Figure 5: Recruitment of CCR4–NOT through CNOT1 is essential for the degradation of endogenous m⁶A-containing RNAs.

(a) Deadenylation assay of BG-PLAC2 and BG-PLAC2-mut on knocking down of endogenous METTL3 or CNOT1 by siRNA. siNC served as a negative control. (b) Deadenylation assay of endogenous ACTB mRNA on knocking down of endogenous METTL3, CNOT1 or CAF1 by siRNA. (c) Deadenylation assay of BG-1boxB mRNA in the presence of λN-FLAG-YTHDF2 on knocking down of endogenous CNOT1 by siRNA. (d–f) Schematic diagrams of YTHDF2-mediated deadenylation of m⁶A-containing RNAs and the dominant-negative effect of YTHDF2-C or CNOT1-SH. m⁶A is recognized by YTHDF2, which further recruits the CCR4–NOT complex by interacting with CNOT1 (d). Overexpressed YTHDF2-C occupies m⁶A sites but is incapable of recruiting the CNOT1 subunit of the CCR4–NOT complex; thus, it impairs the deadenylation and decay of m⁶A-containing RNAs (e). Overexpressed CNOT1-SH binds to endogenous YTHDF2 but is incapable of recruiting the catalytic subunits CAF1 and CCR4A/B; thus, it impairs the deadenylation and decay of m⁶A-containing RNAs (f). (g) Expression level of endogenous target mRNAs measured by quantitative reverse transcriptase–PCR on overexpression of YTHDF2-C, CNOT1-SH or the negative control green fluorescent protein (EGFP). All values were normalized to HPRT1 mRNA, a housekeeping gene previously reported to contain no m⁶A modifications and is not bound by YTHDF2. Error bars, mean±s.d., n=3, biological replicates. **P<0.01, *P<0.05, t-test. (h–j) Half-life of FBXL19, ZBTB7B and HPRT1 mRNA on overexpression of YTHDF2-C, CNOT1-SH or the negative control EGFP after transcription inhibition (TI). All values were normalized to 18S rRNA. Error bars, mean±s.d., n=3, biological replicates.