Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Supersulfide biology and translational medicine for disease control.

For decades, the major focus of redox biology has been oxygen, the most abundant element on Earth. Molecular oxygen functions as the final electron acceptor in the mitochondrial respiratory chain, contributing to energy production in aerobic organisms. In addition, oxygen-derived reactive oxygen species including hydrogen peroxide and nitrogen free radicals, such as superoxide, hydroxyl radical and nitric oxide radical, undergo a complicated sequence of electron transfer reactions with other biomolecules, which lead to their modified physiological functions and diverse biological and pathophysiological consequences (e.g. oxidative stress). What is now evident is that oxygen accounts for only a small number of redox reactions in organisms and knowledge of biological redox reactions is still quite limited. This article reviews a new aspects of redox biology which is governed by redox-active sulfur-containing molecules-supersulfides. We define the term 'supersulfides' as sulfur species with catenated sulfur atoms. Supersulfides were determined to be abundant in all organisms, but their redox biological properties have remained largely unexplored. In fact, the unique chemical properties of supersulfides permit them to be readily ionized or radicalized, thereby allowing supersulfides to actively participate in redox reactions and antioxidant responses in cells. Accumulating evidence has demonstrated that supersulfides are indispensable for fundamental biological processes such as energy production, nucleic acid metabolism, protein translation and others. Moreover, manipulation of supersulfide levels was beneficial for pathogenesis of various diseases. Thus, supersulfide biology has opened a new era of disease control that includes potential applications to clinical diagnosis, prevention and therapeutics of diseases.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction.

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.

Mitochondrial alterations in the cochlea of Cdk5rap1-knockout mice with age-related hearing loss.

Previous studies have revealed that age-related hearing loss (AHL) in Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1)-knockout mice is associated with pathology in the cochlea. Here, we aimed to identify mitochondrial alterations in the cochlea of Cdk5rap1-knockout mice with AHL. Mitochondria in the spiral ganglion neurons (SGNs) and hair cells (HCs) were normal despite senescence; however, the mitochondria of types I, II, and IV spiral ligament fibrocytes were ballooned, damaged, and ballooned, respectively, in the stria vascularis. Our results suggest that the accumulation of dysfunctional mitochondria in the lateral wall, rather than the loss of HCs and SGNs, leads to the onset of AHL. Our results provide valuable information regarding the underlying mechanisms of AHL and the relationship between aberrant tRNA modification-induced hearing loss and mitochondrial dysfunction.

Epitranscriptomics in metabolic disease.

While epigenetic modifications of DNA and histones play main roles in gene transcription regulation, recently discovered post-transcriptional RNA modifications, known as epitranscriptomic modifications, have been found to have a profound impact on gene expression by regulating RNA stability, localization and decoding efficiency. Importantly, genetic variations or environmental perturbations of epitranscriptome modifiers (that is, writers, erasers and readers) are associated with obesity and metabolic diseases, such as type 2 diabetes. The epitranscriptome is closely coupled to epigenetic signalling, adding complexity to our understanding of gene expression in both health and disease. Moreover, the epitranscriptome in the parental generation can affect organismal phenotypes in the next generation. In this Review, we discuss the relationship between epitranscriptomic modifications and metabolic diseases, their relationship with the epigenome and possible therapeutic strategies.

NSUN3-mediated mitochondrial tRNA 5-formylcytidine modification is essential for embryonic development and respiratory complexes in mice.

In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.

CDKAL1 Drives the Maintenance of Cancer Stem-Like Cells by Assembling the eIF4F Translation Initiation Complex.

Cancer stem-like cells (CSCs) have a unique translation mode, but little is understood about the process of elongation, especially the contribution of tRNA modifications to the maintenance of CSCs properties. Here, it is reported that, contrary to the initial aim, a tRNA-modifying methylthiotransferase CDKAL1 promotes CSC-factor SALL2 synthesis by assembling the eIF4F translation initiation complex. CDKAL1 expression is upregulated in patients with worse prognoses and is essential for maintaining CSCs in rhabdomyosarcoma (RMS) and common cancers. Translatome analysis reveals that a group of mRNAs whose translation is CDKAL1-dependent contains cytosine-rich sequences in the 5' untranslated region (5'UTR). Mechanistically, CDKAL1 promotes the translation of such mRNAs by organizing the eIF4F translation initiation complex. This complex formation does not require the enzyme activity of CDKAL1 but requires only the NH2 -terminus domain of CDKAL1. Furthermore, sites in CDKAL1 essential for forming the eIF4F complex are identified and discovered candidate inhibitors of CDKAL1-dependent translation.

p21-activated kinase 1 (PAK1) as a therapeutic target for cardiotoxicity.

