated nucleosides. Subsequently, LC-MS analysis was performed on an Agilent 6460 QQQ mass spectrometer coupled with an Agilent 1260 HPLC system using Multi Reaction Monitoring (MRM) mode for detection. Data acquisition for LC-MS was performed using Agilent Qualitative Analysis software. MRM peaks corresponding to each modified nucleoside were extracted and normalized to the quantity of injected tRNA.

Isolation of mitochondria
To isolate mitochondria from C. elegans, approximately 40,000–60,000 L4 worms were collected using M9 buffer with 0.01% Triton X-100, washed three times, and snap-frozen in liquid nitrogen before storage at −80°C. The collected worm pellets were suspended in a mitochondria isolation buffer (10 mM Hepes-KOH pH 7.5, 210 mM mannitol, 70 mM sucrose, 1 mM EDTA). The worm cuticle was disrupted by grinding using an acid-washed mortar and pestle, followed by further homogenization with a Dounce homogenizer. The homogenate underwent low-speed centrifugation at 200×g for 5 min to remove cellular debris. Subsequently, the resulting supernatant was subjected to a higher-speed centrifugation at 12,000×g for 10 min to isolate the cytosolic fraction in the final supernatant, while the pellet contained the mitochondria. The obtained supernatant and mitochondrial samples were then used for subsequent Western blot analyses.

Western blot analysis
Young adult worms were collected using M9 buffer, washed three times, and then immediately frozen in liquid nitrogen for storage at −80°C. The worm pellets were lysed using RIPA buffer and homogenized twice using a Tissue Lyser at 75 Hz for 6 min at 4°C. This was followed by centrifugation at 10,000×g at 4°C to obtain supernatants. Protein concentrations in these supernatants were quantified using a BCA Protein Assay Kit. Samples were boiled at 95°C for 5 min to denature the proteins prior to electrophoresis on either 6% or 10% SDS-polyacrylamide gels. Following electrophoresis, the separated proteins were transferred onto nitrocellulose membranes. The membranes were then blocked with 5% milk and incubated with primary antibodies against the FLAG (1:3000, Sigma-Aldrich, F1804), HA (1:3,000, Cell Signaling Technology, 3724S), GFP (1:5000, ROCHE, 11814460001), β-actin (1:5,000, Sigma-Aldrich, A1978), or α-tubulin (1:5,000, Cell Signaling Technology, 2125S). The primary antibody was visualized using a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (anti-rabbit, 1:5000, Jackson ImmunoResearch, 111-035-144; anti-mouse, 1:5000, Jackson ImmunoResearch, 115-035-068) and ECL Western Blotting Substrate.

Biotin-labeled Northern Blot analysis
Total RNA samples were analyzed by Northern blot using the Biotin Northern Blot Kit for Small RNA (Beyotime). Briefly, 10 μg of total RNA was denatured, separated on a 15% urea-polyacrylamide gel, and transferred to a nylon membrane. After UV crosslinking, the membrane was hybridized overnight at 42°C with biotin-labeled probes. Following hybridization, the membrane was incubated with HRP-conjugated streptavidin and detected using ECL chemiluminescence. 5S rRNA was used as a reference control for tRNA abundance, and all probe sequences are provided in Supplementary Table 2.

Isolation and lysis of C. elegans sperm
To isolate the sperm, at least 100 virgin males were transferred into a lysis buffer droplet. Incisions were made at one-third of the body length from the tail, between the midpoint and tail, using syringe needles to release the sperm. The broken worms were then carefully removed from the lysis buffer using needles. The buffer containing the sperm was incubated at 65°C for 1 h. The lysed samples were then utilized for subsequent qPCR to measure mtDNA levels.

Measurement of ROS levels
Endogenous ROS levels were evaluated by measuring the fluorescence intensity of 2′7′-Dichlorofluorescein diacetate (H2DCF-DA) according to established protocols. Briefly, young adult worms were exposed to 10 mM H2DCF-DA on OP50 NGM plates for 1 h, then transferred to a fresh OP50 NGM plate for 30 min to eliminate residual H2DCF-DA. Subsequently, the H2DCF-DA labeled animals were mounted on agarose pads and imaged at 20× magnification utilizing a Nikon Ti2-U fluorescence microscope. Each assessment involved a minimum of 30 worms, and each assay was replicated at least twice. The fluorescence intensity in the images was analyzed and quantified using ImageJ software.

Measurement of mtROS
To evaluate mitochondrial ROS levels, MitoSox staining was carried out on worms with minor modifications to an established protocol. Briefly, young adult worms were collected and subjected to triple washing with M9 buffer to remove bacteria before being stained with 5 mM MitoSox in M9 for 30 min. Afterward, the stained worms underwent additional washes in M9 and were mounted on agarose slides for imaging. The head regions of the worms were imaged using a Nikon Ti2-U fluorescence microscope at 20× magnification, and fluorescence intensity measurements were carried out using ImageJ software.

Fluorescent microscopic imaging
To quantify the expression of hsp-6p::gfp, ges-1p::mitoGFP, gst-4p::gfp, and ubl-5p::ubl-5::gfp, at least 30 animals per group were anesthetized with 10 µM levamisole and placed on 2% agar pads for imaging with a Nikon Ti2-U fluorescence microscope. The fluorescence intensity was measured using ImageJ software. For assessing DVE-1::GFP intestine nuclear location, at least 30 dve-1p::dve-1::gfp animals were anesthetized in M9 buffer containing sodium azide (50 mM), mounted onto 2% agarose pads, and intestine cells with DVE-1::GFP signal were counted directly at 200× magnification using a Zeiss Axio Imager Z2 with Apotome.2. To quantify mitochondria in C. elegans sperm, L4 stage male worms were stained overnight with 10 μM MTR and imaged at 1000× magnification using a Zeiss Axio Imager Z2 with Apotome.2 after crawling on fresh OP50 plates for 1 h; fluorescence intensity was quantified using ImageJ software, including at least 10 worms and 3-5 sperm per worm. For imaging and puncta quantification in the seam cells of strain DA2123, animals were immobilized with 10 µM levamisole, and GFP::LGG-1 puncta were directly counted at 630× magnification using a Zeiss Axio Imager Z2 with Apotome.2. For imaging of GFP::NKB-2, males at both L4 and adult stages were anesthetized in M9 buffer containing sodium azide and mounted on agar pads. The entire male germline was then imaged at 630× magnification using a Leica confocal microscope to capture the fluorescence signal. All experiments were conducted at least two times, and statistical analyses were performed using an unpaired Student’s t-test or one-way ANOVA.

C. elegans mtDNA quantification
Approximately 20 L4 stage worms were collected and suspended in 20 μL of lysis buffer before being incubated at 65°C for 60 min. The levels of mtDNA were quantified using qPCR-based methods as previously described. qPCR was conducted using SYBR Green Select Master Mix (RK21203, ABclonal, Wuhan, China) on a LightCycler480 real-time system (Roche, USA), with each assay being replicated three times. The relative levels of mtDNA were determined using primer pairs targeting mtDNA and gDNA, as previously described. The primer sequences are as follows: wild-type mtDNA F, 5′-GCTTTTTCTTTATATGTTTTGTG-3′; wild-type mtDNA R, 5′-TCACCTCAGAAAAATCAAATGG-3′; ges-1 (nuclear genome DNA) F, 5′-AGGCTAAGCCGGGGTAAGTT-3′; ges-1 (nuclear genome DNA) R, 5′-GCCAAAAGCTTAAACTGCGG-3′.

Embryonic lethality assays
The experiments were performed at 25°C following a previously established protocol. L4 larvae were allowed to mature for 12 h before being transferred to fresh plates for a 6-h egg-laying period. Subsequently, the worms were removed from the plates, and the total number of embryos on the plates was counted. After an additional 24-h incubation period, any embryos that had not hatched were recorded as non-viable. The embryonic lethality rate was calculated by dividing the number of non-viable embryos by the total number of embryos counted.

C. elegans sperm numbers analysis
L4 virgin males were isolated onto fresh NGM plates and incubated at 20°C for 18–24 h before staining. Subsequently, the worms were washed with PBST and fixed in 40% isopropanol for 3 min. The samples were then incubated in a DAPI staining solution (10 μg/mL in 40% isopropanol) for 5 min in the dark, followed by a 30 min destaining procedure in PBST. The entire spermatid region was systematically imaged using a Zeiss Axio Imager Z2 microscope at 630× magnification, capturing images from top to bottom in Z-stacks. Total sperm counts were quantified from maximum intensity projections using the ImageJ cell counter plugin.

Male fertility assays
To assess male fertility, a single L4 stage wild-type or alkb-1D247A virgin male was paired with one N2 hermaphrodite at the 24 h post-young adult stage on each 35 mm NGM plate. These pairs were subsequently transferred to fresh plates every 24 h until the hermaphrodite stopped producing eggs. Progeny count was then conducted. Hermaphrodites that failed to mate with males, identified by the absence of male offspring, were excluded from the dataset.

RNA-seq
The RNA-seq libraries were prepared and sequenced at Chi-Biotech (Shenzhen, China). Synchronized young adult worms were harvested using cold PBS buffer for total RNA extraction with AG RNAex PRO reagent (Accurate Biology, Changsha, China). Subsequently, RNA integrity was assessed using the RNA Nano6000 Assay Kit on the Bioanalyzer 2100 system from Agilent Technologies, CA, USA. For library construction and sequencing, 1 μg of RNA per sample was utilized, and sequencing was conducted using the cBot Cluster Generation System with the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) following the manufacturer’s protocols. Differential expression analysis was carried out using the DESeq2 R package (version 1.20.0), with genes showing a fold change greater than two fold and a false discovery rate <0.01 considered as differentially expressed genes (DEGs). The DEGs were functionally annotated using various databases, including Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genomes (KEGG).

