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ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing

Nature Cell Biology volume 23, pages 684–691 (2021)Cite this article

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Abstract

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4,5,6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.

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Fig. 1: Human ALKBH7 regulates mitochondrial m22G methylation in mt-dsRNA.
Fig. 2: ALKBH7 catalyses m22G demethylation.
Fig. 3: ALKBH7-mediated m22G demethylation affects polycistronic mt-RNA processing and alters steady-state mt-RNA levels.
Fig. 4: ALKBH7 loss leads to reduced protein translation and decreased mitochondrial activity.
Fig. 5: Depletion of ALKBH7 in mouse tissues led to altered levels of mature mt-mRNAs and tRNAs.

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Data availability

The sequencing data listed in Supplementary Table 1 are available in the Gene Expression Omnibus database under the accession number GSE148202. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by NIH R01 ES030546 (C.H.) and RM1 HG008935 (C.H.). The ALKBH7 biochemistry work was partially supported by The National Key Research and Development Program of China (2020YFA0803400; R.-J.L.) and The National Natural Science Foundation of China (31971230; R.-J.L.). The Mass Spectrometry Facility of the University of Chicago is funded by the National Science Foundation (CHE-1048528). C.H. is an investigator of the Howard Hughes Medical Institute. We thank A. Andersen at Life Science Editors for editorial assistance. We thank X. Dou and X. Cui for help on sequencing data analysis. We thank P. W. Faber, M. Marchuk and L. Scarpitta at the Genomics Facility at the University of Chicago for generous help on high-throughput sequencing.

Author information

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Li-Sheng Zhang, Chang Liu, Jiangbo Wei, Linda Zhang, Bryan T. Harada, Qing Dai, Xinran Feng, Ziyang Hao, Xueyang Dong, Lulu Hu & Chuan He

  2. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Li-Sheng Zhang, Chang Liu, Jiangbo Wei, Linda Zhang, Bryan T. Harada, Qing Dai, Xinran Feng, Ziyang Hao, Xueyang Dong, Lulu Hu & Chuan He

  3. State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, PR China

    Qing-Ping Xiong, En-Duo Wang & Ru-Juan Liu

  4. Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway

    Sonia Peña Perez, Cassy Le & Arne Klungland

  5. Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Sonia Peña Perez & Arne Klungland

  6. Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Yuru Wang, Tao Pan & Chuan He

  7. School of Life Science and Technology, ShanghaiTech University, Shanghai, PR China

    En-Duo Wang & Ru-Juan Liu

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Contributions

C.H., L.-S.Z. and R.-J.L. conceived the original idea and designed the experiments. L.-S.Z. performed experiments and analysed sequencing data. L.-S.Z. and R.-J.L. analysed experiment results. Q.-P.X. performed ALKBH7 biochemistry with R.-J.L. and E.-D.W. Q.-P.X. and R.-J.L. prepared the ALKBH7-knockout human cell lines. S.P.P. and A.K. provided tissue samples of Alkbh7-knockout mice. S.P.P. and C. Le conducted FAO assays and studied OXPHOS in mouse tissues. C. Liu constructed stable TRMT1-knockdown cell lines and helped with cell biology experiments. J.W. and X.D. helped with RNA experiments. L.Z. helped with animal tissue experiments and manuscript writing. B.T.H. helped with manuscript writing. Q.D. synthesized RNA oligonucleotide probes. X.F. helped with nascent protein synthesis assay. Z.H. helped with Seahorse XF Cell Energy Phenotype experiments. Y.W. and T.P. provided plasmids for the AlkB-D135S protein. L.H. helped with ALKBH7 crystal structure analysis. L.-S.Z., R.-J.L. and C.H. interpreted the results and wrote the manuscript.

Corresponding authors

Correspondence to Ru-Juan Liu or Chuan He.

