Mettl5 is a regulator of Drosophila sleep.

(A) Diagram illustrating CRISPR-Cas knockout of 1 or 9 bases in the Mettl5 gene. (B) Relative expression of Mettl5 mRNA in homozygous Mettl51bp and Mettl59bp mutant male flies compared to control flies. (C) Sleep curve throughout the day for Mettl5 mutant male flies (blue) and control flies (black). (D) Total sleep of Mettl5 mutant male flies and control flies in 24 hours. (E) Total sleep of Mettl5 mutant male flies and control flies within day and night, respectively. (F) Sleep bout duration of Mettl5 mutant male flies and control flies. (G) Number of sleep bouts of Mettl5 mutant male flies and control flies. (H) Percentage of awake for Mettl5 mutant flies and control flies. (I) Sleep curve is tracked throughout the entire day prior to sleep deprivation and during the daytime sleep rebound period. (J) Mettl5 mRNA expression level at different time points. W(wake), SD (sleep deprivation), SR (sleep recovery). (K) Sleep curve is tracked throughout the entire day prior to sleep deprivation and during the daytime sleep rebound period in Mettl5 mutant male flies (blue) and control flies (black). (L) Response to sleep deprivation and performance measures in Mettl5 mutants and controls. Black bars represent the amount of sleep lost during the 24-hour sleep deprivation period, blue bars indicate the amount of sleep regained, whereas the red bars indicate the proportion of sleep recovered (right y-axis). (M) Sleep arousal of Mettl51bp male flies and control flies at ZT19. (N) Sleep curve throughout the day for the following genotypes: w1118(black), Mettl51bp/+ (blue), and Mettl5-Gal4, UAS-Mettl5, Mettl51bp/+(pink). (O) Total sleep of the indicated genotypes. (P) Sleep bout duration of the indicated genotypes. (Q) Number of sleep bouts of the indicated genotypes. (R) Percentage of awake for the indicated genotypes. For * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant. For letter-based annotations, Groups with no significant differences share the same letter, Groups with significant differences are assigned new letters.

Mettl5 regulation of Drosophila sleep was dependent on its methyltransferase activity.

(A) The m6A level in the total RNA of Mettl5 mutant male flies. (B) The m6A level in the 18S rRNA of Mettl5 mutant male flies. (C) The sleep curve throughout the day shows the sleep pattern of induced Trmt112 RNAi male flies and control flies. (D) Total sleep of induced Trmt112 RNAi male flies and control flies. (E) Sleep bout duration in induced Trmt112 RNAi male flies and control flies. (F) Number of sleep bouts in induced Trmt112 RNAi male flies and control flies. (G) Percentage of awake in Trmt112 RNAi and control flies. (H) Sleep curve throughout the day for Mettl5 mutant male flies, induced Mettl5m over expression male flies and control flies. (I) Total sleep of Mettl5 mutant male flies, induced Mettl5m over expression male flies and control flies. (J) Sleep bout duration in Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (K) Number of sleep bouts in Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (L) Percentage of awake in Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. For * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant. For letter-based annotations, Groups with no significant differences share the same letter, Groups with significant differences are assigned new letters.

RNA-seq and Ribo-seq analysis revealed changes in the gene profile of Mettl51bp.

(A) Venn diagram depicting the number of significant differentially expressed genes revealed by RNA-seq and Ribo-seq. (B) Volcano plot representing the differentially expressed genes identified by RNA-seq. Genes that met the criteria of |log2(fold-change)| ≥ 1 and p.adjust < 0.05 were considered significantly expressed, marked in orange for downregulation and green for upregulation, comparing with the controls. (C) Volcano plot representing the differentially expressed genes identified by Ribo-seq. Candidates that satisfied the criteria of |log 2 (fold-change) | ≥ 0.265 and p.adjust <0.05 were regarded as significantly expressed, marked in red for downregulation and blue for upregulation, respectively. (D-K) Gene set enrichment analysis of differentially expressed genes revealed by RNA-seq (D-G) and Ribo-seq (H-K). All the plots are generated using the KEGG gene set database. The bar chart at the bottom of each panel shows the distribution of target genes for each pathway according to their rank position. Each vertical line represents a gene. Genes on the left show positive correlation with Mettl51bp, while, genes on the right show negative correlation with Mettl51bp. The green line indicates the enrichment score (ES), and NES stands for normalized enrichment score. (L) Distribution of the differentially expressed genes revealed by both RNA-seq and Ribo-seq. (M) Cumulative distribution of TE frequencies among w1118 and Mettl51bp (N, O) GO and KEGG enrichment of significantly changed TE-related genes between w1118and Mettl51bp. The color of the bar indicates the enrichment p.adjust value. (P) KEGG network showing the top 10 pathway and associated genes. The size of the dots represents the number of genes in the pathway.