RNC-seq
The RNC-seq libraries were prepared and sequenced at Chi-Biotech (Shenzhen, China). Young adult worms were collected using M9 buffer, washed three times, and rapidly froze in liquid nitrogen. The frozen worms were crushed into a power using a pre-chilled mortar and then mixed with lysis buffer (1% Triton X-100, 20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200 mM KCl, 100 μg/mL cycloheximide, and 2 mM dithiothreitol) for extraction. The resulting lysate was subjected to ultracentrifugation in a 30% sucrose solution. The precipitate was collected, re-suspended, and subjected to chloroform extraction and isopropanol precipitation to isolate total RNA. The integrity of the total RNA was evaluated using the Agilent 4200 Bioanalyzer. mRNA was enriched using oligo(dT) magnetic beads to isolate poly(A) tailed mRNA. The isolated mRNA was fragmented using divalent cations, and these fragments were utilized as templates for synthesizing the first cDNA strand, with random oligonucleotides as primers within the 1st Strand Enzyme reverse transcription system. The second cDNA strand was then synthesized using the 2nd Strand Enzyme and dNTPs. The purified double-stranded cDNA underwent end repair, A-tailing, and adapter ligation. Subsequently, cDNA fragments ranging in size from 200 to 300 bp were selected, PCR amplified, and purified to form the final library. The library concentration was determined using Qubit or qPCR, while the library quality was assessed using the Agilent 4200 Bioanalyzer to verify the insert size. The library was sequenced on the Illumina NovaSeq 6000 platform using PE150 sequencing. To assess changes in translation efficiency, the Translation Ratio (TR) for each gene was calculated as the ratio of its translational abundance (measured by TPM from RNC-seq) to its transcriptional abundance (measured by TPM from RNA-seq), using the formula: TR=(TPM in RNC-seq)/(TPM in RNA-seq). This normalization corrects for changes in mRNA levels that are independent of translation, thereby isolating genuine alterations in translational regulation. Genes exhibiting significant changes in TR between conditions were identified as differential translation efficiency genes. The differential translation efficiency analysis was carried out using DESeq2 with a filter threshold of p-value<0.05 and |log2 FC|> 1. The differential translation transcripts were functionally annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG).

Proteomics analysis
The proteomic experiment was performed by Jingjie PTM BioLab (Hangzhou, China) utilizing 4D label-free proteomics analysis. Young adult worms were collected using M9 buffer, washed, and then snap-frozen in liquid nitrogen before storage at −80°C. Proteins were extracted using lysis buffer containing 8 M urea and 1% protease inhibitor cocktail, followed by trypsin digestion. The resulting tryptic peptides, dissolved in solvent A (0.1% FA in water), were loaded onto an Evotip according to the manufacturer’s protocol. Peptides were separated using the 60-SPD method on the EvoSep One liquid chromatography system, with the mobile phase composed of solvent A and solvent B (0.1% FA in ACN). A gradient increase in solvent B was performed, starting from 4 to 22% over 70 min, then 22 to 30% over 14 min, followed by a quick increase to 80% over 3 min, and a subsequent 3 min equilibration period between gradient changes, all at a flow rate of 450 nL/min. Subsequently, the peptides underwent capillary source analysis followed by analysis using timsTOF Pro mass spectrometry. The timsTOF Pro was operated in dia-PASEF mode with a full MS scan range of 300–1500 m/z and 20 PASEF scans acquired per cycle. The MS/MS isolation window was set at 7 m/z within the range of 400–850 m/z. The DIA data were analyzed using the DIA-NN search engine (v.1.8), and tandem mass spectra were searched against a combined target and decoy database of Blast_Caenorhabditis_elegans_6239_PR_20230529.fasta (26706 entries). Trypsin/P was designated as the cleavage enzyme, allowing for up to one missed cleavage. N-terminal Met excision and carbamidomethyl modification of Cys were indicated as fixed modifications. The false discovery rate (FDR) was adjusted to be less than 1%. Subcellular localization predictions and protein annotation were conducted using WoLF PSORT software.

