Abstract

N 6 ,2’-O-dimethyladenosine (m 6 Am) is a prevalent RNA modification located at the first transcribed nucleotide adjacent to the 5′ cap of mRNAs, where it has been implicated in gene regulation. However, the lack of methods for precise, transcript-specific manipulation of m 6 Am has limited its functional dissection. Here, we develop a programmable RNA-editing platform, termed T argeted m 6 Am M ethylation (TAmM), that enables site-specific installation of m 6 Am on selected cellular transcripts. TAmM is engineered by fusing the catalytically inactive RfxCas13d (dCasRx) with the m 6 Am methyltransferase PCIF1, allowing guided deposition of m 6 Am at cap-proximal nucleotides. Using TAmM, we achieve efficient and specific m 6 Am installation at single-nucleotide resolution, as validated by LC-MS/MS. Targeted m 6 Am editing does not alter steady-state mRNA abundance but modulates protein output in a transcript-dependent manner. Mechanistically, m 6 Am installation enhances polysome association, indicating a direct role in translational regulation. Functional interrogation demonstrates that cap-proximal m 6 Am deposition on CTNNB1 increases β -catenin protein expression, promoting cell proliferation, clonogenicity, and migration in cancer cell models. Importantly, TAmM exhibits high fidelity, with negligible effects on the global m 6 Am landscape, transcriptome, or proteome. Our study establishes TAmM as a precise and versatile platform for programmable m 6 Am manipulation and reveals transcript-specific roles of m 6 Am in gene regulation.