m6Am methyltransferase PCIF1 negatively regulates ciliation by inhibiting BICD2 expression

N6, 2 '-O-dimethyladenosine (m(6)Am) is a widespread RNA modification catalyzed by the methyltransferase PCIF1 (phosphorylated CTD interacting factor 1). Despite its prevalence, the biological functions of m(6)Am in RNA remain largely elusive. Here, we report a critical role of PCIF1-dependent m(6)Am RNA modification in ciliogenesis in RPE-1 cells. Our findings demonstrate that PCIF1 acts as a negative regulator of ciliation through its m(6)Am methyltransferase activity. A quantitative proteomic analysis identifies BICD2 as a downstream target of PCIF1, with PCIF1 depletion resulting in a significant increase in BICD2 levels. BICD2 depletion leads to a significant reduction in ciliation. Crucially, the ciliary phenotype in PCIF1-depleted cells is reversed upon BICD2 knockdown. Further investigations reveal that PCIF1 regulates BICD2 protein levels through its m(6)Am catalytic activity, which reduces the stability and translation efficiency of BICD2 mRNA. Single-base resolution LC-MS analysis identifies the m(6)Am site on BICD2 mRNA modified by PCIF1. These findings establish the essential involvement of PCIF1-dependent m(6)Am modification in ciliogenesis.