Abstract Cdk5 regulatory subunit-associated protein 1-like 1 (Cdkal1) encodes a tRNA-modifying enzyme responsible for thiomethylation generating 2-methylthio-N 6-threonylcarbamoyladenosine (ms2t6A) in the anticodon loop of tRNALys UUU. Genome-wide association studies have identified CDKAL1 variants as risk factors for type 2 diabetes mellitus (DM) and chronic kidney disease (CKD), but whether CKD arises independently of diabetes has remained elusive. Here, we demonstrate that CDKAL1 is required for kidney function and that its dysfunction directly promotes CKD progression independently of diabetes. Systemic and podocyte-specific Cdkal1 knockout in mice leads to CKD phenotypes in later adulthood or after increasing the burden on kidney. Cdkal1-knockout podocytes show reduced lysine-codon translation and decreased levels of lysine-rich proteins, including such that are important for podocyte functions, accompanied by impaired cell migration. These adverse effects on podocytes could be partially reversed by overexpressing CD2AP, a lysine-rich protein. These findings extend the concept of ‘tRNA modopathy’ to kidney disease and provide mechanistic insights into how defective tRNA modification contributes to kidney disease progression.

Abstract Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohn’s disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

Reactive oxygen species (ROS) are a pervasive feature of human cancers, yet the protein targets through which ROS-regulated cell states shape tumor biology remains poorly understood. Here, using cysteine chemical proteomics, we define signatures of protein states under distinct cellular ROS environments that capture protein oxidation and conformational changes. Quantifying these signatures in primary lung tumors and brain metastases revealed a surprising enrichment of oxidative states in metastasis. To determine how these states support fitness, we performed genome-wide CRISPR screens and identified the mitochondrial Complex I subunit NDUFA10 as a key oxidation-dependent vulnerability. Oxidation of NDUFA10•Cys253 supports Complex I function through a previously unrecognized nucleotide kinase activity that maintains mitochondrial DNA levels. Enforcing a reduced conformation in NDUFA10 disrupts brain metastatic colonization in vivo . These findings establish ROS regulated protein states as a functional layer of tumor fitness, providing a framework for identifying redox-dependent mechanisms that support cancer progression.

Mitochondrial tRNAs (mt-tRNAs) are central to energy production by translating essential oxidative phosphorylation subunits. Following transcription, mt-tRNAs undergo diverse processing steps, post-transcriptional modifications, and aminoacylation, which are critical for their functions. In this article, we review how human mt-tRNA-modifying enzymes deposit various post-transcriptional modifications onto mt-tRNAs, encompassing both well-characterized and less-understood marks. We also summarize the principles, peculiarities, and critical roles of mt-tRNA charging and proofreading, and highlight recently uncovered noncanonical functions of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Collectively, these recent findings demonstrate the dynamic regulatory mechanisms of mt-tRNA modification and aminoacylation, the extensive involvement of mt-aaRSs in cellular metabolic pathways, and the promising potential of targeting these enzymes in therapeutics.

Cdk5 regulatory associated protein 1-like 1 (CDKAL1) is one of the most reliable risk genes for type 2 diabetes mellitus (T2DM). Because CDKAL1 controls glucose-induced insulin secretion by KATP channel responsiveness and faithful decoding of Lys codons to prevent mistranslation in pancreatic β-cells, a rescuer of CDKAL1 defects is expected as a new antidiabetes drug. We found that eperisone analogs effectively rescued mistranslation in a MiaB-deficient Escherichia coli dual-luciferase reporter gene system (MiaB is a prokaryotic homologue of eukaryotic CDKAL1). Among them, compounds 1f and 1t demonstrated significant antihyperglycemic efficacy in an oral glucose tolerance test by subcutaneous administration in Wister rats, along with a significant enhancement of insulin secretion in the MIN6 insulinoma cell line without cytotoxicity. These results indicate that CDKAL1 could be a viable molecular target for a new anti-T2DM medication.