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Competing interests

C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc. and a shareholder of Epican Genetech.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Human ALKBH7 effect on mt-RNA methylations by mass spec and the development of DAMM-seq to detect m1A, m3C, m1G and m22G in one sequencing run via misincorporation signature.

a, An overlay of crystal structure of E. coli AlkB and human ALKBH7 on three peripheral loops (two are identical as EcAlkB, one is the ALKBH7 specific loop). b, The unique active site of ALKBH7 resembles an engineered AlkB protein (D135S/L118V) that catalyzes demethylation of m22G. c, Alkbh7 mRNA levels (normalized to mouse Actb) in several mouse tissues. n = 3 biologically independent samples; data are presented as mean values ± SD. d, ALKBH7 mRNA levels (normalized to ACTB) in several human cancer cell lines. e, ALKBH7 knockdown efficiency in HepG2 cells by protein level (compared to β-tubulin). f, ALKBH7 knockdown efficiency in HepG2 cells by mRNA level (normalized to β-actin). For d and f, n = 2 biologically independent samples. g, Modification levels (m22G/G, m1G/G, m6A/A, m1A/A, m7G/G, m2G/G) by LC-MS/MS for mitochondrial tRNA in ALKBH7-depleted HepG2 cells vs. control. Unpaired, two-tailed t-test; n = 4 biologically independent samples; data are presented as mean values ± SD. h, Modification levels by LC-MS/MS for mitochondrial mRNA in ALKBH7-depleted HepG2 cells vs. control. i, Modification levels by LC-MS/MS for mitochondrial rRNA in ALKBH7-depleted HepG2 cells vs. control. For h-i, unpaired, two-tailed t-test; n = 3 biologically independent samples; data are presented as mean values ± SD. j, A flowchart of DAMM-seq library construction pipeline, detecting four types of methylated bases (m22G, m1G, m1A, m3C) by misincorporation signatures in a “one-pot” manner with or without engineered AlkB treatment. k, IGV plot for visualizing the mutation level drop down to zero after demethylase treatment in DAMM-seq.

Source data

Extended Data Fig. 2 DAMM-seq reveals methylated sites in mitochondrial tRNA, and ALKBH7 does not demethylate m22G or other methylated bases (m1A, m3C, m1G) in mature mt-tRNAs.

a, Modification levels by LC-MS/MS in cellular small RNA fraction (input vs. engineered AlkB treatment) to showcase the demethylation efficiency and selectivity in DAMM-seq. P values = 0.0027, 0.0028, 0.4531, 0.0002, 0.6009, 0.5991, 0.5351, 0.5583 for methylated bases respectively; unpaired, two-tailed t-test. n = 3, biologically independent samples. Data are presented as mean values ± SD. For b-e, RT misincorporation frequency (input vs. engineered AlkB treatment) for: b, m1A9 in 14 mitochondrial tRNAs that contain m1A9; c, m3C32 in 2 mitochondrial tRNAs that contain m3C32; d, m1G9 in 5 mitochondrial tRNAs that contain m1G9; e, m1G37 in 4 mitochondrial tRNAs that contain m1G37. For b-e, n = 2 biologically independent samples. f, An example flowchart of library construction pipeline for DAMM-seq revealing methylation fraction change of specific m1A, m3C, m1G, and m22G sites in vivo, with altered TRMT1. g, TRMT1 knockdown efficiency in HeLa cells by mRNA level (below, normalized to GAPDH. n = 2 biologically independent samples) and protein level (above, compared to GAPDH). h, Mutation levels after RT for m22G26 in mt-tRNA in TRMT1-depleted HeLa cells vs. control. i, Mutation levels after RT for m22G26 in mt-tRNA in ALKBH7-depleted HepG2 cells vs. control. j, Mutation levels after RT for m22G26 in mt-tRNA in ALKBH7-overexpressed HeLa cells vs. control. For k-o, Mutation levels after RT (in ALKBH7-depleted HepG2 cells vs. control) for k, 3 mt-tRNAs with m1A58, l, 2 mt-tRNAs with m3C32, m, 14 mt-tRNAs with m1A9, n, 5 mt-tRNAs with m1G9, o, 4 mt-tRNAs with m1G37. For p-t, Mutation levels after RT (in ALKBH7-overexpressed HeLa cells vs. control) for p, 3 mt-tRNAs with m1A58, q, 2 mt-tRNAs with m3C32, r, 14 mt-tRNAs with m1A9, s, 5 mt-tRNAs with m1G9, t, 4 mt-tRNAs with m1G37. For h-t, n = 2 biologically independent samples.

