Endocrine Abstracts

Background and aim: Loss-of-function mutations in TRMT10A, a transfer RNA (tRNA) methyltransferase, cause early onset diabetes and microcephaly. tRNAs play a crucial role in cellular homeostasis and post-transcriptional modifications modulate tRNA function and fragmentation. tRNA-derived halves (tiRNAs, 29-50 nt) and fragments (tRFs, 14-30 nt) are a new class of functional small noncoding RNAs, involved in cellular stress responses. Here we set out to investigate the molecular mechanisms underlying β-cell demise in TRMT10A deficiency.

Methods: Fibroblasts from a TRMT10A-deficient patient and 2 healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs were differentiated into β- like cells using a 7-stage protocol. TRMT10A expression was silenced in human insulinproducing EndoC-βH1 cells by siRNAs. Reactive oxygen species (ROS) were measured using HPF fluorescent probe and mitochondrial function was assessed by Seahorse. qRT-PCR was used to detect guanine-9 methylation (m1G9) in tRNAs. tRNA fragmentation was assessed by Northern blot and qRT-PCR. Synthetic tRNA fragments and tRF inhibitors were transfected by lipofection. Apoptosis was examined by nuclear dyes, Western blot and immunocytochemistry.

Results: iPSCs from controls and TRMT10A diabetic patients were successfully differentiated into β-like cells. The β-like cells expressed insulin mRNA at levels comparable to EndoC-βH1 cells and human islets. In iPSC-β-like cells and TRMT10A-depleted EndoC-βH1 cells (≥70% knockdown, P<0.001) m1G9 methylation was reduced in a subset of cytosolic tRNAs, including tRNAGln (P<0.05, n=6–12). Hypomethylation of tRNAGln resulted in fragmentation and increased 5’-tiRNAGln and 5’-tRFGln in patient-derived cells (1.5±0.5 fold increase vs controls, P<0.05 n=3–6). Transfection of TRMT10A-competent EndoC-βH1 cells with synthetic 5’-tiRNAGln and 5’-tRFGln induced apoptosis. Conversely, transfection of antisense oligonucleotides targeting 5’-tiRNAGln and 5’-tRFGln protected TRMT10A-deficient β-cells from apoptosis (23±2% apoptosis in TRMT10A-silenced cells vs 17±2% following antisense transfection, P<0.05). TRMT10A deficiency induced oxidative stress and mitochondrial dysfunction in β-cells (P<0.05, n=5), triggering the intrinsic pathway of apoptosis. The ROS scavengers Tiron (25 μM) and NAC (1 mM) protected TRMT10Adeficient β-cells from apoptosis (20±2% apoptosis without NAC vs 14±2% with NAC, P<0.05, n=4).

Conclusion: Establishing a novel experimental model based on patient’s iPSC-derived primary β cells, we demonstrated that TRMT10A deficiency induces β-cell demise via oxidative stress, mitochondrial dysfunction and activation of the intrinsic pathway of apoptosis. TRMT10A deficiency leads to hypomethylation and fragmentation of tRNAs, and the 5’-tRNAGln fragments are key mediators of β-cell death. These observations provide unequivocal evidence for the importance of tRNA modifications in human pancreatic β-cells and identify tRNA hypomethylation and fragmentation as a novel mechanism of β-cell demise in human diabetes.