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N6-adenosine methylation controls the translation of insulin mRNA

Nature Structural & Molecular Biology volume 30, pages 1260–1264 (2023)Cite this article

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Abstract

Control of insulin mRNA translation is crucial for energy homeostasis, but the mechanisms remain largely unknown. We discovered that insulin mRNAs across invertebrates, vertebrates and mammals feature the modified base N6-methyladenosine (m6A). In flies, this RNA modification enhances insulin mRNA translation by promoting the association of the transcript with polysomes. Depleting m6A in Drosophila melanogaster insulin 2 mRNA (dilp2) directly through specific 3′ untranslated region (UTR) mutations, or indirectly by mutating the m6A writer Mettl3, decreases dilp2 protein production, leading to aberrant energy homeostasis and diabetic-like phenotypes. Together, our findings reveal adenosine mRNA methylation as a key regulator of insulin protein synthesis with notable implications for energy balance and metabolic disease.

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Fig. 1: Mettl3 is required for glucose balance and energy homeostasis in the insulin-producing cells.
Fig. 2: Mettl3 is required for the translation of dilp2 mRNA.

Data availability

All data are available in the main text or supplementary materials. RNA sequencing reads were deposited in GEO under accession number GSE207547. dilp2 3′ UTR mutants are available upon request. The D. melanogaster genome sequence is available in Ensembl under BDGP6.32, Mus musculus Ins2 is available in GenBank under NM_001185083.2, and Salmo salar ins is available in GenBank under XM_014198195.2. Source data are provided with this paper.

Code availability

All custom scripts for data analysis can be found at https://github.com/dwilinski/m6A-fly-insulin.git.

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Acknowledgements

We thank P. Léopold (Institut Curie) for the kind gift of the dilp2 antibody, P. Callaerts (KU Leuven) for the gift of the dilp3 antibody, R. Seeley and C. Cras-Méneur for mouse islets (University of Michigan), B. Peterson (National Cold Water Marine Aquaculture Center) for salmon tissue, J.-Y. Roignant (University of Lausanne) for Mettl3 mutant flies, and the Bloomington Drosophila Stock Center for other flies used in this study. We are grateful to P. Todd and S. Miller for training and the use of their polysome fractionation equipment, C. Lapointe for thoughtful comments on the manuscript, and C. Duan for advice. We also thank J. Kuhl for designing some of the graphics in this manuscript. This work was supported by National Institutes of Health grants R00 DK-097141 (M.D.), 1DP2DK-113750 (M.D.), T32 DA007268 (D.W.), P30 DK089503 (M.D. and D.W.), and K99 DK128539 (D.W.); the Rita Allen Foundation (M.D.); and National Science Foundation CAREER 1941822 (M.D.).

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Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, The University of Michigan, Ann Arbor, MI, USA

    Daniel Wilinski & Monica Dus

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  1. Daniel Wilinski
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Contributions

Conceptualization: D.W. and M.D. Methodology: D.W. Investigation: D.W. and M.D. Visualization: D.W. and M.D. Funding acquisition: M.D. and D.W. Project administration: M.D. Supervision: M.D. Writing – original draft: D.W. and M.D. Writing – review & editing: D.W. and M.D.

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Correspondence to Monica Dus.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Carolina Perdigoto and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 The effects of Mettl3 KD on energy homeostasis are not developmental.

(a) Quantification of Mettl3 mRNA from heads of control nSyb > w1118CS (n = 3 sets of 20 flies) and nSyb > Mettl3RNAi flies (n = 6 sets of 20 flies). Unpaired two-tailed Student’s t-test, p = 0.045. (b) Triglyceride levels normalized to protein in male control (Tubulin-GAL80ts, dilp2-GAL4 / +) and Tubulin-GAL80ts; dilp2 > Mettl3RNAi flies. n = 8, 6 pools of two flies. Unpaired two-tailed Student’s t-test, p = 0.0003. Error bars are SEM. * p < 0.05, ** p < 0.005.

Source data

Extended Data Fig. 2 Additional phenotyping of Mettl3 mutants.

(a, b, c) Quantification of (a) dilp2, (b) dilp3, and (c) dilp5 mRNA from heads of control (w1118CS) and Mettl3−/− mutant flies. n = 6 sets of 20 flies. Unpaired two-tailed Student’s t-test; p = 0.594 (a), p = 0.778 (b), and p = 0.073 (c). (d) Quantification of insulin cells n = 3 brains. Unpaired two-tailed Student’s t-test, p = 0.374. (e) Representative confocal images of immunofluorescence of dilp3 protein in control (w1118CS) and Mettl3−/− mutant flies. Scale bar, 20um. Quantification of median dilp3 fluorescence of individual insulin-producing cells from (d), n = 6 brains per genotype. Unpaired two-tailed Student’s t-test, p = 0.333. Error bars are SEM. ns = not significant.