Source data

Extended Data Fig. 3 ALKBH7 regulates demethylation of m22G (mt-Ile region) and m1A (mt-Leu1 region) in mitochondrial dsRNA in vivo.

a, DAMM-seq reads from IP-enriched dsRNA (HepG2 wild-type) were predominantly aligned to the mitochondrial genome (normalized to genome length). b, DAMM-seq reads coverage of IP-enriched dsRNA across the mitochondrial genome spanning entire protein coding region (~3.5-16 kb). H-strand are shown as green color and L-strand as red color (both normalized to 18S rRNA). c, A9, G9 and G37 in HeLa mature mt-tRNA compared with those in enriched mt-dsRNA. A9: **P = 0.0074; paired, two-tailed t-test; n = 12 independent m1A sites. G9: P = 0.071; paired, two-tailed t-test; n = 5 independent m1G sites. G37: P = 0.5571; paired, two-tailed t-test; n = 4, independent m1G sites. Each data point represents one methylation site, with the average misincorporation from 2 biologically independent samples. Data are presented as mean values ± SD. d, mt-Ile m22G in mature mt-tRNA compared with that in enriched mt-dsRNA in HeLa cells (shControl), where lentivirus infection elevates m22G methylation level in dsRNA stage. e, IGV visualization of mutation ratio at mitochondrial dsRNA m22G (mt-Ile region) in ALKBH7 knockout HepG2 cells vs. wild-type. Mutation ratio values (majorly G→T + G→C) shown in the parenthesis. f, m22G in mt-dsRNA (mt-Ile region) in ALKBH7-overexpressed HeLa cells vs. control. g, IGV visualization of mutation ratio at mitochondrial dsRNA m1A (mt-Leu1 region) in ALKBH7 knockout HepG2 cells vs. wild-type. Mutation ratio values (majorly A→T) shown in the parenthesis. h, m1A in mt-dsRNA (mt-Leu1 region) in ALKBH7-overexpressed HeLa cells vs. control. i, m22G misincorporation in mt-dsRNA (mt-Ile region) in ALKBH7-depleted HepG2 cells (siALKBH7) can be rescued with the wild-type ALKBH7 protein but not by an ALKBH7 mutant without mitochondrial targeting sequence (MTS). j, m1A misincorporation in mt-dsRNA (mt-Leu1 region) in ALKBH7-depleted HepG2 cells (siALKBH7) can be rescued with the wild-type ALKBH7 protein but not by an ALKBH7 mutant without mitochondrial targeting sequence (MTS). For d, f, and h-j, n = 2 biologically independent samples.

Source data

Extended Data Fig. 4 ALKBH7 demethylation effect on other methylations (such as m1A9, m1G9, and m1G37) within mitochondrial dsRNA is lower than that on Ile-m22G and Leu1-m1A inside cells.