tRNA seq
tRNA m1A seq was performed on the Illumina NextSeq 500 system by Aksomics (Shanghai, China). Total RNA was extracted from C. elegans samples using a standard RNA extraction kit. RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer. Total RNA (≥2 μg) was separated on a 7.5% urea-PAGE gel, and tRNA fragments (60–100 nt) were excised and eluted. To analyze m1A modifications, a modified tRNA-seq approach was employed, incorporating a demethylation (Demethylation (−)) control group. Each sample was divided into two groups: Demethylation (+) and Demethylation (−). For the Demethylation (+) group, the RNA Pretreatment kit (Arraystar) was used to remove m1A modifications, facilitating efficient cDNA reverse transcription. In the Demethylation (−) group, the demethylation step was omitted, allowing for the identification of m1A-associated mismatches or truncations during sequencing and analysis.
tRNA fragments were subjected to partial hydrolysis using a Na2CO3/NaHCO3 buffer to generate 5′-monophosphate ends. Dephosphorylation with calf intestinal alkaline phosphatase (CIP) was followed by rephosphorylation using T4 polynucleotide kinase. Purified tRNA fragments were converted into barcoded small RNA libraries using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs). Library quality and concentration were assessed using an Agilent 2100 Bioanalyzer. Libraries were pooled in equal molar ratios and sequenced on the Illumina NextSeq 500 system.
For tRNA m¹A sequencing data analysis, raw sequencing reads underwent quality assessment using FastQC (v0.11.7) to evaluate base call accuracy and overall read quality, followed by adapter trimming with Cutadapt (v1.14) to remove 5′ and 3′ adapter sequences and filtering to discard reads shorter than 18 nucleotides or longer than 45 nucleotides, retaining only high-quality trimmed reads for downstream analysis. Trimmed reads were aligned to a reference set of mature cytoplasmic and mitochondrial tRNA sequences obtained from GtRNAdb and mitotRNAdb, respectively, using BWA (v0.7.12-r1039) with a maximum of one mismatch allowed per read to ensure high specificity, and the resulting BAM files were used for quantification of m¹A modification sites. To identify and quantify m¹A modification sites, aligned reads were analyzed at each nucleotide position of the reference tRNA sequences: for each position, the total number of mapped reads (coverage) was recorded separately for untreated and treated samples; the number of reads containing a non-reference base (e.g., A→T, A→G, A→C substitutions) or truncations was counted, as these are indicative of potential m¹A modifications due to reverse transcription misincorporation; the modification ratio at each position was calculated as ratio_untreated=Count_untreated/Coverage_untreated and ratio_treated=Count_treated/Coverage_treated; and a differential score was computed for each site as score=ratio_untreated - ratio_treated, reflecting the relative abundance of m¹A modifications assuming chemical treatment reduces misincorporation at modified sites (e.g., a score of 0.1 corresponds to approximately 10% of molecules being modified). To exclude background noise from low-abundance modifications, a change threshold (|ΔScore|>0.05, equivalent to approximately 5% modification ratio change) was applied to filter tRNA species with significantly differential modification levels. Differential analysis was performed using a t-test, with fold change and P-values used to identify differentially modified m¹A sites.
To profile tRNA expression abundance and identify differentially expressed tRNAs, the purified tRNA underwent a critical pre-treatment step involving m¹A and m³C demethylation to reduce sequencing biases, and was then partially hydrolyzed using the Hydro-tRNAseq protocol. These processed fragments were re-phosphorylated and converted into sequencing libraries with the NEBNext® Multiplex Small RNA Library Prep Set for Illumina®. The resulting libraries underwent stringent size selection (~140–155 bp, corresponding to ~19–35 nt tRNA fragments) and were rigorously qualified and quantified on an Agilent 2100 BioAnalyzer prior to being pooled in equimolar amounts for sequencing on an Illumina platform. For bioinformatic analysis, sequencing quality was assessed with FastQC, adapter sequences were trimmed using cutadapt, and the cleaned reads were aligned to both cytoplasmic (GtRNAdb) and mitochondrial (mitotRNAdb) tRNA reference sequences with BWA software, allowing a maximum of two mismatches. Finally, tRNA expression profiles were generated from mapped reads, and differential expression analysis was conducted based on read counts using the R package edgeR.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study investigates how AlkB dioxygenase homologs influence paternal mitochondrial elimination (PME) in Caenorhabditis elegans embryos. The authors present compelling evidence that AlkB homolog 1 (ALKB-1) and its demethylase activity are essential for efficient PME. They also demonstrate that loss of ALKB-1 activity induces the mitochondrial unfolded protein response (mt-UPR), elevates reactive oxygen species (ROS) levels, and activates the stress-response transcription factors ATFS-1 and SKN-1/Nrf2. Notably, RNAi targeting ATFS-1 or SKN-1 rescues the PME phenotype, while ROS scavengers partially rescue it. The majority of experiments are conducted rigorously, with appropriate controls, and the findings reveal a potential link between ALKB-1-dependent mitochondrial homeostasis and PME. However, additional evidence is needed to substantiate the conclusion that ALKB-1 directly regulates PME, rather than its effects being an indirect consequence of mitochondrial dysfunction. Furthermore, several points require clarification to strengthen the study’s conclusions.\nKey Points for Consideration:\n1. Title and Claims:\nThe study concludes that ALKB-1 is an “Epitranscriptomic Regulator of Paternal Mitochondrial Elimination” and “…provides compelling evidence for the critical regulatory role of m1A, a conserved epitranscriptomic modification on tRNAs, in the rapid clearance of paternal mitochondria.”\nWhile the evidence strongly supports that ALKB-1 is necessary for efficient PME, the claim that ALKB-1 regulates PME may be overstated. The observed PME defects could result from indirect effects of mitochondrial homeostasis disruption in ALKB-1 mutant worms, rather than a direct regulatory role. The authors’ model suggests that inefficient PME arises from increased mtDNA levels due to mtDNA amplification triggered by mt-UPR in ALKB-1 mutants. This implies that ALKB-1’s role in PME may be indirect and that any perturbation activating mt-UPR could similarly reduce PME. The study also shows that paraquat-induced ROS elevation is sufficient to impair PME. To address this, the authors should soften their claims by avoiding the term “regulator” in the title and discussion, instead emphasizing that mt-UPR modulates PME efficiency.\n2. Line 130:\nThe authors describe a “demethylase inactive mutant strain, with the D247A substitution,” and note in Supplemental Figure 2i that D247 is an “ion-coordinating residue.” However, it would be more accurate to specify that D247 is an iron-coordinating residue, as AlkB homologs catalyze iron-dependent oxidative demethylation. Additionally, the authors should explain why this specific residue was chosen, referencing prior literature and including a sequence alignment of this residue with other ALKBH1 homologs in Supplemental Figures.\n3. RNA-Seq Analysis:\nThe authors perform ribosome nascent-chain complex sequencing (RNC-Seq) and proteomics on wildtype and ALKB-1 mutant worms but do not conduct standard RNA-Seq to assess global mRNA levels. Standard RNA-Seq is necessary to make claims about translation efficiency, as reduced ribosome association could result from decreased mRNA abundance rather than impaired translation. RNA-Seq would also clarify whether proteomic changes stem from transcriptional or translational alterations.\n4. Line 186:\nThe authors state, “Notably, translation efficiency was reduced for four mtDNA-encoded TCA cycle-associated transcripts (atp-6, ctb-1, ctc-2, nduo-2), in alkb-1D247A worms (Fig. 2g).” However, the RNC-Seq method used is unsuitable for analyzing mitochondrial mRNAs, as it targets cytoplasmic ribosomes. Mitochondrial ribosomes differ in sedimentation properties and are unaffected by cycloheximide, which arrests cytoplasmic ribosomes. The authors should avoid concluding that translation of mitochondrial mRNAs is reduced without performing mitochondrial polysome profiling. Alternatively, reduced transcript levels could reflect decreased mitochondrial transcription due to disrupted homeostasis, which could be tested using RNA-Seq or qRT-PCR.\n5. Line 247:\nThe statement, “Treatment with the antioxidants N-Acetyl-L-cysteine (NAC) and Pyrroloquinoline quinone (PQQ) effectively reversed the PME delay (Fig. 4b, c),” should be revised to: “Treatment with the antioxidants N-Acetyl-L-cysteine (NAC) or Pyrroloquinoline quinone (PQQ) effectively reversed the PME delay.”\n6. Line 255:\nThe observation that SKN-1 knockdown in either the paternal or maternal lineage alleviates PME defects caused by ALKB-1 inactivation, while ATFS-1 knockdown alleviates PME defects only in the paternal lineage, is intriguing. The authors should discuss why SKN-1 plays roles in both paternal and maternal lineages, whereas ATFS-1’s role is restricted to the paternal lineage. This distinction may provide insight into lineage-specific stress responses and their impact on PME.\n7. Lines 299–308:\nThis section would be better placed at the beginning of the Discussion, as it provides context for the study’s findings and sets the stage for interpreting the results.\n8. Line 193:\nThe authors state, “Subcellular localization analysis, using the WoLF PSORT software, identified 15 nuclear-encoded proteins that localize in mitochondria, and were significantly upregulated (Fig. 2j), indicating potential abnormal protein aggregation in mitochondria.” However, increased protein levels do not necessarily indicate abnormal aggregation. Elevated protein levels could result from transcriptional, translational, or post-translational changes, and may even be compensatory. The authors should clarify this point and provide evidence for abnormal aggregation if this is their intended conclusion.\n9. Figure 5:\nThe authors show that overexpression of ATFS-1 using the gain-of-function mutant atfs-1(et17), which constitutively activates mt-UPR, partially restores the delayed PME phenotype in the alkb-1D247A; skn-1 RNAi mutant. However, it would be informative to test PME in embryos from crosses involving atfs-1(et17) mutants alone, without the alkb-1D247A mutation or SKN-1 RNAi. This experiment would reveal whether mt-UPR activation alone is sufficient to impair PME, pinpointing mt-UPR as the primary driver of reduced PME efficiency.\n10. MTS Deletion Strain:\nThe study finds that the MTS deletion strain (alkb-1(ajz102)) can carry out efficient PME similar to wildtype strain crosses, suggesting that mitochondrial localization of ALKB-1 has little impact on PME. However, the authors should validate that deletion of residues 2–35 effectively disrupts ALKB-1 localization to mitochondria in vivo within worms. This could be tested by examining whether mitochondrial tRNA-Met retains the 5-formylcytidine (f5C) modification, as ALKB-1 is required for this formylation. Additionally, the authors should assess whether mt-UPR is activated in the alkb-1(ajz102) strain. If mt-UPR is activated without affecting PME, this would suggest differential effects of mt-UPR on PME in the alkb-1(ajz102) strain versus the D247A substitution (alkb-1(ajz103) strain).