Abstract
Nuclear export of messenger RNAs (mRNAs) through nuclear pore complexes (NPCs) is a critical step in gene expression. Although N6-adenosine methylation (m6A) has been implicated in this process, the underlying mechanism remains obscure. Here we demonstrate, using single-molecule imaging, that m6A markedly accelerates the nuclear export of messenger ribonucleoproteins (mRNPs) by increasing export efficiency and shortening export time through NPCs. We further show that the m6A methyltransferase METTL3 localizes at NPCs and functionally associates with the nucleoporin NUP93 to promote the efficient export of m6A-modified mRNPs. The disruption of this functional association between METTL3 and NUP93 substantially impairs overall mRNP export efficiency. Notably, a steroid-resistant nephrotic syndrome (SRNS)-associated NUP93 variant (c.1162C>T, p.Arg388Trp) fails to associate with METTL3, resulting in the defective nuclear export of key methylated mRNAs required for kidney function. Together, our findings define an m6A–METTL3–NUP93 regulatory axis for nuclear mRNA export with broad implications for human disease.
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Data availability
All the genome-wide datasets generated in this study, including RNA-seq and m6A/MeRIP-seq, can be accessed in the Gene Expression Omnibus (GEO) database under an accession number GSE248589. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the dataset identifier (MSV000100322). All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
References
Das, S., Vera, M., Gandin, V., Singer, R. H. & Tutucci, E. Intracellular mRNA transport and localized translation. Nat. Rev. Mol. Cell Biol. 22, 483–504 (2021).
Voronina, A. S. & Pshennikova, E. S. mRNPs: structure and role in development. Cell Biochem. Function 39, 832–843 (2021).
Khong, A. & Parker, R. The landscape of eukaryotic mRNPs. RNA 26, 229–239 (2020).
Aksenova, V. et al. Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway. Nat. Commun. 11, 4577 (2020).
Chi, B. et al. Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA. Nucleic Acids Res. 41, 1294–1306 (2013).
Yellamaty, R. & Sharma, S. Critical cellular functions and mechanisms of action of the RNA Helicase UAP56. J. Mol. Biol. 436, 168604 (2024).
Dias, A., Dufu, K., Lei, H. & Reed, R. A role for TREX components in the release of spliced mRNA from nuclear speckle domains. Nat. Commun. 1, 97 (2010).
Li, Y. et al. Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore. Proc. Natl. Acad. Sci. USA 118, e2015621118 (2021).
De Magistris, P. The great escape: mRNA export through the nuclear pore complex. Int. J. Mol. Sci. 22, 11767 (2021).
Ma, J., Goryaynov, A., Sarma, A. & Yang, W. Self-regulated viscous channel in the nuclear pore complex. Proc. Natl. Acad. Sci. USA 109, 7326–7331 (2012).
Tingey, M. & Yang, W. Unraveling docking and initiation of mRNA export through the nuclear pore complex. Bioessays 44, e2200027 (2022).
Ribbeck, K. & Görlich, D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21, 2664–2671 (2002).
Tingey, M. & Yang, W. Unraveling docking and initiation of mRNA export through the nuclear pore complex. Bioessays 44, 2200027 (2022).
Li, C., Goryaynov, A. & Yang, W. The selective permeability barrier in the nuclear pore complex. Nucleus 7, 430–446 (2016).
Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).
Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).
Lesbirel, S. et al. The m6A-methylase complex recruits TREX and regulates mRNA export. Sci. Rep. 8, 13827 (2018).
Lesbirel, S. & Wilson, S. A. The m6A‑methylase complex and mRNA export. Biochim. Biophys. Acta Gene Regul. Mech. 1862, 319–328 (2019).
Roundtree, I. A. et al. YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. eLife 6, e31311 (2017).
Ma, J. et al. High-resolution three-dimensional mapping of mRNA export through the nuclear pore. Nat. Commun. 4, 2414 (2013).