a, Mutation levels for m1A9, m1G9, and m1G37 in mt-dsRNA in ALKBH7 knockout HepG2 cells vs. wild-type. m1A9: *P = 0.0398; paired, two-tailed t-test; n = 24, 12 independent m1A sites. m1G9: P = 0.0954; paired, two-tailed t-test; n = 10, 5 independent m1G sites. m1G37: P = 0.38; paired, two-tailed t-test; n = 8, 4 independent m1G sites. b, Mutation levels for m1A9, m1G9, and m1G37 in mt-dsRNA in siALKBH7 HepG2 cells vs. siControl. m1A9: **P = 0.0047; paired, two-tailed t-test; n = 24, 12 independent m1A sites. m1G9: P = 0.1462; paired, two-tailed t-test; n = 10, 5 independent m1G sites. m1G37: *P = 0.0188; paired, two-tailed t-test; n = 8, 4 independent m1G sites. c, Mutation levels for m1A9, m1G9, and m1G37 in mt-dsRNA in ALKBH7-overexpressed HeLa cells vs. control. m1A9: ***P = 0.0002; paired, two-tailed t-test; n = 24, 12 independent m1A sites. m1G9: P = 0.5666; paired, two-tailed t-test; n = 10, 5 independent m1G sites. m1G37: P = 0.4838; paired, two-tailed t-test; n = 8, 4 independent m1G sites. For a-c, each data point represents one methylation site, with the misincorporation from 2 biologically independent samples. Data are presented as mean values ± SD. d, Mutation levels for m1A in mt-ND5 region of mt-dsRNA. Left: ALKBH7 knockout HepG2 cells vs. wild-type; Right: siALKBH7 HepG2 cells vs. siControl. n = 2 biologically independent samples. e, m22G-containing ssRNA probe synthesized by recombinant TRMT1 protein. f, m22G-containing mt-Ile RNA probe in forms of folded tRNA and dsRNA. g, m1A-containing mt-Leu1 RNA probe in forms of ssRNA and dsRNA. h, Modification levels of m1A/A in purified mt-tRNA before and after treatment with recombinant human ALKBH7. P = 0.4048; unpaired, two-tailed t-test. i, Modification levels of m1G/G in purified mt-tRNA before and after treatment with recombinant human ALKBH7. P = 0.2256; unpaired, two-tailed t-test. For h-i, n = 3 biologically independent samples. Data are presented as mean values ± SD.

Source data

Extended Data Fig. 5 Optimized icSHAPE detects one flexible region adjacent to mt-Ile region in mitochondrial dsRNA, in which ALKBH7 depletion and overexpression impact the mt-dsRNA level.

a, A flowchart of library construction pipeline for optimized icSHAPE designed for studying secondary structure within mitochondrial dsRNA. b, IGV visualization of icSHAPE peak region (in H-strand) adjacent to mt-Ile region in mitochondrial dsRNA. For the “pull-down” samples we fed the cells with NAI-N3 (dissolved in DMSO) and enriched NAI-N3 labeled RNAs. For the “input” samples we fed the cells with DMSO and then build the libraries without pull-down enrichment. c, The decreased mitochondrial dsRNA flexible region levels (quantified by RT-qPCR, with gene-specific primer during RT and biotin-tagged spike-in) in ALKBH7-depleted HepG2 cells vs. control. d, The increased mitochondrial dsRNA flexible region level (quantified by RT-qPCR, with gene-specific primer during RT and biotin-tagged spike-in) in TRMT1-depleted HeLa cells vs. control. For c-d, n = 2 biologically independent samples. e, A scheme showing proposed effects of m22G demethylation by ALKBH7 on mitochondrial polycistronic RNA processing. f, ALKBH7-overexpressed cells (o/e ALKBH7) display an increased dsRNA level (in particular the L-strand) compared with o/e Control (normalized to 18S rRNA).

Source data

Extended Data Fig. 6 ALKBH7-depleted cells showed altered levels of steady-state mt-RNA and decreased mitochondrial protein synthesis rate, while mitochondrial transcription was not affected.