\n11. Transgene Testing:\nTo strengthen their conclusions, the authors could test different versions of their ajzIs214 transgene (tbb-2p::ALKB-1::3×Flag). While they show that re-expression of wildtype ALKB-1 in the alkb-1D247A mutant strain rescues the PME defect, testing transgenes expressing the ALKB-ΔMTS mutant or ALKB-D247A mutant could reveal whether these variants can also rescue PME defects in the alkb-1D247A mutant strain.\n12. Figure 2J:\nIn the volcano plot, there is a highly decreased protein in the top left corner. The authors should identify this protein, as its significant difference may provide additional insights.\n13. Figure 6d:\nThere is a typo in the schematic that reads “Delayed parternal mitochondrial elimination.” This should be corrected to “Delayed paternal mitochondrial elimination.”", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This work describes the effect of a putative tRNA demethylase, ALKB-1, that controls the exclusion of paternal mitochondrial DNA in C. elegans. Demethylation of mRNA and tRNA has been described for a while now, but the biological phenotypes and mechanisms directly linked to the demethylation activities have lagged behind. This work shows that ALKB-1, a putative tRNA m1A demethylase, impacts the exclusion of paternal mito-DNA, potentially through its tRNA demethylation activity. ALKB-1 inactivation disrupts translation, mitochondrial proteostasis, and increases ROS. This activates oxidative stress response pathways to result in deficient mt-DNA removal. This study identifies a potential epitranscriptomic mechanism for mitochondrial inherence during reproduction. If true, it would represent an important contribution to our biological understanding of RNA demethylation.\nThe main deficiency of this proposed mechanism is that very little tRNA characterization was done to justify it. The only tRNA work was to use LC/MS in Fig. 2 to measure m1A levels in total tRNA which increased in cells containing a catalytically inactivate ALKB-1 mutant. Specifically:\n1. tRNA-seq (multiple methods available in the literature) should be done to identify specific cytosolic and mitochondrial tRNAs whose m1A levels change between WT and D247A mutant cells.\n2. tRNA-seq can also compare changes in tRNA abundance between these cells. In case significant changes are found for specific tRNAs, these should be validated by Northern blots.\n3. Mammalian ALKBH1 also modifies their mito-tRNA-Met to generate 5-formyl-C (f5C) which impacts mitochondrial activity (PMID 27497299, 28472312). Another mammalian cytosolic f5C tRNA substrate for ALKBH1 is LeuCAA. Does ALKB-1-dependent f5C modification also occur in C. elegans tRNAs? This can be measured by LC/MS and multiple tRNA sequencing methods in the literature). This point is very important, as f5C modification may also in part or even solely account for the observed mito-DNA elimination effects.\n4. Is the recombinant C. elegans ALKB-1 protein active in the tRNA m1A erasing activities in vitro? Only a m6A-DNA substrate was tested here. This can be tested using total tRNA followed by LC/MS.", "reviewer_4": "This manuscript presents a comprehensive and well-executed study uncovering a novel epigenetic mechanism by which the demethylase ALKB-1 promotes paternal mitochondrial elimination (PME) in Caenorhabditis elegans. The authors provide compelling evidence that ALKB-1 limits aberrant tRNA methylation, particularly m¹A, to maintain mitochondrial protein homeostasis. Loss of ALKB-1 demethylase activity triggers mitochondrial stress responses, including UPRmt activation via ATFS-1 and SKN-1/Nrf2, as well as elevated ROS levels. Impressively, genetic suppression of these stress pathways or pharmacological ROS reduction mitigates the PME defect in the alkb-1(D247A) mutant, highlighting a functional link between ALKB-1, mitochondrial quality control, and inheritance.\nThe work is supported by rigorous experiments with appropriate genetic and molecular tools, and the manuscript is clearly written with a logical experimental flow. Furthermore, the authors extend their findings to assess the physiological relevance of PME in the context of sperm function and embryo viability, enhancing the broader significance of the study.\nOverall, this study makes an important contribution to the field of mitochondrial inheritance and epigenetic regulation, and it provides a strong foundation for future mechanistic investigations into the role of RNA modifications in mitochondrial homeostasis.\nHowever, to make it a strong candidate to publish in Nature Communications, a few concerns have to be addressed.\n1. The authors observe slower mtDNA elimination in the alkb-1 knockdown paternal background (Fig. 1A), which could be due to higher initial heteroplasmy levels in mutant males. It would be valuable to directly measure heteroplasmy levels in sperm from wild-type and ALKB-1 KO/KD males to better understand whether this is contributing to the PME defect.\n2. The authors discuss changes in mitochondrial mass and mtDNA copy number in sperm (Fig. S3 and Discussion), but the provided quantifications using whole males (e.g., Fig. 3J) may not fully reflect the changes in sperm specifically. I recommend quantifying mitochondrial mass and mtDNA copy number in mature sperm from the ATFS-1 and SKN-1 mutants to directly link these factors to PME efficiency.\n3. The study would benefit from an investigation into whether ALKB-1 influences sperm development and/or sperm count. This could help clarify whether ALKB-1’s role in PME extends to broader aspects of spermatogenesis.\n4. To further clarify the functional significance of ALKB-1’s demethylase activity in PME, it would be informative to evaluate the rescuing ability of the ajzIs214 transgene (tbb-2p::ALKB-1::3×Flag) in different mutant backgrounds. Specifically, testing whether ajzIs214 can rescue the PME defects in strains carrying either the ALKB-1 ΔMTS or ALKB-1 D247A mutations would help distinguish the respective roles of ALKB-1’s mitochondrial localization and catalytic activity in the regulation of PME.\n5. To better understand whether ALKB-1 acts primarily in sperm or germline cells, the authors could re-express ALKB-1 in a sperm-specific or germline-specific manner in the alkb-1(D247A) mutant background. This would clarify whether ALKB-1’s expression in sperm or the germline is most crucial for regulating PME.\n6. The authors demonstrate that overexpression of ATFS-1 (via the gain-of-function mutant atfs-1(et17)) partially restores the PME defect in the alkb-1(D247A); skn-1 RNAi mutant. However, it would be informative to test PME in embryos from crosses involving only atfs-1(et17) mutants. This experiment would determine whether the activation of UPRmt is sufficient to impair PME and could help pinpoint UPRmt as the key driver of PME dysfunction.\n7. To further strengthen the conclusions regarding the role of UPRmt in PME, I suggest including additional controls, such as testing atfs-1 or skn-1 mutants or RNAi alone, without the alkb-1(D247A) mutation (Fig. 3A, Fig. 5d). This would provide a more comprehensive understanding of the relationship between UPRmt activation and PME efficiency.\n8. The manuscript mentions autophagy regulation by ALKB-1, particularly through the lgg-1::GFP transgene strains. To explore the potential interaction between autophagy and ALKB-1 in PME, it would be insightful to test whether autophagy is required for ALKB-1-mediated PME. Moreover, an epistasis analysis between ALKB-1 and autophagy could clarify how these processes interact during paternal mitochondrial elimination.\n9. In both flies and worms, Endonuclease G was reported to participate in PME. It would be interesting to test the relationship between ALKB-1 and Endonuclease G in the PME process. This could further elucidate the molecular pathways involved and strengthen the overall mechanistic understanding of PME regulation."}, "author_response": "Response: We thank you for your insightful comments and valuable guidance regarding the precise interpretation and appropriate wording of our conclusions. We fully agree that the term “Regulator” may imply a direct mechanism, and we accept the suggestion to soften our claims in the revised manuscript.\nWe have removed the term “Regulator” from the title, abstract, and main discussion sections. The revised title now reads: “ALKB-1 Regulates Proteostasis via tRNA m1A Methylation to Coordinate Paternal Mitochondrial Elimination in C. elegans” This new phrasing avoids the directness of “Regulator” while highlighting the specific upstream signal and the coordinating role of the downstream stress response.\nWe agree that our data show PME defects arise from downstream effects, such as increased mtDNA levels triggered by UPRmt. However, the core of our finding is that the PME impairment observed in the mutant is not simply a generic consequence of UPRmt activation but a specific result of its impairment. We observed that PME was not delayed by various interventions known to strongly activate UPRmt in C. elegans, including pharmacological treatment with doxycycline (Fig. a, c in the following graph, and the corresponding results are shown in Revised Supplemental Figure 10a, b), the atfs-1 (et17) gain-of-function mutants (Fig. b, d in the following graph, and the corresponding results are shown in Revised Supplemental Figure 10c, d), and mtDNA deletion mutants mptDf1 or uaDf5 1 (Fig. e in the following graph, and the corresponding results are shown in Fig. 1a). This suggested that the PME defect is specifically linked to ALKB-1 impairment-induced disruption of mitochondrial homeostasis, particularly via m¹A-driven translational imbalance, rather than a general UPRmt induction. Moreover, comparable UPRmt activation in alkb-1ΔMTS/+ and alkb-1D247A worms, without PME delay (Fig. f-h in the following graph, and the corresponding results are shown in Revised Supplemental Figure 2g-i), supporting a decoupling of UPRmt from PME delay. These findings reinforce that the PME defect induced by ALKB-1 impairment specifically relies on its m¹A-driven translational imbalance, with UPRmt acting as a downstream response rather than a primary driver.\nWe have softened our claims by avoiding the term “Regulator”, we maintain that the pathway initiated by ALKB-1 impairment is a critical, specific, and genetically distinct mechanism that links epitranscriptomic regulation to mitochondrial inheritance.\nReferences:\n2. Line 130:\nThe authors describe a “demethylase inactive mutant strain, with the D247A substitution,” and note in Supplemental Figure 2i that D247 is an “ion-coordinating residue.” However, it would be more accurate to specify that D247 is an iron-coordinating residue, as AlkB homologs catalyze iron-dependent oxidative demethylation. Additionally, the authors should explain why this specific residue was chosen, referencing prior literature and including a sequence alignment of this residue with other ALKBH1 homologs in Supplemental Figures.\nResponse: Thank you for your precise and valuable critique regarding the characterization of the mutant. We have revised the manuscript to enhance the clarity and accuracy of this point and incorporated your suggestion. We fully agree that it is more accurate to specify iron-coordinating residue, as ALKB homologs catalyze iron (Fe (II))-dependent oxidative demethylation. We have corrected the terminology throughout the text.