Tutucci, E., Livingston, N. M., Singer, R. H. & Wu, B. Imaging mRNA in vivo, from birth to death. Annu. Rev. Biophys. 47, 85–106 (2018).
Xiao, Y. et al. An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N6-methyladenosine modification. Angew. Chem. Int. Ed. 57, 15995–16000 (2018).
Yankova, E. et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature 593, 597–601 (2021).
Tagliazucchi, M., Peleg, O., Kroger, M., Rabin, Y. & Szleifer, I. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex. Proc. Natl. Acad. Sci. USA 110, 3363–3368 (2013).
Ori, A. et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol. Syst. Biol. 9, 648 (2013).
Shi, H., Wei, J. & He, C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell 74, 640–650 (2019).
Hong, J., Xu, K. & Lee, J. H. Biological roles of the RNA m6A modification and its implications in cancer. Exp. Mol. Med. 54, 1822–1832 (2022).
Sachdev, R., Sieverding, C., Flötenmeyer, M. & Antonin, W. The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes. Mol. Biol. Cell 23, 740–749 (2012).
Amlacher, S. et al. Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell 146, 277–289 (2011).
Fischer, J., Teimer, R., Amlacher, S., Kunze, R. & Hurt, E. Linker Nups connect the nuclear pore complex inner ring with the outer ring and transport channel. Nat. Struct. Mol. Biol. 22, 774–781 (2015).
Stuwe, T. et al. Architecture of the fungal nuclear pore inner ring complex. Science 350, 56–64 (2015).
Petrovic, S. et al. Architecture of the linker-scaffold in the nuclear pore. Science 376, eabm9798 (2022).
Mosalaganti, S. et al. AI-based structure prediction empowers integrative structural analysis of human nuclear pores. Science 376, eabm9506 (2022).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Yariv, B. et al. Using evolutionary data to make sense of macromolecules with a ‘face-lifted’ ConSurf. Protein Sci. 32, e4582 (2023).
Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).
Pace, C. N. & Scholtz, J. M. A helix propensity scale based on the experimental studies of peptides and proteins. Biophys. J. 75, 422–427 (1998).
Taniguchi, R. et al. Nuclear pores safeguard the integrity of the nuclear envelope. Nat. Cell Biol. 27, 762–775 (2025).
Belin, B. J., Cimini, B. A., Blackburn, E. H. & Mullins, R. D. Visualization of actin filaments and monomers in somatic cell nuclei. Mol. Biol. Cell 24, 982–994 (2013).
Coots, R. A. et al. m6A facilitates eIF4F-independent mRNA translation. Mol. Cell 68, 504–514 e507 (2017).
Stanley, G. J., Fassati, A. & Hoogenboom, B. W. Biomechanics of the transport barrier in the nuclear pore complex. Semin. Cell Dev. Biol. 68, 42–51 (2017).
Tagliazucchi, M., Peleg, O., Kröger, M., Rabin, Y. & Szleifer, I. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex. Proc. Natl. Acad. Sci. 110, 3363–3368 (2013).
Aramburu, I. V. & Lemke, E. A. Floppy but not sloppy: interaction mechanism of FG-nucleoporins and nuclear transport receptors. Semin. Cell Dev. Biol. 68, 34–41 (2017).
Frey, S. et al. Surface properties determining passage rates of proteins through nuclear pores. Cell 174, 202–217 (2018).
Braun, D. A. et al. Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome. Nat. Genet. 48, 457–465 (2016).
Ni, L., Saleem, M. & Mathieson, P. W. Podocyte culture: tricks of the trade. Nephrology 17, 525–531 (2012).
Saleem, M. A. et al. A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J. Am. Soc. Nephrol. 13, 630–638 (2002).
Wang, X. et al. Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex. Nature 534, 575–578 (2016).
Lee, J. H., Jeong, S. A., Khadka, P., Hong, J. & Chung, I. K. Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles. Nucleic Acids Res. 43, 8435–8451 (2015).
Choe, J. et al. mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis. Nature 561, 556–560 (2018).