Transcription of human mitochondrial DNA (mtDNA) begins from specific transcription promoters. In strand-asynchronous mtDNA replication, transcripts from the light-strand promoter serve as primers for leading-strand synthesis at the origin of the H-strand replication (OH). A 7S DNA strand, a presumed aborted replication product, is also synthesized from OH. Transition from RNA synthesis to DNA synthesis at OH is crucial for balancing replication with transcription, yet the mechanism remains unclear. Herein, we examine the role of mitochondrial transcription elongation factor (TEFM) in this process. TEFM knockout results in decreased 7S DNA, strand-asynchronous replication intermediates, and mtDNA copy number, all of which are concordant with downregulation of RNA-to-DNA transition at OH. Conversely, levels of tRNAs encoded near transcription promoters increase, indicating enhanced transcription initiation frequency. Taken together, we propose that, in addition to conferring processivity to the mitochondrial RNA polymerase, TEFM plays a crucial role in maintaining the balance between mitochondrial transcription and replication.

OBJECTIVE:Hepatic steatosis, the early stage of nonalcoholic fatty liver disease (NAFLD), currently lacks targeted pharmacological treatments. G protein-coupled receptors (GPCRs) in hepatocytes differentially regulate lipid metabolism depending on their coupling profile of G protein subtypes. Unlike Gs, Gi, and Gq signaling, the role of G12 signaling in hepatic steatosis remains elusive. The objective of this study was to investigate the effect of G12 signaling on hepatic steatosis and obesity and its mechanisms. METHODS:We generated mice expressing a G12-coupled designer GPCR in a liver-specific manner. We performed phenotypic analysis in the mice under the condition of fasting (acute hepatic steatosis model) or high-fat diet feeding (chronic hepatic steatosis model). RESULTS:In acute and chronic hepatic steatosis models, chemogenetic activation of hepatic G12 signaling suppressed the progression of hepatic steatosis. The treatment led to an increased triglyceride secretion with little effect on mitochondrial respiratory activity, fatty acid oxidation, de novo lipogenesis, and fatty acid uptake. Furthermore, in a high-fat-diet-induced obesity model, activation of the G12-coupled designer GPCR exerted anti-obesity effects with increased whole-body energy expenditure and fat oxidation. Anti-FGF21 antibody treatment showed that the anti-obesity effects of the hepatic G12D activation relied in part on the hepatokine FGF21. CONCLUSIONS:Our findings indicate that the activation of G12 signaling in the liver has the potential to prevent hepatic steatosis and obesity. This discovery provides a strong rationale for the development of drugs targeting G12-coupled GPCRs expressed in the liver.

RNA contains diverse post-transcriptional modifications, and its catabolic breakdown yields numerous modified nucleosides requiring correct processing, but the mechanisms remain unknown. Here, we demonstrate that three RNA-derived modified adenosines, N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), and N6-isopentenyladenosine (i6A), are sequentially metabolized into inosine monophosphate (IMP) to mitigate their intrinsic cytotoxicity. After phosphorylation by adenosine kinase (ADK), they undergo deamination by adenosine deaminase-like (ADAL). In Adal knockout mice, N6-modified adenosine monophosphates (AMPs) accumulate and allosterically inhibit AMP-activated protein kinase (AMPK), dysregulating glucose metabolism. Furthermore, ADK deficiency, linked to human inherited disorders of purine metabolism, elevates levels of the three modified adenosines, resulting in early lethality in mice. Mechanistically, excessive m6A, m6,6A, and i6A impair lysosomal function by interfering with lysosomal membrane proteins, thereby disrupting lipid metabolism and causing cellular toxicity. Through this nucleotide metabolism pathway and mechanism, cells detoxify modified adenosines, linking modified RNA metabolism to human disease.