Clock genes expression mediated the sleep phenotype caused by Mettl5 mutation.

(A-C) Fold changes in clock genes with significant expression level differences between w1118 and Mettl51bp were observed in RNA-seq, Ribo-seq, and translation efficiency analyses. (D) The gene expression levels of per at 4 different timepoints of w1118 and Mettl51bp. (E) Western blot analysis of PER protein and Tubulin protein at 4 different timepoints, (F) along with their ratio quantification. (G) Representative image of PER protein immunofluorescence staining in the small ventral lateral neurons (small LNvs). (H) Statistical analysis of the immunofluorescence intensity for PER in small LNvs. (I) Sleep curve throughout the day for Mettl51bp, per01, double mutant and control flies. (J) Total sleep for Mettl51bp, per01, double mutant and control flies. (K) Percentage of awake time in Mettl51bpflies, partially rescued by double mutant flies. (L) Sleep curve throughout the day for Mettl51bp, per01, double mutant and control flies. (M) Total sleep for Mettl51bp, per01, double mutant and control flies. (N) Percentage of awake time in Mettl51bp flies, partially rescued by double mutant flies. (O) Fold changes in proteasome subunits with significant expression level differences between w1118 and Mettl51bp were observed in RNA-seq, Ribo-seq, and translation efficiency analyses. For statistical significance, * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant.

Circadian rhythm phenotypes of various mutants.

A working model illustrating the role of Mettl5 in Drosophila sleep was presented.

The axon complexity was found to be affected by Mettl51bp

(A–C) Representative confocal micrographs of small ventral lateral neuron (s-LNv) axonal terminals across different genotypes. Presynaptic structures were visualized using syt-eGFP (green), which colocalizes with endogenous synaptic vesicles: (A) control (B) Fmr1 overexpression and (C) Fmr1 null mutant. (D) Quantification of s-LNv axonal terminal volumes corresponding to genotypes in (A– C). (E–H) Representative images of axonal terminal morphology in control flies at four time points: ZT0, ZT6, ZT12, and ZT18. (I–L) Representative images of axonal terminal morphology in Mettl5 mutant flies at ZT0, ZT6, ZT12, and ZT18. (M) Quantitative comparison of axonal terminal volumes between control and Mettl5 mutant flies at different time points. Scale bar: 50 μm. For statistical significance, * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant.

Mettl5 functions in a portion of neurons and glia.

(A-C) Colocalization of Mettl5-YFP with antibodies staining ELAV. Scale bar: 50 μm (main figures) and 20 μm (zoomed regions) (D-F) Colocalization of Mettl5-YFP with antibodies staining REPO. Scale bar: 50 μm (main figures) and 20 μm (zoomed regions). (G) Sleep latency. (H) Climbing index of Mettl51bp male flies and control flies. (I) Sleep curves throughout the day for Mettl5-RNAi and control flies. (J) Total sleep for Mettl5-RNAi and control flies flies. (K) Sleep bout duration of Mettl5-RNAi and control flies. (L)Number of sleep bouts of Mettl5-RNAi and control flies. For statistical significance, * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant

Quality control of RNA-seq and Ribo-seq samples.

(A, D) Meta analysis of gene expression data obtained from RNA-seq and Ribo-seq. (B, E) Principal component analysis (PCA) of different samples for RNA-seq and Ribo-seq. (C, F) Correlation heatmap of normalized RNA and RPFs values between every pair of samples. The color and size of dots represent the Pearson correlation coefficient.

Heatmap of differentially expressed genes (DEGs) between Mettl51bp and w1118.

Heatmap showing the top 100 significantly changed genes identified by RNA-seq (A) and Ribo-seq (B) in Mettl51bp. Red indicates increased expression, while blue indicates decreased expression.

Enrichment analysis of differential expressed genes between Mettl51bp and w1118.

(A-D) GO analysis performed on the differentially expressed genes revealed by RNA-seq (A, C) and Ribo-seq (B, D). The bubble chart shows the top 15 most significantly enriched categories under the themes of biological processes, cellular components and molecular functions. The circle color indicates the enrichment p.adjust, and the dot size indicates the number of differentially expressed genes in the functional class or pathway.

Simplified GO enrichment analysis for differentially expressed genes in RNA-seq and Ribo-seq.