Source data

Extended Data Fig. 3 Reproducibility of biological CLIP replicates.

(a) Correlation plots of log2 normalized reads per CLIP peak. Each dot represents a CLIP peak found in all three biological replicates. Pearson’s correlation coefficient (r). (b) Gene ontology (GO) enrichment analysis of genes that harbor a CLIP peak. Circle size represents the number of genes with CLIP peaks in the corresponding GO categories. The color represents the significance of the enrichment (Benjamini–Hochberg corrected p-value from Gene Set Enrichment Analysis (GSEA)). (c) Metagene plot of CLIP peaks from D. melanogaster head mRNA. Representing the position of 4,506 CLIP peaks. (d) The sequence context of 5,485 cross-linked mutational sites (CIMS) contained within CLIP peaks. (e, f) miCLIP (blue) and input (gray) traces mapped to the dilp3 (e) and dilp5 (f) loci (Reads Per Million mapped reads, RPM).

Source data

Extended Data Fig. 4 Direct RNA sequencing of in vitro transcribed control RNAs and dilp2 RNA.

(a) Schematic of the randomly generated random-1 (rand1) in vitro transcribed RNAs. The RNAs were transcribed with A (gray) and m6A (red). The sequences were identical except for the 6 nt molecular barcode depicted by the green block to distinguish between the unmethylated and methylated RNA unambiguously. (b) Normalized direct RNA sequencing signal derived from in vitro transcribed RNA with A (gray) and with m6A (red). n = 50 reads plotted. Green triangles represent the expected current level based on the base calling mode (see Methods). (c) EpiNano significance trace across the in vitro transcribed RNA sequence. Significant position 239 (red) corresponds to the base following the methylated A (238). Other significant bases labeled ‘barcode’ correspond to the green barcode in (a). (d) Normalized direct-RNA sequencing signal derived from in vitro transcribed dilp2 RNA (left, gray, n = 50 reads) and native dilp2 mRNA from fly heads (right, red n = 50 reads) of the region corresponding to the miCLIP peak in (Fig. 2a). Green triangles represent the expected current level based on the base calling model.

Source data

Extended Data Fig. 5 Creation of dilp2m6A mutant flies.

(a) Diagram of CRISPR strategy to replace 11 AC dinucleotides in 3′ UTR of the dilp2 transcript (dilp2m6A−/− gray). (b) Sanger sequencing of genomic DNA from positive transgenic line 364-4 showing all 11 AC dinucleotides replaced with UC.

Extended Data Fig. 6 Polysome profiles of control and dilp2m6A−/− mutant flies.

(a, b) Representative polysome profile from sucrose gradient of (a) control (w1118) and (b) dilp2m6A−/− mutant fly heads in Fig. 2c. (c) Representative confocal images of immunofluorescence of dilp2 protein in control (w1118CS) and Mettl3−/− mutant flies. Scale bar, 20um. (d) Triglyceride levels normalized to protein in control (w1118) and mutant dilp2m6A−/− flies. n = 8. Error bars SEM. Unpaired two-sided Student’s t-test, p = 0.001. ** p<0.005.

Source data

Extended Data Fig. 7 Vertebrate insulin mRNA is enriched by m6A RIP.

(a, b) Quantification of salmon ins (a) and mouse Ins2 (b) mRNA by qPCR from mock-treated or m6A RIP samples of pancreatic tissue. n = 3 salmon and n = 3 of groups of two male mice. Error bars SEM. Unpaired two-tailed Student’s t-test, p <0.0001 (a) and p = 0.001 (b). (c) Normalized Transcripts Per Kilobase Million (TPM) counts of RNA-sequencing reads from the human insulin (INS) gene6. Paired two-tailed Student′s t-test p <0.0001. *p < 0.05, ** p < 0.005.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Tables 1–4

Supplementary Table 1. Oligonucleotide sequences. DNA sequences used for qPCR primers, Sanger sequencing, CLIP adaptors, and in vitro transcription of control RNAs. Supplementary Table 2. Sequence context of significant direct RNA sequence differences. Sequences are listed for random in vitro transcribed RNA, and regions from native transcripts from each organism tested. Supplementary Table 3. Fly stocks. Supplementary Table 4. Summary of sequencing reads.

Supplementary Data 1

Complete list of peaks from each biological replicate and the union of the data sets. CIMS output of C-to-T transitions.

Source data

Source Data Fig. 1

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 6

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Source Data Extended Data Fig. 7

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Wilinski, D., Dus, M. N6-adenosine methylation controls the translation of insulin mRNA. Nat Struct Mol Biol 30, 1260–1264 (2023). https://doi.org/10.1038/s41594-023-01048-x

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