Site-Specific Mitochondrial RNA N1-Methyladenosine Demethylation via an Engineered MTS-PUF-ALKBH3 Fusion Protein.

Mitochondrial RNA N1-methyladenosine (m(1)A) is a prevalent and reversible epitranscriptomic modification. While the biological roles of cytosolic m(1)A have been increasingly understood, the causal relationship between site-specific mitochondrial m(1)A and phenotypic outcomes remain elusive, partly due to the lack of precise editing tools. Here, a CRISPR-free mitochondrial RNA m(1)A demethylation (MRD) editor is reported, which fuses mitochondria-localized engineered PUF RNA-binding protein with the m(1)A demethylase ALKBH3. Independent cellular assays across multiple sites confirm that MRD editor enables precise demethylation of m(1)A in mitochondrial mRNAs and tRNAs, leading to correlated changes in mitochondrial protein levels with minimal off-target effects. The MRD editor is further employed to systematically investigate how site-specific mitochondrial m(1)A alterations regulate cell proliferation, ATP production, mitochondrial membrane potential (MMP), and mitochondrial respiration. Finally, in vivo application of the MRD editor reveals that demethylation of m(1)A at the A9 position of mitochondrial tRNA-Lys (MT-TK9) induces severe immunodeficiency phenotypes in mice, as evidenced by transcriptomic and histopathological analyses. Collectively, the findings establish MRD as a versatile tool for site-specific mitochondrial RNA m(1)A editing, offering new insights into the functional dissection of these modifications through chemical biology strategies.