Lee, J. H. et al. Regulation of telomere homeostasis and genomic stability in cancer by N6-adenosine methylation (m6A). Sci. Adv. 7, eabg7073 (2021).
Liu, J. et al. N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367, 580–586 (2020).
Bankhead, P. et al. QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878 (2017).
Zhang, G. et al. Optical image collection V.1. J. Clin. Invest. https://doi.org/10.17504/protocols.io.beumjeu6 (2020).
Searle, B. C. et al. Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat. Commun. 9, 5128 (2018).
Gessulat, S. et al. Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning. Nat. Methods 16, 509–518 (2019).
Kall, L., Storey, J. D. & Noble, W. S. Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry. Bioinformatics 24, i42–i48 (2008).
Acknowledgements
We thank all members of the Lee, Xu and Yang laboratories for helpful suggestions. Mass spectrometry analyses were conducted at the University of Texas Health San Antonio (UTHSA) Institutional Mass Spectrometry Laboratory, supported in part by UTHSA, NIH grant no. 5P30 CA054174-27 (Mays Cancer Center Mass Spectrometry Shared Resource) and the University of Texas System Proteomics Core Network for the purchase of the Orbitrap Fusion Lumos mass spectrometer, with the expert technical assistance of S. Pardo and D. Molleur under the direction of S. T. Weintraub. This work is supported by the National Cancer Institute (NCI) grant no. R01 CA279696 to J.L. and the National Institute of General Medical Sciences (NIGMS) grant no. R35 GM122552 to W.Y. and grant no. R01 GM140084 to K.X.
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J.H.L. and K.X. conceptualized and designed the original studies. J.H.L. performed most experiments with help from J.H., B.D., J.-H.J., K.A.J. and M.Y. Z.Z. performed bioinformatics analysis and interpreted the data. Under the supervision of W.Y., M.T. performed image analysis. G.Z., I.M.T. and Q.D.Z. analysed the sections of the mouse kidney. F.B., M.D., N.S., B.S. and F.H. supported SRNS-related studies. J.H.L., M.T., Z.Z., W.Y. and K.X. wrote the paper. All authors approved the final paper.
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Extended data
Extended Data Fig. 1 Confirmation of the firefly luciferase reporter cassettes.
a, Validation of site-specific m6A modification on firefly luciferase reporter cassettes (Fluc-no m6A, Fluc−2x m6A, and Fluc−10x m6A) using SELECT-qPCR. b, Schematic illustration of the dCas13b-ALKBH5-based m6A-editing system with sgRNA targeting m6A sites on firefly luciferase mRNA. c, Western blot analysis of indicated proteins in HeLa cells expressing GFP-POM121, which were transiently transfected with the dCas13b-ALKBH5-based m6A-editing system including control sgRNA (sgCtrl) or three independent target sgRNAs (sgFluc-A, -B, and -C). Blots are representative of two independent experiments with similar results. d,e, m6A-MeRIP-qPCR analysis of Fluc-mRNAs following transient transfection with wild-type dCas13b-ALKBH5 (d) or the catalytically inactive mutant dCas13b-ALKBH5 H204A (e), together with sgCtrl or sgFluc. The percentages of m6A levels on Fluc-mRNAs were obtained by calculating MeRIP signals over input RNA signals. f, Validation of site-specific m6A modification on firefly luciferase reporter cassettes (Fluc-no m6A, Fluc−2x m6A, and Fluc−10x m6A) using SELECT-qPCR in GFP-POM121-expressing HeLa cells treated with DMSO or 10 μM STM2457 for 24 hrs. g, Typical types of nuclear export and trajectories of mRNPs in SPEED microscopy. Classification of Successful and Abortive Export. The centroid of the membrane was determined (red dashed line) via brightfield and used to determine whether a single-molecule trace was successfully exported or aborted. Successful export (left) is defined as having an origin point in the nucleus and an exit point in the cytoplasm. Abortive export (right) is defined as having an origin point in the nucleus, a minimum of one point on the cytoplasmic side of the centroid, and a termination point within the nucleus. Data in (a, d-f) are present as mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Source numerical data, unprocessed gels and blots are provided in the Source Data.
