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

Mitochondrial RNA N1-methyladenosine (m¹A) is a prevalent and reversible epitranscriptomic modification. While the biological roles of cytosolic m¹A have been increasingly understood, the causal relationship between site-specific mitochondrial m¹A and phenotypic outcomes remain elusive, partly due to the lack of precise editing tools. Here, a CRISPR-free mitochondrial RNA m¹A demethylation (MRD) editor is reported, which fuses mitochondria-localized engineered PUF RNA-binding protein with the m¹A demethylase ALKBH3. Independent cellular assays across multiple sites confirm that MRD editor enables precise demethylation of m¹A 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 m¹A 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 m¹A 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 m¹A editing, offering new insights into the functional dissection of these modifications through chemical biology strategies.