Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

Background and aims: Transfer RNA (tRNA) modifications determine translation fidelity and efficiency. It occurs through the action of specific enzymes that modify the nucleotides within the tRNA molecule. Our previous study demonstrated tRNA modopathies and altered queuine-related metabolites in inflammatory bowel diseases. Queuine tRNA-ribosyltransferase catalytic subunit 1 (QTRT1) and QTRT 2 co-localize in mitochondria and form a heterodimeric TGT participating in tRNA Queuosine (tRNA-Q) modification. Human body acquires Queuine/Vitamin Q from intestinal microbiota or from diet. However, the roles of tRNA-Q modifications in the maintenance of intestinal mitochondrial homeostasis and microbiome are still unclear. Methods: We used publicly available human IBD datasets, QTRT1 knockout (KO) mice, cultured cell lines with QTRT1-specific siRNA, and organoids from patients with IBD to investigate the mechanism of tRNA-Q modifications in intestinal mitochondrial homeostasis and therapeutic potential in anti-inflammation. Results: Using publicly available datasets, we identified significantly changes of Vitamin Q-associated bacteria in human IBD, compared to the healthy control. Q-tRNA modification is regulated by QTRT1. In single cell RNA sequencing datasets of human IBD, we identified significant reduced intestinal epithelial QTRT1 expression in the patients with IBD. Qtrt1-/- mice had significant reduction of Q-associated bacteria, e.g., Bacteroides. Intestinal tight junction integrity was impaired in QTRT1-KO mice, as evidenced by reduced ZO-1 and increased Claudin-2 expression. ATP synthesis was significantly decreased in the colon of QTRT1-KO mice, accompanied by severe mitochondrial dysfunction: reduced mitochondrial quality, Cytochrome-C release, and mitochondrial DNA (mtDNA) leakage. Mitochondrial dysfunction contributed to colonic cell death, as shown by elevated expressions of Cleaved Caspase-3 and Cleaved Caspase-1, increased BAX/Bcl-2 ratio, and positive TUNEL signals. Furthermore, Alcian Blue and Mucin-2 staining revealed mucosal barrier damage and disrupted homeostasis, with reduced colonic cell proliferation. Elevated CDC42, CD14, and CD4 levels in QTRT1-KO colon suggested mucosal immune activation and tissue repair responses. QTRT1-deficient CaCO2-BBE cells showed mitochondrial dysfunction. Cytochrome-C and mito-DNA release leading to cell death characterized by elevated expressions of Cleaved Caspase-3 and Caspase-1, increased BAX/Bcl-2 ratio, and higher apoptosis rate. Organoids isolated from patients with IBD showed reduced levels of QTRT1 and dysfunctional mitochondria. Restoring mitochondrial function leads to enhanced QTRT1. Conclusions: These findings underscore the critical role of QTRT1/Q-tRNA modification in maintaining mitochondrial function, mucosal integrity, and microbial homeostasis. QTRT1 deficiency impairs barrier integrity, and induces mitochondrial dysfunction, apoptosis, and dysbiosis, Mechanistically, QTRT1 loss impacts tRNA modification in the intestine, linking to mitochondrial integrity and mucosal homeostasis. Our study highlights the novel roles of tRNA-Q modification in maintaining mucosal barriers and innate immunity in intestinal health.