m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

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N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins1,2,3,4,5,6,7. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir)8,9,10,11,12. In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl)13. However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain3. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models5,7,14, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5′ untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.

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Figure 1: Analysis of Ime4 null mutants and m6A methylation in Drosophila.
Figure 2: m6A methylation is required for Sxl alternative splicing in sex determination and dosage compensation.
Figure 3: Ime4 co-localizes to sites of transcription.
Figure 4: YTH protein YT521-B decodes m6A methylation in Sxl.

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We thank J. Horabin, N. Perrimon and the Bloomington, Harvard and Kyoto stock centres for fly lines, BacPAc for DNA clones, E. Zaharieva and M. L. Li for help with imaging, W. Arlt and R. Michell for comments on the manuscript, and J.-Y. Roignant for communication of results before publication. We acknowledge funding from the BBSRC (BB/M008606/1) to R.F.

Author information

I.U.H. and M.S. performed biochemistry, cell biology and genetic experiments, E.S.M. stained chromosomes, and Z.B., N.A. and R.F. performed biochemistry experiments. N.M. analysed sequencing data. I.U.H., R.F. and M.S. conceived the project and wrote the manuscript with help from N.M. and Z.B.

Correspondence to Matthias Soller.

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Extended data figures and tables

Extended Data Figure 1 m6A levels in unfertilized eggs.

a, b, Thin-layer chromatography from maternal total RNA (a) and mRNA (b) present in unfertilized eggs. The arrow indicates m6A.

Extended Data Figure 2 Ime4 supports Sxl in directing germline differentiation.

ac, Representative ovarioles of wild-type (a), Ime4null/Ime4null (b) and Sxl/+;Ime4null/+ females (c), and a tumerous ovary of a Sxl/+;Ime4null/+ female (d). The tumorous ovary consisting mostly of undifferentiated germ cells in d is indicated with a bracket and the oviduct with an asterisk. Scale bar, 100 μm (applies to all panels).

Extended Data Figure 3 Ime4 is required for female-specific splicing of Sxl, tra and msl-2.

ac, RT–PCR of Sxl (a), tra (b) and msl-2 (c) sex-specific splicing in wild-type males and females, and Ime4null males and females. 100-bp markers are shown on the left. AS, alternative splicing.

Extended Data Figure 4 Alternative splicing of sex-determination genes and differential expression of X-linked genes in Ime4null females.

ac, Sashimi plot depicting Tophat-mapped RNA sequencing reads and exon junction reads below the annotated gene model for sex-specific alternative splicing of tra, fru and dsx. The thickness of lines connecting splice junctions corresponds to the number of junction reads also shown. ss, splice site. d, Significantly (P < 0.05, q < 0.166853) differentially expressed gene expression values expressed as reads per kb of transcript per million mapped reads (RPKM) were log[x + 1]-transformed and Spearman r correlation values determined for X-linked and autosomal genes in wild-type and Ime4null Drosophila. e, The proportion of autosomal and X-linked genes that were significantly either up- or downregulated in Ime4null as compared to wild-type Drosophila were statistically compared using χ2 with Yates’ continuity correction. GraphPad Prism was used for statistical comparisons. Similar results as for the single-read RNA-seq experiment were obtained for the paired-end RNA sequencing experiment.

Extended Data Figure 5 m6A methylation sites map to the vicinity of Sxl binding sites.

a, Schematic of the Sxl alternatively spliced intron around the male-specific exon depicting substrate RNAs used for in vitro m6A methylation. Solid lines depict fragments containing m6A methylation and dashed lines indicate fragments where m6A was absent. b, c, 1D-TLC of in vitro methylated [32P]-ATP-labelled substrate RNAs shown in a. Markers are in vitro transcripts in the absence (M1) or presence (M2) of m6A 32P-labelled after RNase T1 digestion. The right panels in b and c show an overexposure of the same thin-layer chromatography.

Extended Data Figure 6 RT–PCR validation of differential alternative splicing in Ime4null flies.

af, Sashimi plots depicting Tophat-mapped RNA sequencing reads and exon junction reads below the annotated gene model of indicated genes on the left, and RT–PCR of alternative splicing shown on the right using primers depicted on top. The thickness of lines connecting splice junctions corresponds to the number of junction reads also shown.

Extended Data Figure 7 Ime4 affects alternative splicing predominantly in 5′ UTRs in genes with a higher than average number of upstream start codons.

a, b, Classification of differential alternative splicing in Ime4null according to splicing event (a) and location of the event in the mRNA (b). c, Quantification of upstream start codons (AUGs) in all annotated 5′ UTRs (white) or in alternative isoforms differentially spliced between wild-type and Ime4null insects. All Drosophila UTRs were accessed in fasta format from Flybase (version r6.07), (ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/current/fasta/). An R script was used to count the number of ATG sequences in all Drosophila 5′ UTRs and from the genes identified by the Spanki analysis comprising 638 5′ UTRs. A t-test was then used to statistically compare the number of ATGs present in the 638 5′ UTRs of the differentially spliced genes as compared to all 29,822 Drosophila 5′ UTRs. d, e, Classification of differentially alternatively spliced genes in Ime4null according to expression pattern (d) or function (e).

Extended Data Figure 8 Drosophila S2 cells are male.

RT–PCR of Sxl alternative splicing in females, males and S2 cells. 100-bp markers are shown on the left.

Extended Data Figure 9 Preferential binding of the YTH domain of YT521-B to m6A-containing RNA.

a, Coomassie-stained gel depicting the recombinant YTH domain (amino acids 207–423) of YT521-B. b, c, Electrophoretic mobility shift assay of YTH domain binding to Sxl RNA fragment C with or without m6A (50% of adenosine in the transcript methylated) and quantification of RNA bound to the YTH domain shown as mean ± s.e.m. (n = 3). Note that the YTH domain does not form a stable complex with RNA (asterisk) and that this complex falls apart during the run or forms aggregates in the well. d, UV cross-linking of the YTH domain to Sxl RNA fragment C at 0.25 μM, 1 μM, 4 μM and 16 μM (lanes 1–4).

Extended Data Figure 10 YT521-B co-localizes to sites of transcription.

ad, Polytene chromosomes from salivary glands expressing YT521-B::HA stained with anti-Pol II (red, b), anti-HA (green, c) and DNA (DAPI, blue, d), or merged (yellow, a). Scale bars, 5 μm.

Supplementary information

Supplementary Information

This file contains graphical source data, uncropped gels, Western blots and 1D TLCs. (PDF 960 kb)

Supplementary Table 1

This file contains the alternative splicing analysis. (XLS 168 kb)

Supplementary Table 2

This file contains the differential gene expression analysis. (XLS 154 kb)

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Haussmann, I., Bodi, Z., Sanchez-Moran, E. et al. m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature 540, 301–304 (2016) doi:10.1038/nature20577

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