IntroductionAge-related hearing loss (ARHL) is linked to dementia, with mitochondrial dysfunction playing a key role in its progression. Deficient mitochondrial tRNA modifications impair protein synthesis and energy metabolism, accelerating ARHL. Mitochonic acid 5 (MA-5) has shown promise as a therapeutic candidate by improving mitochondrial function, reducing oxidative stress, and stabilizing membrane potential.MethodsIn this study, we investigated the effects of MA-5 on ARHL in cyclin-dependent kinase 5 regulatory subunit-associated protein 1 (Cdk5rap1) knockout (KO) mice, which exhibit early-onset ARHL due to abnormalities in mitochondrial transfer RNA (mt-tRNA) modifications.ResultsMA-5 treatment effectively attenuated ARHL progression in Cdk5rap1-KO mice by improving auditory brainstem response thresholds and distortion product otoacoustic emissions. It also reduced spiral ganglion and outer hair cell loss, while preserving the cochlear structural integrity by preventing mitochondrial degeneration in spiral ligament fibrocytes. Mechanistically, MA-5 upregulated the expression of silent information regulator sirtuin 1 and promoted the nuclear translocation of yes-associated protein, both of which are involved in regulating mitochondrial function and cellular senescence. Metabolomics analysis further demonstrated that MA-5 restored mitochondrial metabolism, reduced lactate accumulation, and maintained mitochondrial integrity.ConclusionThese findings suggest that MA-5 is a viable treatment option for ARHL and other age-related disorders associated with mitochondrial dysfunction.

Since mitochondrial translation leads to the synthesis of the essential oxidative phosphorylation (OXPHOS) subunits, exhaustive and quantitative delineation of mitoribosome traversal is needed. Here, we developed a variety of high-resolution mitochondrial ribosome profiling derivatives and revealed the intricate regulation of mammalian mitochondrial translation. Harnessing a translation inhibitor, retapamulin, our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript, the elongation rate, and the initiation rate. We also surveyed the impacts of modifications at the anticodon stem loop in mitochondrial tRNAs (mt-tRNAs), including all possible modifications at the 34th position, in cells deleting the corresponding enzymes and derived from patients, as well as in mouse tissues. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed mitochondrial translation initiation factor (mtIF) 3-mediated translation initiation from internal open reading frames (ORFs) and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.

Mitochondrial tRNA taurine modifications mediated by mitochondrial tRNA translation optimization 1 (Mto1) is essential for the mitochondrial protein translation. Mto1 deficiency was shown to induce proteostress in embryonic stem cells. A recent finding that a patient with MTO1 gene mutation showed severe anemia led us to hypothesize that Mto1 dysfunctions may result in defective erythropoiesis. Hematopoietic-specific Mto1 conditional knockout (cKO) mice were embryonic lethal and showed niche-independent defect in erythroblast proliferation and terminal differentiation. Mechanistically, mitochondrial oxidative phosphorylation complexes were severely impaired in the Mto1 cKO fetal liver, and this was followed by cytosolic iron accumulation. Overloaded cytosolic iron promoted heme biosynthesis, which induced an unfolded protein response (UPR) in Mto1 cKO erythroblasts. An iron chelator or UPR inhibitor rescued erythroid terminal differentiation in the Mto1 cKO fetal liver in vitro. This mitochondrial regulation of iron homeostasis revealed the indispensable role of mitochondrial tRNA modification in fetal hematopoiesis.

Human cytoplasmic tRNAs contain dihydrouridine modifications at positions 16 and 17 (D16/D17). The enzyme responsible for D16/D17 formation and its cellular roles remain elusive. Here, we identify DUS1L as the human tRNA D16/D17 writer. DUS1L knockout in the glioblastoma cell lines LNZ308 and U87 causes loss of D16/D17. D formation is reconstituted in vitro using recombinant DUS1L in the presence of NADPH or NADH. DUS1L knockout/overexpression in LNZ308 cells shows that DUS1L supports cell growth. Moreover, higher DUS1L expression in glioma patients is associated with poorer prognosis. Upon vector-mediated DUS1L overexpression in LNZ308 cells, 5' and 3' processing of precursor tRNATyr(GUA) is inhibited, resulting in a reduced mature tRNATyr(GUA) level, reduced translation of the tyrosine codons UAC and UAU, and reduced translational readthrough of the near-cognate stop codons UAA and UAG. Moreover, DUS1L overexpression increases the amounts of several D16/D17-containing tRNAs and total cellular translation. Our study identifies a human dihydrouridine writer, providing the foundation to study its roles in health and disease.

