A cohort of mRNAs undergo high-stoichiometry NSUN6-mediated site-specific m5C modification

mRNA modifications are vital in regulating cellular processes. Beyond N6-methyladenosine (m⁶A), most other internal mRNA modifications lack dedicated catalytic machinery and are typically introduced by tRNA-modifying enzymes. The distribution and stoichiometry of these modifications on mRNAs remain debated and require further validation. Furthermore, their precise function remains controversial due to the challenges of excluding the intricate combinational effects of tRNA modifications. Here, we biochemically validate that NSUN6, a tRNA structure-dependent methyltransferase, independently catalyzes 5-methylcytidine (m⁵C) formation with robust activity on mRNA by recognizing the CUCCA motif in a certain stem-loop structure. NSUN6 employs different strategies to recognize tRNA and mRNA substrates. By introducing mutations, we further separate its catalytic capabilities toward mRNA and tRNA revealing that NSUN6 promotes breast cancer cell migration depending on mRNA m⁵C modification. Mechanistically, a cohort of mRNAs involved in cell migration carries high levels of NSUN6-mediated site-specific m⁵C modification, thus being stabilized by the preferential binding of m⁵C readers YBX1 and YBX3. Moreover, introducing a single-site high-level m⁵C can significantly increase the stability of therapeutic mRNAs in cells. Our findings underscore the pivotal role of m⁵C-modified mRNAs in promoting breast cancer cell migration and their potential for therapeutic applications.