ABSTRACT: In this study, we discovered cytosolic and mitochondrial fragments resulting from tRNA and mt-tRNA cleavage, which may act as new regulators of cellular and metabolic functions. We selected the mt-tRF-LeuTAA fragment derived from a tRNA encoded by the mitochondrial genome for further investigation, as its level is reduced in the islets of diabetes-susceptible animal models, while being abundant in ß-cells. mt-tRF-LeuTAA fragment is derived from the cleavage of tRNA-LeuTAA encoded by the mitochondrial genome. We demonstrated that mt-tRF-LeuTAA acts as a key regulator of mitochondrial OXPHOS functions, mitochondrial membrane potential, the insulin secretory capacity of ß-cells, and the insulin sensitivity of myotube muscle cells. We sought to investigate the downstream mechanisms activated by this fragment. To gain a comprehensive understanding, we conducted proteomic analyses on rat islets with silenced mt-tRF-LeuTAA for 72 hours. Inhibiting mt-tRF-LeuTAA led to significant differential expression of 642 proteins, cut-off adjusted p ≤ 0.05. To further investigate the cellular rearrangement associated with the inhibition of mt-tRF-LeuTAA, we conducted enrichment analysis using Gene Ontology Molecular Function terms on mass spectrometry data. At the protein level, there was a significant enrichment of mitochondrial pathways, such as oxidoreductase activity, ATPase activity, hydrogen transport, NADH dehydrogenase activity, cytochrome-c oxidase activity, and oxygen transport. To elucidate the mechanisms by which mt-tRF-LeuTAA operates, we also conducted pull-down experiments in insulin-secreting INS832/13 cells, followed by mass spectrometry using 3'-biotinylated mimic sequence oligonucleotides of mt-tRF-LeuTAA. Our analysis unveiled interactions between mt-tRF-LeuTAA and 24 proteins, meeting the criteria of a fold change ≥ 6 and an adjusted p-value ≤ 0.05. Notably, among these proteins, 13 are localized within the mitochondria and play significant roles in mitochondrial oxidative functions. Some of these key proteins include Suclg2, Nme3, Sdha, Lrpprc, and Ndufa12. Pathway enrichment analysis of the binding partners associated with the mitochondrial fragment mt-tRF-LeuTAA indicates an over-representation of signaling pathways crucial to maintaining mitochondrial metabolism. These pathways include oxidative phosphorylation (OXPHOS), ROS-induced stress responses, the tricarboxylic acid (TCA) cycle, calcium homeostasis regulation, lipid metabolism, RNA splicing, and mitochondrial import, which all contribute fundamentally to the maintenance of mitochondrial metabolism. These findings collectively provide insights into the essential mechanisms underlining the functionality of mitochondrially-encoded tRNA-derived fragments with the view to sustaining mitochondrial metabolism.
Patrice Waridel 