Abstract GBM stands as the most aggressive brain tumor in adults. The median overall survival is less than 2 years despite conventional treatments. Recent advancements in DNA analysis have delineated the molecular characteristics of gliomas, leading to revisions in the WHO classification based on DNA profiles. However, the role of RNA modifications, epitranscriptomics, remains poorly understood in glioma. In this study, we underscore the significance of tRNA modifications targeting adenosine at position 37 in GBM. Transfer RNA isopentenyl transferase 1 (TRIT1) is identified as an isopentenyl transferase catalyzing the conversion of A into i6A at position 37 of tRNA, a nucleotide adjacent to the anticodon region. Additionally, i6A is further modified to 2-methylthio i6A (ms2i6A) by cyclin-dependent kinase 5 regulatory subunit associated-protein 1 (CDK5RAP1) within mitochondrial tRNA. Analysis of TCGA data reveals elevated TRIT1 mRNA expression levels in GBM compared to non-tumor samples. Furthermore, a low expression group of TRIT1 in gliomas demonstrates improved survival outcomes according to data from the CGGA, implicating TRIT1 in glioma progression. Functional assays involving TRIT1 knockdown in human GBM cell lines result in decreased cell viability, indicating its functional significance. Moreover, our data suggest that this phenotype may be mediated through translational control of selenoprotein W (SEPW1) via tRNA^(Ser)Sec modification. GO analysis of TRIT1 and SEPW1 in TCGA data further supports this proposed mechanism. At position 37 of tRNA, we also found that CDK5RAP1 in mitochondrial tRNAs is indispensable for sustaining glioblastoma initiating cell (GIC)-related traits. CDK5RAP1 maintains the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs, independent of translational control of mitochondrial proteins. Notably, CDK5RAP1 abrogates the antitumor effect of i6A by converting i6A to ms2i6A and shields GICs from excessive autophagy triggered by i6A. This work underscores the critical role of tRNA modifications in glioma pathogenesis. We will report these findings alongside RNA modification profile data and clinical data from glioma patients.

SUMMARY RNA contains diverse post-transcriptional modifications and its catabolic breakdown yields numerous modified nucleosides that must be properly processed, but the molecular mechanism is largely unknown. Here, we show that three RNA-derived modified adenosines, N 6 -methyladenosine (m 6 A), N 6 , N 6 -dimethyladenosine (m 6,6 A), and N 6 -isopentenyladenosine (i 6 A), are sequentially metabolized to inosine monophosphate (IMP) to prevent their intrinsic cytotoxicity. These modified adenosines are phosphorylated by adenosine kinase (ADK) followed by adenosine deaminase-like (ADAL)-mediated deamination in both plants and animals. ADAL knockout mice accumulate N 6 -modified AMPs that allosterically inhibit AMP-activated protein kinase (AMPK), leading to dysregulation of glucose metabolism. Furthermore, ADK deficiency, reported in patients with severe metabolic defects, induces aberrant elevation of m 6 A/m 6,6 A/i 6 A, disrupting lipid metabolism and causing early death in mouse models. The findings unveil a fundamental mechanism by which cells alleviate the toxicity of modified adenosines, and that connects modified adenosines to human disease. In brief RNA catabolism yields diverse nucleosides with modifications attached. Three RNA-derived modified adenosines (m 6 A, m 6,6 A, and i 6 A) are intrinsically toxic and subjected to sequential metabolism to yield inosine monophosphate. Dysregulation of this pathway impairs energy balance and contributes to metabolic diseases. Highlights Cytotoxic RNA catabolism-derived modified adenosines undergo sequential metabolism m 6 A, m 6,6 A, and i 6 A are phosphorylated by ADK then deaminated by ADAL to IMP m 6 AMP, m 6,6 AMP, and i 6 AMP allosterically inhibit AMP-mediated AMPK activation Loss of ADK causes the accumulation of m 6 A, m 6,6 A, and i 6 A, and abnormal lipid metabolism, which can lead to genetic disease