Fig. 4: METTL1 promotes miR-760 processing in an m⁷G-dependent manner.

A Schematic of how to identify downstream targets. B Volcano plot showed differentially expressed miRNAs upon METTL1 depletion from miRNA-seq. (|log₂FC| > 1 and P value < 0.05) 53 miRNAs downregulated (blue) and 44 miRNAs upregulated (red). C Heatmap showed 53 downregulated miRNA arranged by log₂FC from smallest. D KEGG enrichment analysis of differentially expressed miRNAs upon METTL1 depletion from miRNA-seq. E Venn diagram showed the overlap of downregulated miRNAs (log₂FC < −1 and P value < 0.05) in UM-UC3 cells upon the depletion of METTL1 from miRNA-seq and miRNAs downregulated by METTL1 knocked down in A549 cells from MeRIP-seq. F RT-qPCR assay showed that downregulated expression of METTL1 led to decreased expression of miR-760 and upregulated expression of METTL1 led to increased expression of miR-760. U6 was used for the normalization control. The average of three independent biological replicates ±SDs is shown (*P < 0.05, ***P < 0.001). G Dot blot assay showing downregulation of METTL1 led to decreased m⁷G level and upregulated expression of METTL1 led to increased m⁷G level. H Dot blot assay showed m⁷G level in total RNA was unchanged by pMETTL1 with catalytic center inactivation (c.i.). I RT-qPCR assay showed the expression of miR-760 was unchanged by pMETTL1 with catalytic center inactivation (c.i.). U6 was used for the normalization control. The average of three independent biological replicates ±SDs is shown. J RIP-RT-qPCR assay showed pri-miR-760 was enriched by METTL1 antibody indicating METTL1 bond to pri-miR-760 in T24 and UM-UC3 cells. The average of three independent immunoprecipitation reactions ±SDs is shown (*P < 0.05). K MeRIP-RT-qPCR assay showed the decreased pri-miR-760 and pre-miR-760 enriched by m⁷G antibody upon the METTL1 depletion, suggesting the direct methylation of METTL1 in UM-UC3. The average of three independent immunoprecipitation reactions ±SDs is shown (**P < 0.01).