Extended Data Fig. 2 Removal of m6A modification or inhibition of m6A deposition leads to the nuclear retention of m6A-modified reporter mRNAs.
a, Validation of site-specific m6A modification on firefly luciferase reporter mRNAs (Fluc-no m6A, Fluc−2x m6A, and Fluc−10x m6A) using SELECT-qPCR in GFP-POM121-expressing HeLa cells, which were transiently transfected with wild-type dCas13b-ALKBH5 or the H204A mutant together with sgCtrl or sgFluc-A. b-d, qPCR analysis of Fluc-mRNA abundance in total (b), nuclear (c) and cytosolic (d) fractions under the conditions described in (a). Relative RNA levels were normalized to GAPDH (total), MALAT1 (nuclear), and 18S rRNA (cytosolic). e, m6A MeRIP-qPCR analysis of Fluc-mRNAs following treatment with DMSO or 10 μM STM2457 for 24 hrs. The percentages of m6A levels on Fluc-mRNAs were obtained by calculating MeRIP signals over input RNA signals. f-h, qPCR analysis of Fluc-mRNA abundance following treatment with DMSO or 10 μM STM2457 for 24 hrs. Data are present as mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Source numerical data are provided in the Source Data.
Extended Data Fig. 3 METTL3 interacts with NUP93 at NPCs.
a,b, Reciprocal IP assays using anti-GFP (a) or anti-HA (b) antibodies in total lysates from HeLa cells exogenously expressing GFP-tagged NUP93 and HA-tagged METTL3. c, IP assays showing that the interaction of METTL3 and NUP93 is mediated by protein-protein contacts. Treatment with RNase A or DNase I during IP did not disrupt the interaction. d, In vitro GST pull-down assay demonstrating a direct interaction between METTL3 and NUP93. GST-NUP93 proteins (amino acids 197-552) were used. e, IP assays showing that the interaction of METTL3 and NUP93 occurs in multiple cell types. f, An in situ proximity ligation assay (PLA) showing interaction between METTL3 and NUP93 at the peripheral nuclear membranes in multiple cell types. g, An in situ PLA using transcription factors that translocate through nuclear pore complexes (MYC and ETS1), RNA polymerase II (RNAPII), or METTL14 as negative controls showed no detectable PLA signal with NUP93, supporting the specificity of the METTL3-NUP93 interaction. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale in (f, g) bar, 2 μm. All panels show representative images from three independent experiments. Source unprocessed gels and blots are provided in the Source Data.
Extended Data Fig. 4 Highly conserved α-helical domains of NUP93 are required for its interaction with METTL3.
a, IP assays using anti-HA antibody in HeLa cells transiently expressing GFP-tagged NUP93 and HA-tagged METTL3 (full-length or truncation mutants), whose domain structures are shown in (a, top). The C-terminal region of METTL3 (amino acids 351-580) mediates interaction with NUP93. b, IP assays using anti-GFP antibody in HeLa cells exogenously expressing HA-tagged METTL3 and GFP-tagged full-length or truncated NUP93 forms, as depicted in (b, top). The middle region of NUP93 (amino acids 197-552) mediates interaction with METTL3. c, IP assays using anti-HA antibody to detect endogenous METTL3 in immunoprecipitates from HeLa cells transiently expressing HA-tagged full-length NUP93 or a series of deletion NUP93 constructs, as illustrated in (c, top). Amino acids 301-400 of NUP93 are required for binding to endogenous METTL3. d,e, NUP93 structure (PDB:7R5K) from PDB (Protein Data Bank) (d) and predicted NUP93 structure from AlphaFold Protein Structure Database (e). The resolved and predicted structure are highly similar. The METTL3-interacting region (amino acids 301-400) is highlighted in red. f, Evolutionary conservation analysis of the middle region (amino acids 301-400) of NUP93 protein. Five conserved α-helices are highlighted in light red. Highly conserved residues are marked in rose red at the bottom of the sequence alignment, and mutated alanine (A), arginine (R), and lysine (K) residues are shown in red. g, IP assays using anti-HA antibody to detect endogenous METTL3 in immunoprecipitates from HeLa cells transiently expressing HA-tagged NUP93 or the indicated single-point mutants. **, immunoglobulin heavy chain; *, immunoglobulin light chain. All panels show representative images from three independent experiments. Source unprocessed gels and blots are provided in the Source Data.