\nWe selected the residue for site-directed mutagenesis based on strong evolutionary conservation and established functional evidence. The catalytic activity of human and mouse demethylases ALKBH1 depends on a conserved aspartate residue (D233 in the human homolog), which is essential for coordinating the ferrous iron cofactor 1, 2, 3, 4, 5. Multiple studies have demonstrated that mutating this aspartate residue to alanine (D233A) abolishes their enzymatic activity entirely 1, 2, 3, 5. Through cross-species sequence alignment of homologs, we confirmed that this critical iron-coordinating residue is evolutionarily conserved in C. elegans ALKB-1, corresponding to D247A. Based on this conservation and functional evidence, we selected the residue to generate a catalytically inactive variant (D247A) of ALKB-1.\nTo visually substantiate this justification, we have supplemented the manuscript with a sequence alignment (following graph, and the corresponding results are shown in Revised Supplemental Figure 3a). This alignment compares ALKB-1 with other ALKBH1 homologs and clearly highlights the conserved iron-coordinating residue D247.\nReferences:\n3. RNA-Seq Analysis:\nThe authors perform ribosome nascent-chain complex sequencing (RNC-Seq) and proteomics on wildtype and ALKB-1 mutant worms but do not conduct standard RNA-Seq to assess global mRNA levels. Standard RNA-Seq is necessary to make claims about translation efficiency, as reduced ribosome association could result from decreased mRNA abundance rather than impaired translation. RNA-Seq would also clarify whether proteomic changes stem from transcriptional or translational alterations.\nResponse: We appreciate you raising this important question regarding the necessity of RNA-seq for the rigorous interpretation of data.\nWe would like to clarify that we did, in fact, perform standard on the corresponding samples concurrently with (as is standard practice for translation efficiency studies) and the original analysis of translation efficiency was based on the ratio. However, we apologize that the RNA-seq data itself was not presented separately, which led to the misunderstanding that global mRNA levels were not assessed.\nTo definitively address your concern and clarify whether changes stem from transcriptional or translational alterations, we have now explicitly detailed the joint analysis results in the revised manuscript. We confirmed that and were used in a joint analysis to identify gene sets with significant changes in translation efficiency (TE) between the two groups while ensuring no underlying differences in abundance. The results revealed significant alterations in the TE transcripts of alkb-1D247A worms compared to wild-type N2 (471 transcripts upregulated and 140 downregulated). These findings confirm that the observed changes are due to impaired translation efficiency, supporting our conclusion that tRNA m1A hypermethylation primarily functions to alter the efficiency of protein synthesis, rather than affecting gene transcription or abundance. We have updated the figure legends and methods to make this RNA-seq/RNC-seq joint analysis absolutely clear (Page 36, line 781-787), where we say,\n“To assess changes in translation efficiency, the Translation Ratio (TR) for each gene was calculated as the ratio of its translational abundance (measured by TPM from RNC-seq) to its transcriptional abundance (measured by TPM from RNA-seq), using the formula: TR = (TPM in RNC-seq) / (TPM in RNA-seq). This normalization corrects for changes in mRNA levels that are independent of translation, thereby isolating genuine alterations in translational regulation. Genes exhibiting significant changes in TR between conditions were identified as differential translation efficiency genes.”\n4. Line 186:\nThe authors state, “Notably, translation efficiency was reduced for four mtDNA-encoded TCA cycle-associated transcripts (atp-6, ctb-1, ctc-2, nduo-2), in alkb-1D247A worms (Fig. 2g).” However, the RNC-Seq method used is unsuitable for analyzing mitochondrial mRNAs, as it targets cytoplasmic ribosomes. Mitochondrial ribosomes differ in sedimentation properties and are unaffected by cycloheximide, which arrests cytoplasmic ribosomes. The authors should avoid concluding that translation of mitochondrial mRNAs is reduced without performing mitochondrial polysome profiling. Alternatively, reduced transcript levels could reflect decreased mitochondrial transcription due to disrupted homeostasis, which could be tested using RNA-Seq or qRT-PCR.\nResponse: We thank the reviewer for this insightful comment, which highlights an important methodological consideration regarding the analysis of mitochondrial mRNAs using RNC-Seq. We fully agree that RNC-Seq, which relies on cycloheximide arrest of cytoplasmic ribosomes, is not suitable for assessing mitochondrial translation due to the distinct sedimentation properties and cycloheximide insensitivity of mitochondrial ribosomes. Accordingly, we have removed the statement regarding reduced translation efficiency of mtDNA-encoded transcripts (previously in Fig. 2g and associated text) to avoid unsupported conclusions in this regard. Instead, we have performed additional analyses to address potential impacts on mitochondrial homeostasis, as suggested.\nRe-analysis of our RNA-seq data revealed widespread upregulation of mtDNA-encoded transcripts in alkb-1D247A worms, a finding consistently validated by our qRT-PCR results (following graph, and the corresponding results are shown in Revised Supplemental Figure 6a, b). These results align with established literature indicating that inhibition of mitochondrial translation triggers a compensatory increase in mtDNA transcription as a stress response to maintain bioenergetic homeostasis 1, 2, 3.\nReferences:\n5. Line 247:\nThe statement, “Treatment with the antioxidants N-Acetyl-L-cysteine (NAC) and Pyrroloquinoline quinone (PQQ) effectively reversed the PME delay (Fig. 4b, c),” should be revised to: “Treatment with the antioxidants N-Acetyl-L-cysteine (NAC) or Pyrroloquinoline quinone (PQQ) effectively reversed the PME delay.”\nResponse: We appreciate the reviewers’ suggestions and have incorporated the corresponding revisions in the revised manuscript.\n6. Line 255:\nThe observation that SKN-1 knockdown in either the paternal or maternal lineage alleviates PME defects caused by ALKB-1 inactivation, while ATFS-1 knockdown alleviates PME defects only in the paternal lineage, is intriguing. The authors should discuss why SKN-1 plays roles in both paternal and maternal lineages, whereas ATFS-1’s role is restricted to the paternal lineage. This distinction may provide insight into lineage-specific stress responses and their impact on PME.\nResponse: We thank the reviewer for this insightful comment, highlighting the intriguing asymmetry in lineage-specific contributions of SKN-1 and ATFS-1 to PME rescue upon ALKB-1 inactivation.\nThe bilateral rescue of PME defects by SKN-1 knockdown is consistent with its maternal deposition in oocytes and early embryos, which enables transgenerational activation of ROS detoxification and mitochondrial biogenesis pathways in both parental lineages 1. In contrast, although ATFS-1-dependent UPRᵐᵗ activation and mtDNA copy number expansion occurs in both germlines, ATFS-1 knockdown alleviates PME defects specifically in the paternal lineage. This paternal restriction suggests that SKN-1 offers broad antioxidant and mitochondrial buffering capabilities, contributing to PME regulation in both maternal and paternal lineages, whereas ATFS-1, although induced in both gametes, likely acts as a mitochondria-centric, sperm-biased surveillance mechanism, predominantly regulating PME in the paternal lineage. We agree that this distinction offers valuable insights into lineage-specific stress responses and their influence on PME. Future studies exploring the underlying mechanisms driving these differential roles would be highly interesting and informative.\nIn light of the above discussion, we have incorporated a corresponding section in the revised manuscript to enhance our insights regarding these results (Page 15, line 301-305), where we say,\n“These findings support a model in which SKN-1 offers broad antioxidant and mitochondrial buffering capabilities, contributing to PME regulation in both maternal and paternal lineages, whereas ATFS-1, although induced in both gametes, likely acts as a mitochondria-centric, sperm-biased surveillance mechanism, predominantly regulating PME in the paternal lineage.”\nReferences:\n7. Lines 299–308:\nThis section would be better placed at the beginning of the Discussion, as it provides context for the study’s findings and sets the stage for interpreting the results.\nResponse: We thank the reviewer for this valuable suggestion. Accordingly, we have integrated this content into the opening paragraph of the Discussion section. The revised text can be found on page 17, line 357-364 of the updated manuscript.\n8. Line 193:\nThe authors state, “Subcellular localization analysis, using the WoLF PSORT software, identified 15 nuclear-encoded proteins that localize in mitochondria, and were significantly upregulated (Fig. 2j), indicating potential abnormal protein aggregation in mitochondria.” However, increased protein levels do not necessarily indicate abnormal aggregation. Elevated protein levels could result from transcriptional, translational, or post-translational changes, and may even be compensatory. The authors should clarify this point and provide evidence for abnormal aggregation if this is their intended conclusion.\nResponse: We thank the reviewer for this constructive comment. We agree that upregulated mitochondrial protein levels alone do not conclusively demonstrate abnormal aggregation and may reflect compensatory mechanisms. To address this, we have revised the text in the revised manuscript.\nWe have also added the new experiments to the revised manuscript. We employed a well-established NS-mtGFP reporter system in C. elegans by generating a transgenic strain with the construct “myo-3p::SDHB-1 MTS::gfp (S65C) (no stop codon)::unc-54 3'UTR AAAAn”, a method validated in yeast and mammalian models 1, 2, 3. Imaging revealed punctate mitochondrial aggregates in alkb-1D247A body wall muscle, contrasting with the uniform GFP distribution in controls (following graph, and the corresponding results are shown in Revised Supplemental Figure 6c, d), providing direct evidence of aberrant protein aggregation. This data strengthens our conclusion that ALKB-1 loss leads to mitochondrial proteotoxic stress via impaired translation and aggregation, rather than simply upregulation.\nReferences:\n9. Figure 5:\nThe authors show that overexpression of ATFS-1 using the gain-of-function mutant atfs-1(et17), which constitutively activates mt-UPR, partially restores the delayed PME phenotype in the alkb-1D247A; skn-1 RNAi mutant. However, it would be informative to test PME in embryos from crosses involving atfs-1(et17) mutants alone, without the alkb-1D247A mutation or SKN-1 RNAi. This experiment would reveal whether mt-UPR activation alone is sufficient to impair PME, pinpointing mt-UPR as the primary driver of reduced PME efficiency.