Genetics and Genomics

Mettl5 coordinates protein production and degradation of PERIOD to regulate sleep in Drosophila

Xiaoyu Wu
Xingzhuo Yang
Tiantian Fu
Yikang S Rong
Juan Du author has email address

Department of Entomology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China
MOE Key Lab of Rare Pediatric Diseases, Hengyang College of Medicine, University of South China, Hengyang, China

https://doi.org/10.7554/eLife.103427.1

Open access
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Figures and data

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 Mettl5^1bp and Mettl5^9bp mutant male flies compared to control flies. (C) Sleep curve throughout the day for Mettl5 mutant male flies and control flies. (D) Total sleep of Mettl5 mutant male flies and control flies. (E) Sleep bout duration of Mettl5 mutant male flies and control flies. (F) Number of sleep bouts of Mettl5 mutant male flies and control flies. (G) Percentage of awake for Mettl5 mutant flies and control flies. (H) Sleep curve throughout the day for Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (I) Total sleep of Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (J) Sleep bout duration of Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (K) Number of sleep bouts of Mettl5 mutant male flies, induced Mettl5 over expression male flies and control flies. (L) Percentage of awake for Mettl5 mutant flies, Mettl5 over expression male flies and control flies. (M) Sleep curve is tracked throughout the entire day prior to sleep deprivation and during the daytime sleep rebound period. (N) Mettl5 mRNA expression level at different time points. W(wake), SD (sleep deprivation), SR (sleep recovery). (O) The daytime sleep recovery during the six hours following sleep deprivation in Mettl5 mutant male flies. For * stands for p<0.05, ** stands for p<0.01, *** stands for p<0.001, ns stands for not significant.

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.

RNA-seq and Ribo-seq analysis revealed changes in the gene profile of Mettl5^1bp.
(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. (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 Mettl5^1bp, while, genes on the right show negative correlation with Mettl5^1bp. 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 Mettl5^1bp (N, O) GO and KEGG enrichment of significantly changed TE-related genes between w¹¹¹⁸ and Mettl5^1bp. The color of the bar indicates the enrichment p.adjust value. (P) KEGG network showing the top 5 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 w¹¹¹⁸and Mettl5^1bp were observed in RNA-seq, Ribo-seq, and translation efficiency analyses. (D-E) PER protein immunofluorescence staining in small LNvs (ventral lateral neurons) and its immunofluorescence intensity statistics. (F-G) Western blot analysis of PER protein and Tubulin protein, along with their grayscale quantification. (H) Sleep curves throughout the day for Mettl5^1bp, CLK^JRK, double mutant and control flies. (I) Total sleep for Mettl5^1bp, CLK^JRK, double mutant and control flies. (J) Percentage of awake time in Mettl5^1bpflies, partially rescued by double mutant flies. (K) Sleep curve throughout the day for Mettl5^1bp, per⁰¹, double mutant and control flies. (L) Total sleep for Mettl5^1bp, per⁰¹, double mutant and control flies. (M) Percentage of awake time in Mettl5^1bp flies, partially rescued by double mutant flies. (N) Fold changes in proteasome subunits with significant expression level differences between w¹¹¹⁸ and Mettl5^1bp 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 Mettl5^1bp
(A-D) Examples of small LNvs axonal terminals labeled by syt-eGFP (green). A rectangular box indicates the location where LNvs axons exit the posterior optic tract and the first axonal bifurcation to the tip of the terminal region, whose volume was measured. (E) Mean volume measurements of small LNvs axonal terminals labeled by syt-eGFP.

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