Silencing of mt-tRF-LeuTAA fragment in pancreatic rat islets and investigation by RNA-sequencing of the downstream mechanisms activated by this fragment

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 for further investigation, as its level is altered in the islets 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. To gain a comprehensive understanding, we conducted transcriptomic and proteomic analyses on rat islets with silenced mt-tRF-LeuTAA for 72 hours. We transfected rat islet cells with an antisense oligonucleotides (antisense oligonucleotides to reduce mt-tRF-LeuTAA levels versus control oligos). These oligos specifically decreased the levels of the fragment (anti-tRF-LeuTAA) by more than 95% without affecting the host full-length tRNA mt-tRNA-LeuTAA. Inhibiting mt-tRF-LeuTAA led to significant differential expression of 4843 mRNA transcripts, 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 RNA-sequencing. At the transcriptomic level, we observed enrichment of pathways related to ion transport and various enzymatic activities, including mitochondrial oxidoreductase activity, when the mitochondrial fragment was repressed in rat islets. In order to characterize the gene expression profile of rat islet cells upon mt-tRF-LeuTAA inhibition, we transfected dispersed rat islet cells for 72hrs with antisense oligonucleotides to reduce mt-tRF-LeuTAA levels (anti-tRF-LeuTAAn n=6) versus control oligos (anti-control, n=6). Afterwards we isolated total RNA and performed RNA-sequencing.