Cap-Specific m(6)Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m(6)A Methyltransferase Activity.

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent N(6),2'-O-dimethyladenosine (m(6)Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered N(6) methyltransferase PCIF1. However, its role in gene expression remains unclear due to conflicting reports on its effects on mRNA stability and translation. In this study, we investigated the impact of siRNA-mediated transient suppression of PCIF1 on global mRNA expression in HeLa cells. We identified a subset of differentially expressed genes (DEGs) that exhibited minimal overlap with previously reported DEGs. Subsequent validation revealed that PCIF1 positively and negatively regulates RAB23 and CNOT6 expression, respectively, at both the mRNA and protein levels. Mechanistic analyses demonstrated that PCIF1 regulates the stability of these target mRNAs rather than their transcription, and rescue experiments confirmed the requirement of PCIF1's methyltransferase activity for these regulations. Furthermore, MeRIP-qPCR analysis showed that PCIF1 suppression significantly reduced the m(6)A levels of RAB23 and CNOT6 mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m(6)A modification, providing new insights into the role of m(6)Am in the regulation of gene expression.