The worldwide prevalence of type 2 diabetes (T2D), which is caused by a combination of environmental and genetic factors, is increasing. With regard to genetic factors, variations in the gene encoding Cdk5 regulatory associated protein 1–like 1 (Cdkal1) have been associated with an impaired insulin response and increased risk of T2D across different ethnic populations, but the molecular function of this protein has not been characterized. Here, we show that Cdkal1 is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-N⁶-threonylcarbamoyladenosine (ms²t⁶A) in tRNA^Lys(UUU) and that it is required for the accurate translation of AAA and AAG codons. Mice with pancreatic β cell–specific KO of Cdkal1 (referred to herein as β cell KO mice) showed pancreatic islet hypertrophy, a decrease in insulin secretion, and impaired blood glucose control. In Cdkal1-deficient β cells, misreading of Lys codon in proinsulin occurred, resulting in a reduction of glucose-stimulated proinsulin synthesis. Moreover, expression of ER stress–related genes was upregulated in these cells, and abnormally structured ER was observed. Further, the β cell KO mice were hypersensitive to high fat diet–induced ER stress. These findings suggest that glucose-stimulated translation of proinsulin may require fully modified tRNA^Lys(UUU), which could potentially explain the molecular pathogenesis of T2D in patients carrying cdkal1 risk alleles.

TRNA molecules are post‐transcriptionally modified by tRNA modification enzymes. Although composed of different chemistries, more than 40 types of human tRNA modifications play pivotal roles in protein synthesis by regulating tRNA structure and stability as well as decoding genetic information on mRNA. Many tRNA modifications are conserved among all three kingdoms of life, and aberrations in various human tRNA modification enzymes cause life‐threatening diseases. Here, we describe the class of diseases and disorders caused by aberrations in tRNA modifications as ‘tRNA modopathies’. Aberrations in over 50 tRNA modification enzymes are associated with tRNA modopathies, which most frequently manifest as dysfunctions of the brain and/or kidney, mitochondrial diseases, and cancer. However, the molecular mechanisms that link aberrant tRNA modifications to human diseases are largely unknown. In this review, we provide a comprehensive compilation of human tRNA modification functions, tRNA modification enzyme genes, and tRNA modopathies, and we summarize the elucidated pathogenic mechanisms underlying several tRNA modopathies. We will also discuss important questions that need to be addressed in order to understand the molecular pathogenesis of tRNA modopathies.

A subset of mitochondrial tRNAs (mt-tRNAs) contains taurine-derived modifications at 34U of the anticodon. Loss of taurine modification has been linked to the development of mitochondrial diseases, but the molecular mechanism is still unclear. Here, we showed that taurine modification is catalyzed by mitochondrial optimization 1 (Mto1) in mammals. Mto1 deficiency severely impaired mitochondrial translation and respiratory activity. Moreover, Mto1-deficient cells exhibited abnormal mitochondrial morphology owing to aberrant trafficking of nuclear DNA-encoded mitochondrial proteins, including Opa1. The mistargeted proteins were aggregated and misfolded in the cytoplasm, which induced cytotoxic unfolded protein response. Importantly, application of chemical chaperones successfully suppressed cytotoxicity by reducing protein misfolding and increasing functional mitochondrial proteins in Mto1-deficient cells and mice. Thus, our results demonstrate the essential role of taurine modification in mitochondrial translation and reveal an intrinsic protein homeostasis network between the mitochondria and cytosol, which has therapeutic potential for mitochondrial diseases.

Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m⁵C) at positions 48–50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m⁵C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m⁵C at positions 48–50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m⁵C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

N6-Methyladenosine (m6A) modification is the major chemical modification in mRNA that controls fundamental biological processes, including cell proliferation. Herein, we demonstrate that fat mass and obesity-associated (FTO) demethylates m6A modification of cyclin D1, the key regulator for G1 phase progression and controls cell proliferation in vitro and in vivo. FTO depletion upregulates cyclin D1 m6A modification, which in turn accelerates the degradation of cyclin D1 mRNA, leading to the impairment of G1 progression. m6A modification of cyclin D1 oscillates in a cell-cycle-dependent manner; m6A levels are suppressed during the G1 phase and enhanced during other phases. Low m6A levels during G1 are associated with the nuclear translocation of FTO from the cytosol. Furthermore, nucleocytoplasmic shuttling of FTO is regulated by casein kinase II-mediated phosphorylation of FTO. Our results highlight the role of m6A in regulating cyclin D1 mRNA stability and add another layer of complexity to cell-cycle regulation.