Extended Data Fig. 5 Disruption of the METTL3-NUP93 interaction does not affect nuclear pore complex (NPC) integrity.
a, Immunofluorescence assays in HeLa cells in which endogenous NUP93 was replaced with HA-tagged wild-type NUP93 (WTR) or the indicated mutants (A333PR or R388WR). b,c, Representative images (b) and quantification (c) of PLA signals in HeLa cells infected with control (shCtrl) or NUP93-specific (shNUP93) shRNA and reconstituted with empty vector, wild-type NUP93 (WTR) or the indicated mutants (A333PR or R388WR). Data in (c) are based on PLA signals at the NE per nucleus (three biological replicates; n = 31, 34, 30, and 32 cells per group; data are presented as mean ± s.e.m.). P values calculated by two-sided t-test. Box plots indicate the first and third quartiles, the median (center line), and the minimum and maximum non-outlier values (whiskers). d, Immunofluorescence analysis of nuclear import of GFP-3×NLS (nuclear localization signal) proteins. Numbers indicate the proportion of cells showing successful nuclear import. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. All panels show representative images from three independent experiments. Source numerical data is provided in the Source Data.
Extended Data Fig. 6 METTL3 preferentially binds to m6A-modified RNAs and mediates their association with NUP93, thereby enhancing RNA export.
a,b, In vitro RNA-protein binding assay (b) by mixing the synthesized RNA molecules, sequences of which are displayed in (a), with recombinant His6-tagged METTL3 and/or GST-tagged NUP93 fragment (amino acids 197-552). CBB, Coomassie brilliant blue staining. c, Western blot analysis (top) and quantification of band intensities (bottom) from in vitro RNA-protein binding assay using the synthesized RNAs in (a) with increasing amounts of recombinant His6-tagged METTL3. d, Western blot analysis of in vitro RNA-protein binding assays using synthesized RNAs incubated with recombinant METTL3, METTL14, and NUP93. e,f, In vitro GST-pulldown assay (f) by mixing GST-tagged NUP93 fragment (amino acids 197-552) and His6-tagged METTL3 (wild-type; RNA-binding domain-deleted mutant, ΔRBD; RNA-binding domain-substituted mutant with 6 Alanine loop, 6A) illustrated in (e). g, In vitro RNA-protein binding assay using the synthesized RNAs shown in (a) incubated with GST-tagged NUP93 (amino acids 197-552) and the indicated METTL3 described in (e). CBB, Coomassie brilliant blue staining. h, RNA immunoprecipitation (RIP)-qPCR showing MCP-mCherry or MCP-METTL3 binding to Fluc-mRNAs lacking m6A modifications. i-k, qPCR analysis of Fluc-mRNA abundance in total (i), nuclear (j), and cytosolic (k) fractions. Relative RNA levels were normalized to GAPDH (total), MALAT1 (nuclear), and 18S rRNA (cytosolic). Data are present as mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. All panels in (b-d, f, g) show representative images from three independent experiments. Source numerical data, unprocessed gels and blots are provided in the Source Data.