The p21-activated kinase 1 (PAK1), an effector protein of the small G protein Rac and cell division cycle protein 42 (Cdc42), is highly expressed in cardiac tissue. Although a large number of studies have explored the molecular basis and biological function of PAK1, research on PAK1 as a therapeutic target for cardiotoxicity remains in a stage of continuous innovation, and further clarification of its role in cardiotoxicity is required. In this review, we examine the important role of PAK1 in the programmed death (apoptosis, autophagy, and pyroptosis) of cardiomyocytes, and its involvement in oxidative stress and inflammatory responses, which are based on mitochondrial dysfunction and calcium homeostasis imbalance. We also summarize the related signaling pathways through which PAK1 may cause oxidative stress and inflammatory response in cardiotoxicity, and discuss the PAK1-mediated contributions of the gut microbiome and micro RNAs to cardiotoxicity. We propose that PAK1 holds great promise for novel therapeutic strategies to facilitate improvements in the treatment of complex and diverse cardiovascular diseases.

t6A and ms2t6A Modified Nucleosides in Serum and Urine as Strong Candidate Biomarkers of COVID-19 Infection and Severity.

SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.

Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation.

Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.

Extracellular N6 -isopentenyladenosine (i6A) addition induces cotranscriptional i6A incorporation into ribosomal RNAs.

N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Subtilase cytotoxin from Shiga-toxigenic Escherichia coli impairs the inflammasome and exacerbates enteropathogenic bacterial infection.

Subtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1ß and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-ß/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.

Protocol for preparation and measurement of intracellular and extracellular modified RNA using liquid chromatography-mass spectrometry.

About 150 modifications have been identified in RNA species. Besides their regulatory roles in the intracellular gene expression, abundant modified RNA nucleosides are catabolized from RNA and released into extracellular fluids, which can impact extracellular signaling as ligands for receptors. Here, we describe a protocol to prepare samples from biological specimens, including cultured cells, extracellular fluid, and tissues, to measure both intracellular and extracellular RNA modifications using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Ogawa et al. (2021).

Cooperative methylation of human tRNA3Lys at positions A58 and U54 drives the early and late steps of HIV-1 replication.

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2'-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.

Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

Cdk5 regulatory subunit-associated protein 1 knockout mice show hearing loss phenotypically similar to age-related hearing loss.

Mitochondrial dysfunction is associated with aging and age-related hearing loss (AHL). However, the precise mechanisms underlying the pathophysiology of hearing loss remain unclear. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) enables efficient intramitochondrial translation by catalyzing the deposition of 2-methylthio modifications on mitochondrial tRNAs. Here we investigated the effect of defective mitochondrial protein translation on hearing and AHL in a Cdk5rap1 deficiency C57BL/6 mouse model. Compared to control C57BL/6 mice, Cdk5rap1-knockout female mice displayed hearing loss phenotypically similar to AHL from an early age. The premature hearing loss in Cdk5rap1-knockout mice was associated with the degeneration of the spiral ligament and reduction of endocochlear potentials following the loss of auditory sensory cells. Furthermore, cultured primary mouse embryonic fibroblasts displayed early onset of cellular senescence associated with high oxidative stress and cell death. These results indicate that the CDK5RAP1 deficiency-induced defective mitochondrial translation might cause early hearing loss through the induction of cellular senescence and cochlear dysfunction in the inner ear. Our results suggest that the accumulation of dysfunctional mitochondria might promote AHL progression. Furthermore, our findings suggest that mitochondrial dysfunction and dysregulated mitochondrial tRNA modifications mechanistically cause AHL. Understanding the mechanisms underlying AHL will guide future clinical investigations and interventions in the attempt to mitigate the consequences of AHL.

N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.

Posttranscriptional modifications in mitochondrial tRNA and its implication in mitochondrial translation and disease.

A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. Mitochondrial DNA encodes 22 tRNAs that translate mitochondrial mRNAs to 13 polypeptides of respiratory complexes. Various chemical modifications have been identified in mitochondrial tRNAs via complex enzymatic processes. A growing body of evidence has demonstrated that these modifications are essential for translation by regulating tRNA stability, structure and mRNA binding, and can be dynamically regulated by the metabolic environment. Importantly, the hypomodification of mitochondrial tRNA due to pathogenic mutations in mitochondrial tRNA genes or nuclear genes encoding modifying enzymes can result in life-threatening mitochondrial diseases in humans. Thus, the mitochondrial tRNA modification is a fundamental mechanism underlying the tight regulation of mitochondrial translation and is essential for life. In this review, we focus on recent findings on the physiological roles of 5-taurinomethyl modification (herein referred as taurine modification) in mitochondrial tRNAs. We summarize the findings in human patients and animal models with a deficiency of taurine modifications and provide pathogenic links to mitochondrial diseases. We anticipate that this review will help understand the complexity of mitochondrial biology and disease.