Mbd5 preferentially binds to m5C-modified mRNAs and regulates target transcript abundance. (A) Schematics showing the analyses of nucleic acids crosslinked to Mbd5 by UV irradiation. (B) Immunoblotting showing the detection of biotinylated nucleic acids crosslinked to Mbd5 upon RNase or DNase treatments. The arrow points to the Mbd5 protein band. The two bands detected in the RNase A-treated and RNase A- & DNase I-treated samples represent endogenous biotinylated proteins. (C) Quantification of m5C abundance in immunoprecipitated Mbd5-bound RNA. As a control, Mbd5 does not enrich m6A-modified RNA. (D) EMSA assay analyzing binding preference of purified recombinant Mbd5-GFP to RNA oligos (top) or dsDNA (bottom) end-labeled with Alexa Fluor 594 dye. (E) Enrichment of RNA m5C signal on Mbd5 binding sites. Density plots were generated by plotHeatmap function provided by deeptools package, using the Mbd5 CLIP-seq bigWig files and bed files from the published RNA m5C bisulfite sequencing (44). (F) Scatter plot showing the differentially expressed genes (left) and Mbd5 direct targets (right) upon mbd5 MO treatment in zebrafish larvae (76 hpf). P values were determined by DESeq2 algorithm with a Wald test and were corrected for multiple testing using the Benjamini and Hochberg method. (G) Boxplot showing the average m5C modification fractions of downregulated (Down) or upregulated (Up) genes in the 76 hpf morphants. **** P < 0.0001. Student's t-test. (H) Heatmaps showing the number of Mbd5 binding peaks revealed by CLIP-seq (#Mbd5 peaks) and changes in RNA abundance upon mbd5 MO treatment (Log2 fold change of RNA) on selective genes important in autism and erythrocyte development. (I) IGV tracks showing the binding of Mbd5 to selective genes (hbae3, hbbe1.1, hbbe2).
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