\nResponse: We appreciate the reviewers’ suggestions. To determine whether activation of UPRmt causes PME defects, we examined the PME process in atfs-1 (et17). The results revealed no significant difference in the PME process between atfs-1 (et17) and the wild-type N2. These results demonstrate that UPRmt activation per se is insufficient to disrupt PME. Constitutive ATFS-1 activation via atfs-1 (et17) partially restores the PME delay in alkb-1D247A; skn-1 RNAi backgrounds (Fig. 5a, b), confirming SKN-1's epistatic role upstream of UPRmt. Importantly, atfs-1 (et17) alone does not affect PME (following graph, and the corresponding results are shown in Revised Supplemental Figure 10c, d), demonstrating that UPRmt induction is necessary but not sufficient to drive PME defects. Rather, ALKB-1 inactivation specifically generates translation-dependent proteotoxic stress that maladaptively engages UPRmt, leading to mitochondrial accumulation and delayed elimination.\n10. MTS Deletion Strain:\nThe study finds that the MTS deletion strain (alkb-1(ajz102)) can carry out efficient PME similar to wildtype strain crosses, suggesting that mitochondrial localization of ALKB-1 has little impact on PME. However, the authors should validate that deletion of residues 2–35 effectively disrupts ALKB-1 localization to mitochondria in vivo within worms. This could be tested by examining whether mitochondrial tRNA-Met retains the 5-formylcytidine (f5C) modification, as ALKB-1 is required for this formylation. Additionally, the authors should assess whether mt-UPR is activated in the alkb-1(ajz102) strain. If mt-UPR is activated without affecting PME, this would suggest differential effects of mt-UPR on PME in the alkb-1(ajz102) strain versus the D247A substitution (alkb-1(ajz103) strain).\nResponse: We thank the reviewer for the insightful and constructive comments. We have added new experiments and revised the text accordingly to fully address these points.\nIn the original manuscript, we demonstrated ALKB-1 mitochondrial localization in C. elegans via cell fractionation (Supplementary Fig. 2b) and identified residues 2-35 as the mitochondrial targeting sequence (MTS) using HeLa cell ectopic expression (Supplementary Fig. 2e, f). As suggested, we generated transgenic strains expressing tbb-2p::ALKB-1ΔMTS::3×Flag (lacking residues 2-35). Cell fractionation confirmed complete loss of mitochondrial ALKB-1 in these worms (Fig. a in the following graph, and the corresponding results are shown in Revised Supplemental Figure 2b), validating the MTS function in vivo.\nLC-MS analysis of total tRNA revealed extremely low f5C abundance, with no significant difference between alkb-1D247A and N2 (Fig. b in the following graph). Although minor mitochondrial-specific changes cannot be ruled out, the overall scarcity suggests limited functional relevance. Combined with the normal PME observed in alkb-1ΔMTS/+ (Supplementary Fig. 3e, f), this supports the conclusion that mitochondrial ALKB-1 and mt-tRNA f5C are dispensable for the PME delay phenotype. We note that a previous report detected f5C modification in mt-tRNA in C. elegans 1, reporting higher abundance than observed here, likely due to their use of isolated mt-tRNAMet, whereas our analysis utilized total tRNA. Given the low abundance and its apparent limited relevance, we have not included a detailed discussion of f5C in the main text.\nUPRmt is activated in alkb-1ΔMTS/+ to a similar as in alkb-1D247A, yet PME remains normal (Fig. c-e in the following graph, and the corresponding results are shown in Revised Supplemental Figure 2g-i). This decoupling of UPRmt from PME delay is consistent with atfs-1 (et17) alone not affecting PME (Fig. f, g in the following graph, and the corresponding results are shown in Revised Supplemental Figure 10c, d). Thus, UPRmt is not sufficient to impair PME. Differential outcomes likely reflect distinct stress modalities: tRNA m¹A hypermethylation and translational imbalance in alkb-1D247A vs. compensatory responses in alkb-1ΔMTS/+. Both loss of enzymatic activity and mitochondrial localization can trigger UPRmt via separate impacts on mitochondrial function.\nReferences:\n11. Transgene Testing:\nTo strengthen their conclusions, the authors could test different versions of their ajzIs214 transgene (tbb-2p::ALKB-1::3×Flag). While they show that re-expression of wildtype ALKB-1 in the alkb-1D247A mutant strain rescues the PME defect, testing transgenes expressing the ALKB-ΔMTS mutant or ALKB-D247A mutant could reveal whether these variants can also rescue PME defects in the alkb-1D247A mutant strain.\nResponse: We appreciate the reviewer’s constructive suggestion to strengthen the rescue experiments. Following this recommendation, we introduced the transgenic constructs “tbb-2p::ALKB-1D247A::3×Flag::tbb-2 3'UTR” (expressing the catalytically inactive variant) and “tbb-2p::ALKB-1ΔMTS::3×Flag::tbb-2 3'UTR” (expressing the mitochondrial localization-deficient variant) into the alkb-1D247A worms and assessed their ability to rescue PME defects through microscopic analyses. Our results showed that re-expression of ALKB-1ΔMTS significantly restored PME efficiency in alkb-1D247A worms (comparable to wild-type ALKB-1 rescue), whereas re-expression of ALKB-1D247A had no rescue effect (following graph, and the corresponding results are shown in Revised Supplemental Figure 4a, b). These findings reinforce that ALKB-1 demethylase activity, rather than its mitochondrial localization, is critical for PME.\n12. Figure 2J:\nIn the volcano plot, there is a highly decreased protein in the top left corner. The authors should identify this protein, as its significant difference may provide additional insights.\nResponse: We appreciate the reviewer’s suggestion to identify the highly downregulated protein in the volcano plot, as its significant difference may offer additional insights. We have re-analyzed the proteomics data and updated the manuscript accordingly.\nThe most significantly downregulated protein in alkb-1D247A worms (top left of volcano plot, Fig. 2h) is VF13D12L.3. VF13D12L.3 is a predicted oxidoreductase with homology to TCA cycle components and is implicated in mitochondrial metabolism (WormBase: WBGene00022247). However, due to the limited existing reports on VF13D12L.3 and its unclear precise localization, we have annotated it in the figure but have not included a detailed description in the main text. Its notable reduction is consistent with disrupted TCA cycle function and mitochondrial homeostasis following ALKB-1 inactivation, aligning with our RNC-seq findings of overall OXPHOS/TCA pathway suppression (Fig. 2e, f).\n13. Figure 6d:\nThere is a typo in the schematic that reads “Delayed parternal mitochondrial elimination.” This should be corrected to “Delayed paternal mitochondrial elimination.”\nResponse: We thank the reviewer for their careful reading. The typo “Delayed parternal mitochondrial elimination” in Fig. 6d has been corrected to “Delayed paternal mitochondrial elimination” in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate the time and expertise invested by both the primary reviewer and the co-reviewer in evaluating our manuscript.\nReviewer #3 (Remarks to the Author):\nThis work describes the effect of a putative tRNA demethylase, ALKB-1, that controls the exclusion of paternal mitochondrial DNA in C. elegans. Demethylation of mRNA and tRNA has been described for a while now, but the biological phenotypes and mechanisms directly linked to the demethylation activities have lagged behind. This work shows that ALKB-1, a putative tRNA m1A demethylase, impacts the exclusion of paternal mito-DNA, potentially through its tRNA demethylation activity. ALKB-1 inactivation disrupts translation, mitochondrial proteostasis, and increases ROS. This activates oxidative stress response pathways to result in deficient mt-DNA removal. This study identifies a potential epitranscriptomic mechanism for mitochondrial inherence during reproduction. If true, it would represent an important contribution to our biological understanding of RNA demethylation.\nThe main deficiency of this proposed mechanism is that very little tRNA characterization was done to justify it. The only tRNA work was to use LC/MS in Fig. 2 to measure m1A levels in total tRNA which increased in cells containing a catalytically inactivate ALKB-1 mutant. Specifically:\n1. tRNA-seq (multiple methods available in the literature) should be done to identify specific cytosolic and mitochondrial tRNAs whose m1A levels change between WT and D247A mutant cells.\nResponse: We appreciate the reviewers’ professional guidance and suggestions regarding the tRNA m1A modification section. We have conducted tRNA m¹A sequencing using established methods and added the new data to the revised manuscript. To further elucidate the role of ALKB-1 in tRNA m1A modification, we performed tRNA m1A sequencing. This analysis revealed significantly elevated m¹A levels in 11 tRNA isodecoders and marked reductions in 4 others. Given that ALKB-1 functions as an m¹A demethylase, the observed decreases likely arise from indirect effects. Further examination of the upregulated isodecoders showed that they comprised 9 cytosolic and 2 mitochondrial tRNAs, with 8 of the 9 cytosolic species being tRNA-Gly (TCC/GCC) isoacceptors, indicating ALKB-1 preferentially demethylates m¹A in tRNA-Gly (following graph, and the corresponding results are shown in Revised Figure. 2e, and Supplemental Figure 5j, k). Collectively, these findings indicate that ALKB-1 demethylates m¹A on both cytosolic and mitochondrial tRNAs, with a primary preference for cytosolic substrates.\n2. tRNA-seq can also compare changes in tRNA abundance between these cells. In case significant changes are found for specific tRNAs, these should be validated by Northern blots.\nResponse: We appreciate the reviewer’s suggestion to assess tRNA abundance changes via tRNA-seq and validate by Northern blot. We have performed these analyses and added the data to the revised manuscript.\ntRNA-seq revealed no significant difference in tRNA abundance between wild-type N2 and alkb-1D247A worms across all identified tRNA species (Fig. a in the following graph, and the corresponding results are shown in Revised Supplemental Figure 5l). To validate, we selected six representative tRNAs with high m¹A scores or mitochondrial relevance (Gly-TCC, iMet-CAT, Asp-GTC, Gln-TTG, Lys-CTT, Ser-AGA) for biotin-labeled Northern blot. These confirmed comparable tRNA levels in both genotypes (Fig. b in the following graph, and the corresponding results are shown in Revised Supplemental Figure 5m). These results demonstrate that ALKB-1 inactivation does not alter tRNA abundance, reinforcing that PME defects arise specifically from m¹A hypermethylation and its impact on translation efficiency, not tRNA expression.\n3. Mammalian ALKBH1 also modifies their mito-tRNA-Met to generate 5-formyl-C (f5C) which impacts mitochondrial activity (PMID 27497299, 28472312). Another mammalian cytosolic f5C tRNA substrate for ALKBH1 is LeuCAA. Does ALKB-1-dependent f5C modification also occur in C. elegans tRNAs? This can be measured by LC/MS and multiple tRNA sequencing methods in the literature). This point is very important, as f5C modification may also in part or even solely account for the observed mito-DNA elimination effects.\nResponse: We thank the reviewer for raising this important point regarding the potential role of ALKB-1 in f5C modification in C. elegans tRNAs.