The landscape of mitochondrial transfer RNA post-transcriptional modifications reveals GTPBP3-responsive mo⁵U in...

Mitochondrial dysfunction is central to acute ischemic stroke (AIS), but the contribution of mitochondrial transfer RNA (mt-tRNA) post-transcriptional modification changes to ischemic stress remains insufficiently defined. Therefore, a mass-spectrometry-based workflow was utilized to generate an exploratory map of mt-tRNA post-transcriptional modifications in AIS models and to functionally evaluate a candidate GTPBP3/5-methoxyuridine (mo⁵U) axis in OGD/R-injured PC12 cells.

Chemical labeling-assisted liquid chromatography-tandem mass spectrometry was applied to mt-tRNAs obtained both in vivo and vitro models of AIS. Candidate modification-associated nucleosides and RNase digestion fragments were profiled. To test the functional relevance of GTPBP3, PC12 cells were transfected with empty pcDNA vector or pcDNA-GTPBP3, followed by measurement of GTPBP3 abundance, mo⁵U signal, cell viability, ROS, mitochondrial membrane potential (MMP), and ATP production.

This method achieved over 60% sequence coverage and uncovered 38 distinct modification types spanning 237 nucleotide positions. Compared with prior studies (18 modifications at 137 sites), 20 previously unannotated modification types were identified, including novel sites within underexplored variable loop regions. Remarkably, 15.33% of all nucleotide positions were modified. Both the A[mo⁵U]AGp digestion fragment from the anticodon loop of mt-tRNA^Ile(GAU) and the mo⁵U modification level were significantly reduced both in vitro and in vivo AIS models, accompanied by decreased GTPBP3 abundance. Curcumin treatment in PC12 cells partially restored cell viability, lowered ROS, improved MMP, and was associated with recovery of GTPBP3 abundance and A[mo5U]AGp/mo5U-related signals. Importantly, pcDNA-GTPBP3 overexpression increased GTPBP3 abundance and mo5U levels, and significantly attenuated OGD/R-induced loss of cell viability, ATP depletion, ROS accumulation, and MMP dissipation.

These findings provide a landscape of mammalian mt-tRNA modifications and identify candidate epitranscriptomic changes associated with AIS. Specifically, we highlight a possible GTPBP3-dependent mo5U axis in ischemia-induced mitochondrial dysfunction, which represents a promising neuroprotective target warranting further functional validation.