Fig. 6: DHX36 facilitates YTHDF1 binding to decrease mRNA stability on selected targets.

A Genome tracks showing RNA abundance in chromatin fraction and whole cell, DHX36 binding sites, m⁶A sites, YTHDF1 binding sites, and SHAPE reactivity of the DHX36 binding sites in mRNAs EZR, MEPCE, PHF23, and ZNF768. B The folded structures of the DHX36 binding sites within the above mRNAs in WT and DHX36-KO cells. m⁶A and YTHDF1 binding sites are highlighted using yellow lines. Nucleotides are color-coded based on the SHAPE reactivity scores. C The abundances of the above mRNAs in WT and DHX36-KO cells were quantified by RT-qPCR (n = 3 biological replicates). D mRNA stability of the above mRNAs was determined by quantifying the mRNA abundance 0, 4, and 8 h after actinomycin D treatment (n = 3 biological replicates). E Schematic illustration showing the construction of the EGFP reporters. F The stability of the EGFP mRNAs fused with DHX36 3’UTR binding site of each selected mRNA was determined by quantifying the EGFP mRNA abundance 0, 2, and 6 h after actinomycin D treatment (n = 3 biological replicates). G The half-life of the above mRNAs in WT and YTHDF1-KO. H RIP assay was performed in WT and DHX36-KO cells with a YTHDF1 antibody. The enrichment of the immunoprecipitated YTHDF1 and GAPDH proteins was assessed by Western blotting. IgG was used as a negative control. I The enrichment of the representative mRNAs in the above RIP was detected by RT-qPCR. The relative enrichment was normalized by input (n = 3 biological replicates). J Western blot confirmed the unaltered protein levels of YTHDF1 in WT and DHX36-KO cells. Data are presented as mean values ± SD in (C, D, F, G, I). The statistical significances in (C, D, F, G, I) were calculated by a two-sided Student’s t-test. Source data are provided as a Source Data file.