\nOur LC-MS analysis of total tRNA revealed extremely low f5C abundance, with no significant difference between alkb-1D247A and N2 (figure below). Although minor mitochondrial-specific changes cannot be ruled out, the overall scarcity suggests limited functional relevance. Combined with the normal PME observed in alkb-1ΔMTS/+ (Supplementary Fig. 3e, f), this supports the conclusion that mitochondrial ALKB-1 and mt-tRNA f5C are dispensable for the PME delay phenotype. We note that a previous report detected f5C modification in mt-tRNA in C. elegans 1, reporting higher abundance than observed here, likely due to their use of isolated mt-tRNAMet, whereas our analysis utilized total tRNA. Given the low abundance and its apparent limited relevance, we have not included a detailed discussion of f5C in the main text.\nReferences:\n4. Is the recombinant C. elegans ALKB-1 protein active in the tRNA m1A erasing activities in vitro? Only a m6A-DNA substrate was tested here. This can be tested using total tRNA followed by LC/MS.\nResponse: We appreciate the reviewer’s valuable suggestion to test ALKB-1 tRNA m¹A demethylation activity in vitro. We have performed the assay using a synthetic stem-loop tRNA probe containing m¹A and added the data to the revised manuscript.\nIn vitro demethylation followed by anti-m¹A dot blot showed that recombinant wild-type ALKB-1 robustly reduced m¹A levels, while ALKB-1D247A exhibited negligible activity (following graph, and the corresponding results are shown in Revised Supplemental Figure 5i). Consistent RNA loading was confirmed by pre- and post-reaction quantification (A260). Methylene blue staining was not used because the short stem-loop probe (~40 nt) yields poor signal, a common limitation in the literature for small RNA substrates1, 2, 3. Thus, we relied on quantitative dot blot with input normalization, which is standard, sensitive, and modification-specific for validating demethylase activity on defined RNA substrates.\nAlthough LC-MS of total tRNA was suggested, the probe-based dot blot is more appropriate here, it directly tests catalytic specificity on a known m¹A-containing substrate, avoiding background from endogenous tRNAs and confirming ALKB-1 acts directly on m¹A in tRNA, consistent with in vivo data (Fig. 2a-d).\nReferences:\nReviewer #4 (Remarks to the Author):\nThis manuscript presents a comprehensive and well-executed study uncovering a novel epigenetic mechanism by which the demethylase ALKB-1 promotes paternal mitochondrial elimination (PME) in Caenorhabditis elegans. The authors provide compelling evidence that ALKB-1 limits aberrant tRNA methylation, particularly m¹A, to maintain mitochondrial protein homeostasis. Loss of ALKB-1 demethylase activity triggers mitochondrial stress responses, including UPRmt activation via ATFS-1 and SKN-1/Nrf2, as well as elevated ROS levels. Impressively, genetic suppression of these stress pathways or pharmacological ROS reduction mitigates the PME defect in the alkb-1(D247A) mutant, highlighting a functional link between ALKB-1, mitochondrial quality control, and inheritance.\nThe work is supported by rigorous experiments with appropriate genetic and molecular tools, and the manuscript is clearly written with a logical experimental flow. Furthermore, the authors extend their findings to assess the physiological relevance of PME in the context of sperm function and embryo viability, enhancing the broader significance of the study.\nOverall, this study makes an important contribution to the field of mitochondrial inheritance and epigenetic regulation, and it provides a strong foundation for future mechanistic investigations into the role of RNA modifications in mitochondrial homeostasis.\nHowever, to make it a strong candidate to publish in Nature Communications, a few concerns have to be addressed.\n1. The authors observe slower mtDNA elimination in the alkb-1 knockdown paternal background (Fig. 1A), which could be due to higher initial heteroplasmy levels in mutant males. It would be valuable to directly measure heteroplasmy levels in sperm from wild-type and ALKB-1 KO/KD males to better understand whether this is contributing to the PME defect.\nResponse: We thank the reviewer for this helpful suggestion. To rule out elevated initial heteroplasmy in alkb-1-deficient males as a contributor to delayed PME, we directly quantified mtDNA heteroplasmy in sperm using nested PCR with mptDf1 standards. Standard samples from gradient-diluted mptDf1/WT mtDNA confirmed that band intensity ratios reliably reflect heteroplasmy proportions. Analysis of sperm from alkb-1 RNAi males showed no change in mptDf1/WT ratios vs. controls (following graph, and the corresponding results are shown in Revised Supplemental Figure 1i, j). Thus, ALKB-1 RNAi does not increase paternal heteroplasmy, and PME delay reflects impaired elimination kinetics, not higher starting mtDNA load.\n2. The authors discuss changes in mitochondrial mass and mtDNA copy number in sperm (Fig. S3 and Discussion), but the provided quantifications using whole males (e.g., Fig. 3J) may not fully reflect the changes in sperm specifically. I recommend quantifying mitochondrial mass and mtDNA copy number in mature sperm from the ATFS-1 and SKN-1 mutants to directly link these factors to PME efficiency.\nResponse: We thank the reviewer for this valuable suggestion. To directly link SKN-1 and ATFS-1 to sperm-specific changes in mitochondrial mass and mtDNA copy number, we isolated mature sperm from the indicated genotypes and performed targeted quantifications.\nIn the original manuscript, sperm from alkb-1D247A males showed increased mitochondrial mass (MitoTracker) and mtDNA copy number (qPCR) vs. N2 (previously in Supplementary Fig. 3a-c). We now extend this to ATFS-1/SKN-1 RNAi in alkb-1D247A males: both RNAi treatments suppressed the elevation in sperm mitochondrial mass and mtDNA copy number (Fig. a, b in the following graph, and the corresponding results are shown in Revised Supplemental Figure 4e, f). These data confirm that ALKB-1 inactivation increases sperm mitochondrial load in an ATFS-1- and SKN-1-dependent manner, directly contributing to PME defects.\n3. The study would benefit from an investigation into whether ALKB-1 influences sperm development and/or sperm count. This could help clarify whether ALKB-1’s role in PME extends to broader aspects of spermatogenesis.\nResponse: We thank the reviewer for this excellent suggestion, which helps clarify whether ALKB-1’s role in PME extends to broader aspects of spermatogenesis.\nWe labeled male gonads with GFP::NKB-2 (Na⁺/K⁺-ATPase β-subunit, plasma membrane marker of spermatocytes/spermatids). No differences were observed in spermatocyte differentiation (L4 stage) or spermatid number per spermatocyst (adult stage) between N2, alkb-1 RNAi, and alkb-1D247A males (Fig. a, b in the following graph, and the corresponding results are shown in Revised Supplemental Figure 9a, b).\nAdult males were DAPI-stained and imaged (Zeiss). Sperm were identified by compact nuclei within spermatocysts. Sperm quantity was unchanged in alkb-1D247A vs. WT in both N2 and him-5(e1467) (high-incidence male) backgrounds (Fig. c-f in the following graph, and the corresponding results are shown in Revised Supplemental Figure 9c-f).\nThese data indicate that ALKB-1 inactivation does not affect sperm development or count, and PME defects arise specifically from impaired paternal mitochondrial elimination, not altered spermatogenesis.\n4. To further clarify the functional significance of ALKB-1’s demethylase activity in PME, it would be informative to evaluate the rescuing ability of the ajzIs214 transgene (tbb-2p::ALKB-1::3×Flag) in different mutant backgrounds. Specifically, testing whether ajzIs214 can rescue the PME defects in strains carrying either the ALKB-1 ΔMTS or ALKB-1 D247A mutations would help distinguish the respective roles of ALKB-1’s mitochondrial localization and catalytic activity in the regulation of PME.\nResponse: We thank the reviewer for this insightful suggestion to further clarify the functional significance of ALKB-1's demethylase activity and mitochondrial localization in PME.\nIn direct response to this point, we have performed additional genetic crosses and experiments as suggested. Firstly, as we originally presented in the manuscript, the ajzIs214 transgene (expressing wild-type ALKB-1) successfully rescues the PME defects in alkb-1D247A worms (Fig. 1h, j). This finding already established that the demethylase activity of ALKB-1 is essential for its role in PME.\nFurthermore, and crucially, to directly address the reviewer's question regarding mitochondrial localization, we introduced the ajzIs214 transgene into the alkb-1ΔMTS/+ mutant, as recommended. Consistent with our initial findings that the alkb-1ΔMTS/+ mutation itself does not cause PME defects, the expression of wild-type ALKB-1 in this background also showed normal PME, identical to the alkb-1ΔMTS/+ single mutant (following graph, and the corresponding results are shown in Revised Supplemental Figure 2h, i).\nTogether, these results strongly support the conclusion that ALKB-1 regulates PME through its catalytic demethylase activity in a manner that is independent of its mitochondrial localization.\n5. To better understand whether ALKB-1 acts primarily in sperm or germline cells, the authors could re-express ALKB-1 in a sperm-specific or germline-specific manner in the alkb-1(D247A) mutant background. This would clarify whether ALKB-1’s expression in sperm or the germline is most crucial for regulating PME.\nResponse: We thank the reviewer for this valuable suggestion to clarify cell-type-specific roles of ALKB-1 in PME regulation.\nTo determine whether ALKB-1 acts primarily in sperm or germline, we generated transgenic lines expressing ALKB-1::3×Flag under sperm-specific (trp-3) or germline-specific (pie-1) promoters via MOSCI. These were crossed into alkb-1D247A mutants and assessed for PME rescue by MitoTracker imaging. Sperm-specific ALKB-1 rescue significantly alleviated PME defects, while germline-specific rescue showed a trend toward improvement but lacked statistical significance (following graph, and the corresponding results are shown in Revised Supplemental Figure 4c, d). These results confirm ALKB-1 catalytic activity is required in both lineages, with a more critical role in sperm, consistent with paternal-lineage dominance in PME defects (Fig. 1j, k).\n6. The authors demonstrate that overexpression of ATFS-1 (via the gain-of-function mutant atfs-1(et17)) partially restores the PME defect in the alkb-1(D247A); skn-1 RNAi mutant. However, it would be informative to test PME in embryos from crosses involving only atfs-1(et17) mutants. This experiment would determine whether the activation of UPRmt is sufficient to impair PME and could help pinpoint UPRmt as the key driver of PME dysfunction.\nResponse: We appreciate the reviewers’ suggestions. To determine whether activation of UPRmt causes PME defects, we examined the PME process in atfs-1 (et17). The results revealed no significant difference in the PME process between atfs-1 (et17) and the wild-type N2. These results demonstrate that UPRmt activation per se is insufficient to disrupt PME. Constitutive ATFS-1 activation via atfs-1 (et17) partially restores the PME delay in alkb-1D247A; skn-1 RNAi backgrounds (Fig. 5a, b), confirming SKN-1’s epistatic role upstream of UPRmt. Importantly, atfs-1 (et17) alone does not affect PME (following graph, and the corresponding results are shown in Revised Supplemental Figure 10c, d), demonstrating that UPRmt induction is necessary but not sufficient to drive PME defects. Rather, ALKB-1 inactivation specifically generates translation-dependent proteotoxic stress that maladaptively engages UPRmt, leading to mitochondrial accumulation and delayed elimination.