miCLIP in METTL14 R298P mutation knock-in cell clones

N6-methyladenosine (m6A) is the major type of RNA modi'cation that regulates RNA stability and gene expression, characterized by a conserved motif including 5'-(m6A)C-3'. However, the functional significance of the specific motif for m6A modification is unclear. Here, we focused on a single amino acid substitution in the m6A 'writer' complex, METTL14 R298P, and created mutation knock-in cells. m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) revealed that METTL14R298P/R298P but not METTL14WT/R298P cells had an aberrant sequence motif, 5'-(m6A)[C/T]-3'. This study reveals a mechanism underlying sequence-specific m6A modification. Overall design: miCLIP analysis was performed in METTL14 R298P mutation knock-in cell clones. METTL14 R298P mutation knock-in cell clones were established from HEC108 cell line. miCLIP analysis was performed on poly(A)-purified mRNA in HEC108_ki042 clone (METTL14+/+), HEC108_ki004 clone (METTL14+/R298P), and HEC108_ki047 clone (METTL14 R298P/R298P).