Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (tRF overexpression). Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (tRF overexpression)

Transfer RNAs (tRNAs) play a central and well recognized role in protein synthesis. Recent studies revealed that these molecules can be cleaved to generate tRNA fragments (tRFs) with regulatory functions. Here, we studied the contribution of tRFs to pancreatic β-cell loss during the initial phases of type 1 diabetes (T1D), an autoimmune disorder characterized by the invation of immune cells in the pancreas and progressive loss of insulin-secreting cells. Small RNA-profiling showed that the pool of tRFs present in pancreatic β-cells is altered in non-obese diabetic (NOD) mice, a mouse model used to study T1D. We found that part of these changes is triggered by the exposure of β-cells to proinflammatory cytokines released during the autoimmune reaction while others result from the direct transfer of tRFs from autoreactive T lymphocytes to insulin-secreting cells via extracellular vesicles. Indeed, using an RNA-tagging approach, we could demonstrate that a group of tRFs are transferred in vivo in from CD4+CD25- T lymphocytes to pancreatic β-cells, upon T cell adoptive transfer in NOD scid mice. Morevoer, the up-regulation of selected tRFs associated with the autoimmune reaction triggers β-cell apoptosis and gene expression changes that affect the immune regulatory capacity of β-cells. Our data point to tRFs as novel players in type 1 diabetes and potentially in other autoimmune disorders. Overall design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. Through an RNA-tagging approach we were able to show both in vitro and in vivo that a fraction of the tRFs displaying elevated levels during insulitis are directly transferred from autoreactive T cells to β-cells via EVs. To gain more insights into the role of the tRFs transferred in β-cells, we performedbulk RNA-seq analysis in mouse islet cells overexpressing mimic oligonucleotides of LysCTT-5’, PheGAA-5’ and SerGCT-3’, for 48h. A scramble sequence of the mimic tRFs was used in the control condition (CT).

转运RNA（transfer RNAs，tRNAs）在蛋白质合成过程中发挥核心且广受认可的关键作用。近期研究显示，这类分子可被切割产生具有调控功能的tRNA片段（tRNA fragments，tRFs）。本研究旨在探究tRFs在1型糖尿病（type 1 diabetes，T1D）发病初期对胰腺β细胞丢失的调控贡献。1型糖尿病是一种自身免疫性疾病，其病理特征为胰腺内免疫细胞浸润以及胰岛素分泌细胞的进行性丢失。小RNA谱分析显示，在用于T1D研究的非肥胖糖尿病（non-obese diabetic，NOD）小鼠模型中，胰腺β细胞内的tRFs库发生了显著改变。研究发现，此类改变部分源于β细胞暴露于自身免疫反应中释放的促炎细胞因子，其余则源自自身反应性T淋巴细胞通过细胞外囊泡（extracellular vesicles，EVs）向胰岛素分泌细胞直接转运tRFs。事实上，通过RNA标记技术，我们证实了在向NOD-scid小鼠进行T细胞过继转移后，一组tRFs可在体内从CD4+CD25- T淋巴细胞转运至胰腺β细胞。此外，与自身免疫反应相关的特定tRFs的上调可诱导β细胞凋亡，并引发影响β细胞免疫调控能力的基因表达改变。本研究数据表明，tRFs是1型糖尿病乃至其他自身免疫性疾病中潜在的新型调控因子。

实验整体设计：以NOD小鼠作为T1D模型，我们在疾病初期的胰岛细胞中检测到了特异性的tRF表达特征。通过RNA标记技术，我们在体外及体内实验中均证实，在胰岛炎阶段表达上调的部分tRFs可通过EVs从自身反应性T细胞直接转运至β细胞。为深入探究转运至β细胞内的tRFs的功能，我们对过表达LysCTT-5’、PheGAA-5’及SerGCT-3’模拟寡核苷酸的小鼠胰岛细胞进行了48小时的批量RNA测序（bulk RNA-seq）分析。实验对照组则使用模拟tRFs的乱序序列（CT）。