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Role of genetic and environmentally mediated tRNA fragmentation in monogenic and polygenic diabetes development

par Arroyo, Maria Nicol
Président du jury Deplus, Rachel
Promoteur Igoillo Esteve, Mariana
Publication Non publié, 2024-05-13

Thèse de doctorat

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Résumé :

tRNAs are essential molecules that play an important role in protein translation. They frequently undergo multiple post-transcriptional modifications that are carried out by highly specific tRNA-modifying enzymes in their mature or immature state. These modifications are crucial to preserve cellular function. Loss-of-function mutations in these enzymes have been associated with neurological disorders, mitochondrial diseases and diabetes. Previous reports suggest that pancreatic β-cells are highly sensitive to alterations in tRNA modifications. In 2013, our group was the first to report a new syndrome of young onset diabetes and microcephaly caused by a non-sense mutation in the TRMT10A gene, encoding a tRNA methyltransferase that modifies guanosine in position 9 of particular cytosolic tRNAs. Afterwards, our group also showed that the lack of TRMT10A leads to hypomethylation of its tRNA substrates, fragmentation of the glutamine tRNA (tRNAGln), and that TRMT10A-deficiency sensitizes pancreatic β-cells to apoptosis, increases oxidative stress and activates the intrinsic pathway of apoptosis, probably as a result of mitochondrial dysfunction.Against this background, the main aim of this thesis was to study the impact of TRMT10A deficiency and tRNA fragmentation in pancreatic β-cell development, function, and survival in the context of monogenic and type 1 diabetes (T1D), and to elucidate the mechanisms involved.Using isogenic control and CRISPR-Cas9-mediated TRMT10A-KO induced pluripotent stem cells (iPSC) differentiated into human islet (HI)-like aggregates, in the first section of this thesis we demonstrated that TRMT10A deficiency does not affect β-cell development, in good concordance with the fact that TRMT10A patients do not have neonatal diabetes but develop the disease at a young age. Functional analysis performed on TRMT10A-deficient HI-like aggregates, unveiled altered mitochondrial functionality and reduced insulin secretion in TRMT10A-depleted cells compared to controls, suggesting that increased apoptosis and impaired β-cell function are the main contributors to the development of diabetes in TRMT10A patients. A proteomic analysis performed in TRMT10A-silenced EndoC-βH1 cells revealed dysregulated pathways potentially affecting insulin secretion, mitochondrial function, and cell survival. Moreover, a small RNA sequencing performed in the same model allowed us to identify new tRNA-derived fragments generated under TRMT10A-deficiency that may play an important role in gene and protein expression regulation in pancreatic β-cells. Several of these novel tRFs were validated in our newly developed model of TRMT10A-KO iPSCs.In the second section of the thesis, we studied the role played by TRMT10A deficiency in the development of T1D. T1D is characterized by progressive β-cell demise due to an autoimmune assault. Recent evidence suggests that TRMT10A expression is reduced in -cells from T1D individuals, and that environmental conditions associated with the development of polygenic T1D such as IFNalpha, reduce TRMT10A expression. Here, we confirmed the TRMT10A downregulation by IFNalpha, and showed that Coxsackie virus strain B1 (CVB1) infections, considered as a key environmental factor triggering T1D in genetically predisposed individuals, also reduce TRMT10A levels and enhance tRNA fragmentation in β-cells. These very interesting findings suggest that, besides being a hallmark or a monogenic form of diabetes, TRMT10A deficiency and tRNA fragmentation may also contribute to the pathogenesis of T1D. Experiments of viral infections on TRMT10A-KO iPSC-derived HI-like cells performed in the context of this thesis suggest that viral replication could be impaired by TRMT10A-deficiency and that 18 nucleotides-long tRNA fragments derived from the 5’ end of the tRNAGln may be involved in this regulation. In conclusion, during this thesis we have investigated the repercussions of TRMT10A deficiency on the proteomic and small RNA landscape of pancreatic β-cells, and we have studied the implications of this deficiency in the functionality of β-cells employing a CRISPR-Cas9 TRMT10A-KO iPSC model. Despite normal β-cell development, TRMT10A-deficiency induces apoptosis, oxidative stress, and impaired insulin secretion, emphasizing its role in diabetes pathogenesis. Novel tRFs identified through small RNA sequencing hint at regulatory functions in gene and protein expression. The study also unveils potential connections between TRMT10A deficiency, tRNA fragmentation, and environmental triggers in T1D. This research enhances our understanding of TRMT10A-related diabetes and identifies potential therapeutic avenues for pancreatic β-cell dysfunction.