GO enrichments for biological process (A, D), cellular components (B, E), and molecular function (C, F) were shown. The bottom right clusters, which lack word cloud annotations, include all other small clusters with fewer than 5 terms. The color shades represent the similarity of GO terms based on binary cut clustering.

Gene set enrichment analysis (GSEA) for differentially expressed genes found in RNA-seq and Ribo-seq.

GSEA were performed on the differentially expressed genes identified in RNA-seq (A, B) and Ribo-seq (C, D). The top 5 terms or pathways found in GSEA based on GO (A, C) and KEGG (B, D) databases are shown. Genes were sorted according to their signed NES values. The circle color indicates the enrichment p.adjust, and the dot size indicates the number of differentially expressed genes in the respective functional class or pathway.

GO and KEGG enrichment analysis of genes that were significantly changed at both transcriptional and translational levels (showed in figure3L).

(A, B) Top 10 GO and KEGG terms for genes with the same direction of change at transcriptional and translational levels. (C, D) Top 10 GO and KEGG terms for genes with the opposite direction of change at transcriptional and translational levels. (E, F) Top 10 GO terms for genes that were only changed at the transcriptional level; (G, H) Top 10 GO terms for genes that were only changed at the translational level. The circle color indicates the enrichment p.adjust, and the dot size indicates the number of differentially expressed genes in the respective functional class or pathway. Changes with |log 2 (fold change) |≥ 0.265 of translational levels and |log2(fold change) |≥ 1 of transcriptional levels were considered significant.

TE correlation and P-site offsets

(A-F) TE correlation between the three biological replicates of two group showing high reproducibility. Different colors represent the density distribution of different translated genes. (G-L) Metagene analysis of individual 28nt reads mapped to their 5’ ends to determine P-site offsets. The distribution of 28nt RPF reads around the ribosomal P site in w1118 (G-I) and Mettl5 (J-L). The horizontal axis represents the distance from the start-codon or stop-codon, and the vertical axis represents the read counts. The P-site positions are colored according to the reading frame.

Global scan of RPFs distribution

(A-F) Distribution of RPFs periodicity among translation start codons and translation end codons in w1118(A-C) and Mettl51bp (D-F). (G-L) Distribution of RPFs on different coding frames, including frame 0, frame 1 and frame 2, for the 5’UTR, CDS and 3’UTR in w1118 (G-I) and Mettl51bp (J-L). The plot illustrates the distribution of ribosome-protected fragments (RPFs) across different coding frames (frame 0, frame 1, and frame 2) on the x-axis, with the y-axis representing the percentage of RPFs in each coding frame.

Ribo-seq has revealed that the Mettl51bp leads to changes in global translation features.

(A) The distribution of RPF length among different samples in Mettl51bp and w1118 was compared. (B) A comparison was made between Mettl51bp and w1118 regarding the statistics on active translated ORFs. The distribution of different types of ORFs based on their relative location to associated coding sequences (CDS) was illustrated in the comparison between Mettl51bp and w1118. The categories include "annotated" (ORFs that overlap with annotated CDS and have the same stop codon as the annotated CDS), "uORF" (ORFs located upstream of annotated CDS, not overlapping annotated CDS), "dORF" (ORFs located downstream of annotated CDS, not overlapping annotated CDS), "Overlap_uORF" (ORFs located upstream of annotated CDS, overlapping annotated CDS), "Overlap_dORF" (ORFs located downstream of annotated CDS, overlapping annotated CDS), "Internal" (ORFs located within annotated CDS but in a different frame relative to the annotated CDS), and "novel" (ORFs located in non-coding genes or non-coding transcripts of coding genes). The percentages of each ORF category were compared between Mettl51bp and w1118, providing insights into the differences in ORF distribution between the two analyzed samples. (C) A comparison was made between Mettl51bp and w1118 regarding the length of translated and untranslated uORFs. (D) The count of dORFs was compared between Mettl51bp and w1118. (E) The count of translated uORFs was compared between Mettl51bp and w1118. (F, G) Motif analysis was performed separately on the translated and untranslated uORFs between w1118 and Mettl51bp. (H) The correlation of codon occupancy (A-site) between Mettl51bp and w1118 was analyzed. (I-L) The cumulative changes in RPFs for codon UCC (I), GAC (J), GAU (K) and amino acid ASP (L) were compared between Mettl51bpand w1118. The statistical analysis was performed using the Kolmogorov-Smirnov test (KS test). (M-O) A metagene plot was generated for RPFs on whole CDS region and around the CDS start and end regions in group case study. The CDS was divided into 100 equal bins.

Morphology of clock neurons in Mettl5 mutant.

Ventral lateral neurons labeled by PDF staining in Mettl51bp and control flies across different time points.