Mettl3-dependent m6A modification attenuates the brain stress response in Drosophila

N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain’s susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5′UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain’s biological response to stress.


Supplementary Figure 1 Validation of m 6 A knockdown
a Dot blot of m 6 A on polyA+ RNA levels of basal and 30 min HS from heads of the eya allele pinhead (which is eyeless). These flies have reduced eye and head tissue. n =1 biological replicate, 200 heads per replicate per condition.
Source data and statistical analysis are provided as a Source Data file.

Supplementary Figure 2 The brain has a distinct stress response
a Time course analysis of Hsp70 protein levels in basal, HS 38.5 °C (HS) for 30 min, HS 30 min with 6 h recovery, HS 30 min with 24 h recovery from control fly (BL5905) brains, heads, and whole fly tissue. Tubulin used as the loading control. n=3 biological replicates per tissue type, 15 brains or heads per replicate, 6 whole flies per replicate. Data are presented as mean ± SD, **p<0.01; ****p<0.0001; one-way ANOVA with Dunnett's test across tissue type, compared to basal. For brain p=0.0053, p<0.0001, for head p<0.0001, p<0.0001, for whole fly p<0.0001, p<0.0001.
b Hsp70 protein levels in dissected brain, head, or head tissue with brain removed (outer cuticle) in basal conditions. No significant changes in Hsp70 protein per tissue type. n= 2 biological replicates, 15 flies per replicate. Data are presented as mean ± SD, one-way ANOVA with Tukey's test.
c Hsp70 protein levels in dissected brain, head, or head tissue with brain removed (outer cuticle) after HS 30 min. Significant increase in Hsp70 levels in heads and heads minus brains compared to brain tissue. n= 2 biological replicates, 15 flies per replicate. Data are presented as mean ± SD, *p<0.05; **p<0.01; one-way ANOVA with Tukey's test.
d-e Time course analysis of DnaJ-1 (HSP-40) and stv (BAG3) protein levels in basal, HS 30 min at 38.5 °C, HS 30 min and 6 h rec, HS 30 min and 24 h rec from brains, heads, and whole fly tissue. n=3 biological replicates per tissue type, 15 brains or heads per replicate, 6 whole flies per replicate. Data are presented as mean ± SD *p<0.05; **p<0.01; one-way ANOVA with Dunnett's test across tissue type. For DnaJ-1, for brain ns= not significant, for head p= 0.0059, for whole fly p= 0.0207. For stv, for brain ns= not significant, for head p= 0.0353, for whole fly p= 0.0126, p=0.0056.
f-h RT-qPCR analysis of Hsp70, DnaJ-1, and stv RNA levels from brains, heads, and whole fly tissue at basal, HS 30 min, HS 30 min + 6 h rec. RNA levels relative to basal no stress, normalized to b-tubulin. For Hsp70 and DnaJ-1 n=6 biological replicates per tissue type for control and HS conditions and n=4 biological replicates for 6 h rec, for stv n=3 biological replicates for all conditions. 15 brains or heads per replicate, 6 whole flies per replicate. Data are presented as mean ± SD, *p<0.05, ****p<0.0001; one-way ANOVA with Tukey's test across tissue type. For Hsp70, p<0.0001, for DnaJ-1 p<0.0001, for stv p<0.0001, p= 0.0344.
Source data and statistical analysis are provided as a Source Data file. b Mettl3 ΔCatalytic domain mutant crossed to BL5905 (ΔCat/+) or BL5905 (+/+) were HS for 1.5 hour at 38.5 °C and scored for survival after 24 h recovery. n=4 biological replicates, each data point represents percent survival in vial of 20 flies per replicate. Data are presented as mean ± SD, **p<0.05, student's t-test. p= 0.002. c Mettl3 protein levels from control BL5905 brains dissected in basal, HS 30 min at 38.5 °C, HS + 6 h recovery, or HS + 24 h recovery. n= 3 biological replicates, 15 brains per replicate. Quantification of biological replicate immunoblots showed no significant change in Mettl3 protein levels with HS. Data are presented as mean ± SD, one-way ANOVA, ns=not significant.
Source data and statistical analysis are provided as a Source Data file.

Supplementary Figure 4 m 6 A-IP seq using two antibodies (NEB and SYS).
a Schematic diagram of the m 6 A-IP sequencing protocol and experimental design (Genotype: DaGal4> mCherry RNAi; DaGal4> Mettl3 RNAi).
b Overlap of genes with m 6 A peaks from NEB and SYS m 6 A -IP in basal conditions and HS 30 min conditions. c De novo motif discovery from both antibody IP experiments (NEB and SYS), and from Mettl3-dependent m 6 A genes. Full Motif analysis and p-values found in Supplementary Data 1.
d Example genome browser tracks of m 6 A genes that show increase m6A upon HS: csw, pnt, RasGAP1, cic in basal and HS 30 min from control and Mettl3 RNAi brains. The yellow box highlights the Mettl3-dependent m 6 A peak in the 5'UTR.
f Overlap of m 6 A peaks from head tissue m 6 A-IP (SYS antibody, this study) with miCLIPseq m 6 A peaks from head tissue (SYS) 39 .
g Overlap of m 6 A-IP determined m 6 A peaks from head tissue (SYS antibody, this study) with miCLIP-seq from S2 cells (SYS) 40 .
h Genome wide correlation of log(m6A/input) read coverage comparing rep 1 to rep 2 of m 6 A-IP seq experiments from SYS antibody. Pearson correlation shown for each comparison (left). Comparison of genes with m 6 A peaks called from each individual replicate m 6 A-IP seq in each condition (right). For MetPeak and RADAR peak calling both replicates are taken into account and call peaks/ differential peaks common to both reps.
Source data and statistical analysis are provided as a Source Data file.

Supplementary Figure 5 Analysis of m 6 A genes
a-b GO term and KEGG pathway enrichment of all genes expressed in the brain that are not m 6 A modified. The -log10(pval) enrichment of genes in each category is shown, as well as the number of genes that fall into each category. Full Go-term list and p-values provided in Supplementary Data 3.
Source data and statistical analysis are provided as a Source Data file.