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.本研究中，我们发现了由转运RNA（transfer RNA, tRNA）和线粒体转运RNA（mitochondrial tRNA, mt-tRNA）切割产生的胞质与线粒体片段，这类片段可作为细胞功能与代谢功能的新型调控因子。我们选取线粒体转运RNA衍生片段（mitochondrial tRNA-derived fragment, mt-tRF）-LeuTAA开展后续研究，因该片段在糖尿病易感动物模型的胰岛中表达水平发生显著改变，且在β细胞中富集。mt-tRF-LeuTAA片段源自线粒体基因组编码的tRNA-LeuTAA的切割产物。我们证实，mt-tRF-LeuTAA是线粒体氧化磷酸化（oxidative phosphorylation, OXPHOS）功能、线粒体膜电位、β细胞胰岛素分泌能力以及肌管细胞胰岛素敏感性的关键调控因子。为全面解析其功能机制，我们对沉默mt-tRF-LeuTAA达72小时的大鼠胰岛开展了转录组与蛋白质组学分析。我们向大鼠胰岛细胞转染反义寡核苷酸（antisense oligonucleotides, ASO）以敲低mt-tRF-LeuTAA的表达水平，同时设置转染对照寡核苷酸的对照组。该类寡核苷酸可特异性降低该片段（anti-tRF-LeuTAA）的表达水平达95%以上，且不会影响其宿主全长tRNA mt-tRNA-LeuTAA的表达。抑制mt-tRF-LeuTAA可导致4843个mRNA转录本出现显著差异表达，筛选阈值为校正后P值≤0.05。为进一步探究抑制mt-tRF-LeuTAA所介导的细胞重编程过程，我们基于RNA测序（RNA sequencing）数据，采用基因本体（Gene Ontology, GO）分子功能术语开展富集分析。在转录组层面，当大鼠胰岛中的该线粒体片段被抑制时，我们观察到与离子转运及多种酶活性（包括线粒体氧化还原酶活性）相关的通路出现显著富集。