A reduction in the level of reduced m
6A modification results in diminished binding affinity for reader proteins, which affects various biological processes such as mRNA export, stability, and translation efficiency. To investigate the molecular mechanisms underlying retinal degeneration after
Mettl3 deletion, we performed proteomic analysis on 2.5-month-old control and RKO mice, which started to exhibit retinal degeneration (Fig. S7). A total of 133 up-regulated proteins and 136 down-regulated proteins were identified in RKO mice compared with controls (foldchange >1.5 or < 1/1.5;
P < 0.05) (
Fig. 6A). The top 30 significant differentially expressed proteins (DEPs) are shown as radar plots (
Fig. 6B). Gene ontology (GO) enrichment analysis of the 269 differentially expressed genes revealed that the majority of these genes are associated with photoreceptor outer and inner segments, as well as visual perception pathways (
Fig. 6C, highlighted in red). By integrating the genes highlighted in the pathways, we identified a total of 15 genes closely related to photoreceptor function (
Fig. 6C, bottom). The reference DEPs mapped to KEGG enrichment analysis pathways, including several key genes within photoreceptors, was illustrated (
Fig. 6D). Using previous m
6A sequencing data in
Mettl14 rod specific knockout mice,
35 we compared genes with downregulated m
6A modification with genes downregulated in protein expression after
Mettl3 knockout and identified a group of 32 genes. Similarly, we found five intersecting genes between downregulated m
6A-modified genes and protein upregulated after
Mettl3 knockout (
Fig. 6E). Further analysis revealed that all 15 genes identified in
Figure 6C are among the 32 genes downregulated in m
6A modification and downregulated in protein expression after
Mettl3 knockout (
Fig. 6E). A majority of these 15 target genes of METTL3 overlapped with 18 target genes of METTL14 as previously identified,
35 and the overlapped 10 genes (
Rho, Prph2, Pde6b, Gnat1, Guca1b, Arl3, Unc119, RGS9, Rgs9bp, Rs1) were assigned as the most probable METTL3 target genes (
Fig. 6F). Heatmap analysis of these 10 selected genes showed a consistent downregulation trend in both mRNA levels in METTL14 transcriptome sequencing and protein levels in METTL3 proteome sequencing (
Fig. 6G).