a, Relative mRNA levels of 13 mt-mRNAs (normalized to ACTB) in ALKBH7-depleted HEK 293T cells vs. control. P values = 0.0001, 0.0002, <0.0001, <0.0001, 0.0004, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001 for 13 mt-mRNAs, respectively; unpaired, two-tailed t-test. b, ALKBH7-overexpressed HeLa cells vs. control. P values = 0.275, 0.0941, 0.0042, 0.0075, 0.0011, 0.0008, 0.013, 0.009, 0.0124, 0.0008, 0.0209, 0.003, 0.0033 for 13 mt-mRNAs, respectively; unpaired, two-tailed t-test. c, Relative levels of 12S and 16S rRNAs (normalized to 18S rRNA) in ALKBH7-depleted HepG2 cells vs. control. P values = 0.0027 and 0.0003 for two rRNAs, respectively; unpaired, two-tailed t-test. For a-c, n= 4 biologically independent samples. Data are presented as mean values ± SD. NS, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. d, The transcription rate for mt-mRNA regions with and without ALKBH7 depletion. RT-qPCR quantification was conducted on isolated EU-labeled RNA at early time points of 1-min, 2-min, 3-min and 5-min, respectively, from siALKBH7 vs. siControl. Normalized levels of mt-mRNA region are shown. e, The normalized level changes of tRNA-mRNA junctions in nascent mt-RNA with and without ALKBH7 depletion. RT-qPCR quantification was conducted on isolated EU-labeled RNA at early time points of 1-min, 2-min, 3-min and 5-min, respectively, from siALKBH7 vs. siControl. Normalized levels of junction site are shown. For d-e, n= 3 biologically independent samples. Data are presented as mean values ± SD.

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Extended Data Fig. 7 The faster nascent mt-RNA processing led to the decreased overall levels of nascent mt-tRNA and mt-mRNA.

For a-c, Reads coverage of 10-min, 20-min and 30-min EU-labeled nascent RNA (from HepG2 WT cells) across the mitochondrial genome spanning entire protein coding region (~3.5-16 kb, normalized to 18S rRNA). H-strand genes are shown in green color and L-strand in red color. d, Above: IGV reads coverage at the mRNA-tRNA junction of mt-ND1/mt-Ile in mt-dsRNA from dsRNA-IP; below: IGV reads coverage at this mt-ND1/mt-Ile junction in nascent mt-RNA from 10-min EU-labeling. e, Above: IGV reads coverage at the mRNA-tRNA junction of mt-Leu1/mt-ND1 in mt-dsRNA from dsRNA-IP; below: IGV reads coverage at this mt-Leu1/mt-ND1 junction in nascent mt-RNA from 10-min EU-labeling. f, Relative reads coverage comparison at each tRNA-mRNA junction in mt-dsRNA (from dsRNA-IP) vs. in nascent mt-RNA from 10-min EU labeled RNA. The tRNA-mRNA junction ratio is defined by pre-tRNA/mRNA reads coverage ratio, which was calculated by the reads sum in pre-tRNA region (~70 nt) compared to the reads sum within its adjacent mt-mRNA region (within a 200-nt window). The ratio of an unprocessed junction is normalized to 1.0. Many regions were already decayed 10 min after transcription. g, Left: m22G in 10-min EU-labeled RNA (mt-Ile region) in siALKBH7 HepG2 cells vs. siControl. Right: m22G in 10-min EU-labeled RNA (mt-Ile region) in ALKBH7 knockout HepG2 cells vs. wild-type. h, Left: m1A in 10-min EU-labeled RNA (mt-Leu1 region) in siALKBH7 HepG2 cells vs. siControl. Right: m1A in 10-min EU-labeled RNA (mt-Leu1 region) in ALKBH7 knockout HepG2 cells vs. wild-type. For f-h, n= 2 biologically independent samples. i, Normalized mt-mRNA levels in 30-min EU-labeled mitochondrial nascent RNA extracted from siALKBH7 HepG2 cells vs. siControl. P values = 0.0127, 0.0049, 0.0192, 0.3494, 0.8318, 0.5296, 0.0003, 0.0807, 0.0062, 0.0004, 0.0065 for 11 mt-mRNAs, respectively; unpaired, two-tailed t-test. j, Normalized mt-mRNA levels in 60-min EU-labeled mitochondrial nascent RNA extracted from siALKBH7 HepG2 cells vs. siControl. P values = <0.0001, 0.0013, 0.0006, 0.0004, 0.0004, 0.0004, 0.0135, <0.0001, <0.0001, 0.0009, <0.0001 for 11 mt-mRNAs, respectively; unpaired, two-tailed t-test. For i-j, n = 4 biologically independent samples. NS, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

