Cap-Specific m6Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m6A Methyltransferase Activity

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent <i>N</i>⁶,2'-<i>O</i>-dimethyladenosine (m⁶Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered <i>N</i>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 m⁶A levels of <i>RAB23</i> and <i>CNOT6</i> mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m⁶A modification, providing new insights into the role of m⁶Am in the regulation of gene expression.