Extended Data Fig. 7 Silencing of METTL3 or NUP93 leads to global nuclear retention of m6A-modified mRNAs.
a,b, Cumulative distribution curves (left) and box plots (right) showing the log2fold change in nuclear mRNA abundance between METTL3-knockdown (shMETTL3)(a) or NUP93-knockdown (shNUP93) (b) and control (shCtrl) HeLa cells. Nuclear mRNAs were grouped as m6A-modified or unmodified, or further stratified by the number of m6A marks (0, 1, 2-4, or +5). Numbers in parentheses indicate the number of transcripts in each group. P values were calculated by a nonparametric two-tailed Wilcoxon-Mann-Whitney test. c-d, Volcano plots of genes differentially expressed in the nuclear fraction of METTL3-knockdown (shMETTL3) (c) or NUP93-knockdown (shNUP93) (d) cells relative to control (shCtrl) HeLa cells (adjusted P < 0.05 and |Log2fold change | > 0.58). Nuclear-enriched and m6A-modified transcripts are shown as yellow-filled red (upregulated) or blue (downregulated) circles. Expression levels were normalized to ERCC spike-in using a linear regression method. e-i, Western blot analysis with the indicated antibodies (e), cellular m6A levels in total (f), expression of m6A regulators (writers and erasers) (g), abundance of selected target mRNAs in the nuclear fraction (h), and total mRNA abundance (i) in LNCaP cells transduced with control (shCtrl) or shNUP93 and reconstituted with empty vector, shNUP93-resistant wild-type NUP93 (WTR), or the A333P mutant (A333PR). rRNA-depleted RNA was used for measurement of cellular m6A levels in (f). Relative mRNA levels were normalized to MALAT1 (nuclear) or GAPDH (total). Data in (f-i) represent mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Box plots indicate the first and third quartiles, the median (center line), and the minimum and maximum non-outlier values (whiskers). Transcriptome-wide data represent integration of at least two independent biological replicates. Source numerical data, unprocessed gels and blots are provided in the Source Data.
Extended Data Fig. 8 Removal of m6A modification and disruption of METTL3-NUP93 axis leads to the nuclear retention of m6A-modified RNAs.
a, Integrative Genomics Viewer (IGV) profiles showing m6A peaks on selected m6A-modified mRNAs (SKI and PHF13). m6A peaks were called from two published m6A-seq datasets (GSE147891 and GSE98623). MeT-DB v2.0 was further utilized to guide the design of sgRNAs targeting m6A sites on SKI and PHF13 mRNAs. Locations of targeting sgRNAs are indicated in red. b,c, m6A signal intensity measured by m6A-MeRIP-qPCR (b) and site-specific m6A signals measured by SELECT-qPCR (c) on SKI and PHF13 mRNAs in HeLa cells transiently transfected with a dCas13b-ALKBH5-based m6A-editing system. Percentages of m6A levels on SKI and PHF13 mRNAs in (b) were obtained by calculating MeRIP signals over input mRNA signals. d-f, qPCR analysis of total mRNA expression (d), nuclear mRNA abundance (e), and pre-mRNA levels (f) of SKI and PHF13 mRNAs under the conditions described in (b). Relative RNA levels were normalized to GAPDH (d) or MALAT1(e and f). g, Measurement of nuclear mRNA half-life following transient transfection of wild-type dCas13b-ALKBH5 together with sgCtrl or targeting sgRNAs (sgSKI or sgPHF13) in HeLa cells treated with 5 μg/ml actinomycin D (ActD). Nuclear mRNAs were collected at the indicated time points and analyzed by qPCR. h, Validation of site-specific m6A signals on SKI and PHF13 mRNAs by SELECT-qPCR in HeLa cells transduced with control (shCtrl) or shNUP93 and reconstituted with empty vector, shNUP93-resistant wild-type NUP93 (WTR) or the A333P mutant (A333PR). i, Measurement of nuclear mRNA half-life in HeLa cells in which endogenous NUP93 was replaced with shNUP93-resistant wild-type NUP93 (WTR) or the A333P mutant (A333PR). Nuclear mRNAs were collected at the indicated time points and analyzed by qPCR. (j) IGV profiles showing NUDT5 mRNA that lacks annotated m6A sites based on the published m6A-seq data (GSE147891 and GSE98623) and MeT-DB v2.0. Data in (b-i) represent mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Source numerical data is provided in the Source Data.