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Extended Data Fig. 8 ALKBH7 loss leads to decreased mitochondrial activity and reduced protein synthesis.

a, Decreased OCR in ALKBH7-depleted HepG2 cells (siALKBH7) vs. siControl. b, Decreased ECAR in ALKBH7-depleted HepG2 cells (siALKBH7) vs. siControl. For a-b, n = 6 biologically independent samples; data are presented as mean values ± SD. c, Western blotting shows protein levels of mitochondrial marker VDAC, SDHA, HSP60, and cellular TRMT1 in siALKBH7 vs. siControl (normalized to β-tubulin). d, The metabolic labeling assay for mitochondrial protein synthesis. Using 3-hour AHA (L-Azidohomoalanine) labeling together with emetine (cytoplasmic translation inhibitor), biotinylated-alkyne-assisted click reaction enables western blotting to reveal mitochondrial nascent protein levels in siALKBH7 vs. siControl. Reduced protein synthesis was observed with ALKBH7 depletion. For c-d, two rounds of immunoblots for biologically independent samples were performed, with similar results obtained.

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Extended Data Fig. 9 Loss of ALKBH7 in mouse tissues led to altered mt-mRNA and mt-tRNA levels, with decreased FAO activity observed in the fat tissue.

a, Relative levels of 13 mt-mRNAs (normalized to Actb) in the kidney isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. P values = 0.0408, 0.018, 0.0229, 0.0618, 0.0256, 0.0428, 0.074, 0.0157, 0.0138, 0.0192, 0.0126, 0.0067, 0.0254 for 13 mt-mRNAs, respectively. b, Relative levels of 13 mt-mRNAs (normalized to Actb) in the heart isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. P values = 0.0084, 0.0026, 0.0008, 0.025, 0.0048, 0.0056, 0.0043, 0.006, 0.0046, 0.0062, 0.0004, 0.0061, 0.0004 for 13 mt-mRNAs, respectively. c, Relative levels of ten mt-tRNAs in the BAT isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. P values = <0.0001, 0.0001, 0.7191, 0.0745, 0.4192, 0.053, 0.1789, 0.0005, 0.4229, 0.4391 for ten mt-tRNAs, respectively. d, Relative levels of ten mt-tRNAs in the liver isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. P values = 0.0221, 0.0485, 0.1482, 0.0484, 0.0086, 0.5072, 0.2898, 0.1218, 0.036, 0.1803 for ten mt-tRNAs, respectively. e, Relative levels of ten mt-tRNAs in the kidney isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. P values = <0.0001, 0.0056, 0.9016, 0.0086, <0.0001, 0.455, 0.0752, 0.0029, 0.0022, 0.0012 for ten mt-tRNAs, respectively. For c-e, mouse U6 snRNA was used for normalization. For a-e, n = 6 biologically independent samples; unpaired, two-tailed t-test; data are presented as mean values ± SD. f, Western blotting of mitochondrial marker Vdac (normalized to β-tubulin) in the lysate from BAT, liver and kidney isolated from Alkbh7 knockout mice (Alkbh7-/-) vs. wild-type. Two rounds of immunoblots for biologically independent samples were performed, with similar results obtained. g-h, Mitochondrial fatty acid oxidation (FAO) assay on five mouse tissues, where the fresh tissue lysate was incubated with FAO substrate octanoyl-CoA for 60 minutes (g) and 90 minutes (h), which is further oxidized in mitochondria. For g, P values = 0.1519, 0.0196, 0.1916, 0.2827, 0.4914 for 5 tissues, respectively; for h, P values = 0.7372, 0.0068, 0.0879, 0.8232, 0.0389 for 5 tissues, respectively. For g-h, n = 8 biologically independent samples; unpaired, two-tailed t-test; data are presented as mean values ± SD. For a-e and g-h, NS P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

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Zhang, LS., Xiong, QP., Peña Perez, S. et al. ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing. Nat Cell Biol 23, 684–691 (2021). https://doi.org/10.1038/s41556-021-00709-7

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