\n7. To further strengthen the conclusions regarding the role of UPRmt in PME, I suggest including additional controls, such as testing atfs-1 or skn-1 mutants or RNAi alone, without the alkb-1(D247A) mutation (Fig. 3A, Fig. 5d). This would provide a more comprehensive understanding of the relationship between UPRmt activation and PME efficiency.\nResponse: We thank the reviewer for highlighting the need for comprehensive controls to clarify the specificity of UPRmt activation. Using the hsp-6p::GFP reporter, we found that ALKB-1 inactivation robustly induced UPRmt in both males and hermaphrodites (following graph, and the corresponding results are shown in Revised Fig. 3a, b), while atfs-1 or skn-1 RNAi in alkb-1D247A completely abolished this activation (following graph, and the corresponding results are shown in Revised Fig. 5d). Critically, in wild-type N2, atfs-1 or skn-1 RNAi alone had no effect on UPRmt (following graph, and the corresponding results are shown in Revised Fig. 3a, b, and Fig. 5d), as basal activity is undetectable, consistent with prior reports.\n8. The manuscript mentions autophagy regulation by ALKB-1, particularly through the lgg-1::GFP transgene strains. To explore the potential interaction between autophagy and ALKB-1 in PME, it would be insightful to test whether autophagy is required for ALKB-1-mediated PME. Moreover, an epistasis analysis between ALKB-1 and autophagy could clarify how these processes interact during paternal mitochondrial elimination.\nResponse: We thank the reviewer for this insightful suggestion to probe the interaction between ALKB-1 and autophagy in PME. We performed epistasis analysis by knocking down lgg-1 (the C. elegans Atg8/LC3 ortholog) in both N2 and alkb-1D247A backgrounds, with PME quantified via MitoTracker imaging. In the alkb-1D247A background, lgg-1 RNAi further exacerbated the PME delay, yielding a phenotype indistinguishable from lgg-1 RNAi alone (following graph, and the corresponding results are shown in Revised Supplemental Figure 12c, d). These results indicate that LGG-1 functions downstream of ALKB-1 in PME regulation, with ALKB-1 inactivation triggering translational stress that converges on autophagy-dependent clearance as a common effector for paternal mitochondrial elimination.\n9. In both flies and worms, Endonuclease G was reported to participate in PME. It would be interesting to test the relationship between ALKB-1 and Endonuclease G in the PME process. This could further elucidate the molecular pathways involved and strengthen the overall mechanistic understanding of PME regulation.\nResponse: We thank the reviewer for this insightful suggestion to explore the relationship between ALKB-1 and Endonuclease G (CPS-6) in PME. We performed microscopic PME assays in alkb-1D247A, cps-6 (tm3222), and the double mutant alkb-1D247A; cps-6 (tm3222). All mutants exhibited significant PME defects. Notably, cps-6 (tm3222) and the double mutant showed more severe delays than alkb-1D247A alone, with the double mutant phenocopying cps-6 single mutant severity (following graph, and the corresponding results are shown in Revised Supplemental Figure 12a, b). These results indicate that CPS-6 functions downstream of ALKB-1 in PME. ALKB-1 inactivation increases sperm mitochondrial and mtDNA load, which overwhelms CPS-6-mediated degradation post-fertilization, resulting in delayed elimination. This aligns with CPS-6 acting after sperm-egg fusion to degrade paternal mtDNA."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript presents new results that strengthen the conclusion that ALKB-1 is essential for efficient PME. The authors have also addressed previous concerns by making corrections and enhancing the clarity of the manuscript. Overall, the revision represents a significant improvement compared to the original submission.\nHowever, we believe the new title remains overstated, despite the authors removing the term “Regulator.” The current title, “ALKB-1 Regulates Proteostasis via tRNA m1A Methylation to Coordinate Paternal Mitochondrial Elimination in C. elegans,” implies that ALKB-1 directly modulates proteostasis. This interpretation is not fully supported by the data. The conclusion that ALKB-1 regulates proteostasis is based on observations that ALKB-1 knockout leads to impaired proteostasis. However, this impairment appears to result from mitochondrial translation defects caused by ALKB-1 deficiency, which subsequently disrupt protein synthesis and proteostasis. While many proteins can cause proteostasis defects when lost, this does not necessarily qualify them as regulators of proteostasis.\nWe strongly recommend that the authors revise the title and avoid language suggesting a regulatory role for ALKB-1 in proteostasis or in paternal mitochondrial elimination. For instance, the title could be rephrased as:\n• “tRNA m1A Methylation by ALKB-1 is Essential for Efficient Paternal Mitochondrial Elimination”\nOr\n• “ALKB-1-Dependent tRNA Methylation is Required for Efficient Paternal Mitochondrial Elimination.”\nAside from this issue, the authors have satisfactorily addressed nearly all concerns raised in the initial review.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors adequately addressed my comments.", "reviewer_4": "The authors have satisfactorilly addressed all my concerns and I recommend publication of the paper.\nThis study presents a compelling and timely investigation into the epitranscriptomic regulation of paternal mitochondrial elimination (PME) in C. elegans. The authors identify ALKB-1, an evolutionarily conserved demethylase, as a critical regulator of PME through its tRNA m¹A demethylase activity. The mechanistic depth is impressive: ALKB-1 inactivation causes tRNA m¹A hypermethylation, which disrupts protein translation, perturbs mitochondrial proteostasis, and elevates ROS levels. This cascade activates SKN-1/Nrf2 and triggers the mitochondrial unfolded protein response (UPRmt) via ATFS-1, ultimately leading to mitochondrial and mtDNA accumulation in sperm and delayed PME.\nThe novelty of this work is substantial. While extensive research has focused on autophagy, mitophagy, and ubiquitin-proteasome pathways in PME, this is the first study to characterize the role of epitranscriptomic modifications in this fundamental process. The elegant dissection of ALKB-1's substrate specificity—demonstrating that m¹A demethylation rather than 6mA demethylation underlies PME regulation—represents a significant conceptual advance. The epistatic relationship established between SKN-1 and ATFS-1 provides a coherent framework for understanding how oxidative stress signals converge on mitochondrial quality control.\nThis work makes an important contribution to reproductive biology, mitochondrial genetics, and epitranscriptomics. The findings have broad implications for understanding mitochondrial diseases, infertility, and transgenerational inheritance."}, "author_response": "Response: We are grateful for the reviewer’s positive assessment of our revised manuscript. We appreciate the acknowledgment that the new results strengthen our conclusions and that the revisions have significantly improved the clarity of the manuscript.\nWe have carefully considered the reviewer’s critique regarding the title. We agree that the previous title (“ALKB-1 Regulates Proteostasis via tRNA m1A Methylation to Coordinate Paternal Mitochondrial Elimination in C. elegans”) was overstated, as it implied a direct regulatory role for ALKB-1 in proteostasis that is not fully supported by our data. As the reviewer correctly pointed out, the proteostasis defects observed are likely a downstream consequence of mitochondrial translation defects.\nTo address this concern and strictly align the title with our experimental findings, we have removed the term “Regulates” and the reference to proteostasis. We have revised the title to focus on the essential role of ALKB-1-mediated tRNA modification.\nThe new title is: “ALKB-1-Dependent tRNA Methylation is Required for Efficient Paternal Mitochondrial Elimination”.\nWe believe this revised title accurately reflects the content of the paper and avoids overinterpretation of the data.\nWe thank the reviewer again for their constructive comments, which have helped us improve the quality of this manuscript.\nReviewer #2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate the time and expertise invested by both the primary reviewer and the co-reviewer in evaluating our manuscript.\nReviewer #3:\nThe authors adequately addressed my comments.\nResponse: We thank the reviewer for the positive evaluation and are pleased to know that we have adequately addressed their comments.\nReviewer #4:\nThe authors have satisfactorilly addressed all my concerns and I recommend publication of the paper.\nThis study presents a compelling and timely investigation into the epitranscriptomic regulation of paternal mitochondrial elimination (PME) in C. elegans. The authors identify ALKB-1, an evolutionarily conserved demethylase, as a critical regulator of PME through its tRNA m¹A demethylase activity. The mechanistic depth is impressive: ALKB-1 inactivation causes tRNA m¹A hypermethylation, which disrupts protein translation, perturbs mitochondrial proteostasis, and elevates ROS levels. This cascade activates SKN-1/Nrf2 and triggers the mitochondrial unfolded protein response (UPRmt) via ATFS-1, ultimately leading to mitochondrial and mtDNA accumulation in sperm and delayed PME.\nThe novelty of this work is substantial. While extensive research has focused on autophagy, mitophagy, and ubiquitin-proteasome pathways in PME, this is the first study to characterize the role of epitranscriptomic modifications in this fundamental process. The elegant dissection of ALKB-1's substrate specificity—demonstrating that m¹A demethylation rather than 6mA demethylation underlies PME regulation—represents a significant conceptual advance. The epistatic relationship established between SKN-1 and ATFS-1 provides a coherent framework for understanding how oxidative stress signals converge on mitochondrial quality control.\nThis work makes an important contribution to reproductive biology, mitochondrial genetics, and epitranscriptomics. The findings have broad implications for understanding mitochondrial diseases, infertility, and transgenerational inheritance.\nResponse: We are deeply grateful to the reviewer for the enthusiastic endorsement of our work and the recommendation for publication. We greatly appreciate the comprehensive summary of our study, which accurately highlights the novel connections we have uncovered between epitranscriptomic regulation, mitochondrial quality control, and paternal mitochondrial elimination (PME).\nIt is particularly rewarding to have our mechanistic dissection of ALKB-1, specifically its role as a tRNA m¹A demethylase, recognized as a significant conceptual advance. We also value the reviewer’s acknowledgment of the clarity provided regarding the epistatic relationship between SKN-1 and ATFS-1 in mediating these stress responses.\nWe are thrilled that the reviewer finds our work to be a compelling and important contribution to the fields of reproductive biology and mitochondrial genetics. We hope that the publication of this study will indeed provide valuable insights relevant to mitochondrial diseases and infertility.\nThank you again for your constructive feedback and support throughout the review process."}]

Decision letter