Extended Data Fig. 9 m6A modification promotes translational output through METTL3/NUP93-mediated nuclear mRNA export.
a,b, CRISPR-mediated removal of m6A (a) or pharmacological inhibition of METTL3 with STM2457 (b) resulted in a marked reduction in luciferase activity of m6A-containing Fluc reporters. c, Global proteomic profiling following METTL3 or NUP93 knockdown in Hela cells revealed reduced protein abundance for m6A-modified transcripts accumulated in the nucleus. d, Gene Ontology (GO) analysis of genes with nuclear accumulated m6A-modified RNAs (Log2FC > 0.58) and reduced proteins (Log2FC < −0.26). Data in (a, b) represent mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. Source numerical data is provided in the Source Data.
Extended Data Fig. 10 Disruption of METTL3-NUP93 interaction by mutation on NUP93 driving SRNS induces nuclear accumulation of SRNS-associated mRNAs.
a, Sanger sequencing traces validating NUP93 genotypes at residue R388 in patient with NS and his family. p, paternal (A2403-11) with heterozygous R388W mutation; m, maternal (A2403-12) with heterozygous G591V mutation; WT, wild type; h, heterozygous. SRNS patient (A2403-21) harbors a compound mutation with Arg388Trp (R388W) and Gly591Val (G591V). b, IP assays using anti-NUP93 antibody or IgG control in total lysates from skin fibroblasts derived from the SRNS patient and parents described in (a). Skin fibroblasts from a healthy donor (wild-type) were included as a control. c,d, IP assays showing that NUP93 R388W (c) does not affect wild-type NUP93 binding to METTL3. Another single point mutant from arginine 388 to proline (R388P) in (d), which also disrupts the interaction between NUP93 and METTL3, further supports the critical role of residue R388 in mediating the interaction. e, Schematic illustration of the experimental workflow for MeRIP/m6A-seq in differentiated human podocytes. f-h, Metagene profiles of m6A peaks across transcripts (f), distribution of m6A peaks across transcript regions (g), and enriched sequence motifs at m6A sites (h) identified from MeRIP/m6A-seq data generated as in (e). 5’ (3’)-UTR, 5’ (3’) untranslated region; CDS, coding sequence. i, qPCR analysis of nuclear (top) and total (bottom) expression of indicated SRNS-associated mRNAs in skin fibroblasts from members of the A2403 family described in (a). Relative RNA levels were normalized to MALAT1 (nuclear) or GAPDH (total). Data in (i) are present as mean ± s.d. from three independent experiments. P values were calculated by two-sided student’s t-test. P values in (h) were calculated by Fisher’s exact test. All panels in (b-d) show representative images from three independent experiments. Transcriptome-wide data represent integration of at least two independent biological replicates. Source numerical data, unprocessed gels and blots are provided in the Source Data.
Supplementary information
Supplementary Video 1 (download ZIP )
Successful nuclear export in HeLa-GFP-POM121_no m6A_2xm6A_10xm6A.
Supplementary Video 2 (download ZIP )
Successful nuclear export in HeLa-GFP-POM121 expressing shNUP93-resistant NUP93 WT_no m6A_2xm6A_10xm6A.
Supplementary Video 3 (download ZIP )
Successful nuclear export in HeLa-GFP-POM121 expressing shNUP93-resistant NUP93 A333P_no m6A_2xm6A_10xm6A.
Source data
Source Data Figs. 2, 5 and 6 and Extended Data Figs. 1, 3, 4, 6, 7 and 10 (download PDF )
All unprocessed western blots and/or gels.
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Lee, J.H., Tingey, M., Zhang, Z. et al. N6-adenosine methylation enhances nuclear mRNA export through METTL3 and NUP93. Nat Cell Biol 28, 553–566 (2026). https://doi.org/10.1038/s41556-026-01882-3
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DOI: https://doi.org/10.1038/s41556-026-01882-3
