Figure 6.

METTL3-dependent m⁶A modification positively regulates NOTCH signaling. (A) The genomic tracks for Notch pathway genes–DLL1, DLL3, NOTCH1, NOTCH2, NOTCH3, JAG2 and HES1 are depicted in both shNT vs. shMETTL3 m⁶A RIP-Seq and RNA-Seq. Normalized sequence coverage of m⁶A RIP peaks and RNA-Seq reads is indicated above the gene architecture in UCSC format. Thin black boxes represent the 5′ and 3′UTRs and thick black boxes represent the coding sequences and thin lines represent introns. (B) HES-1 promoter luciferase activity was measured after silencing (left panel) and over expression of METTL3 (right panel). Luciferase activity was plotted as percentage after normalization to the control cells. Silencing of METTL3 reduced NOTCH activation while over-expression induced the NOTCH pathway. (C) Transcript levels of NOTCH pathway members, which have multiple METTL3-dependent m⁶A RIP-Seq peaks, were measured post-METTL3silencing. Differential expression was measured compared to siNT condition by real-time qPCR and plotted as log₂ ratio. (D) Differential m⁶A enrichment of NOTCH 1, NOTCH 2, NOTCH 3, NOTCH 4 and HES1 genes were measured by m⁶A RIP-PCR after METTL3-silencing and were plotted as log₂ ratio. The enrichment was normalized to siNT condition, (E) Enrichment of NOTCH1 and HES1 genes were measured by METTL3 RIP-PCR and were plotted as log₂. Anti-METTL3 immunoprecipitation (IP) was normalized to IgG control IP. (F) NOTCH1 (top) and HES1 (bottom) transcripts were measured at indicated time points post actinomycin D (5 μg/mL) treatment in siNT and siMETTL3 condition by reverse transcription-qPCR. The log₂ ratio of remaining NOTCH1 was plotted using linear regression after normalizing to the 0th hour of respective condition. (G) Protein levels of full-length NOTCH1 and Hes1 after METTL3 knockdown. The p-value was analyzed by Student’s t-test. *** p-value <0.001, ** p-value <0.01, * p-value <0.05. Error bars represent standard deviation.