Ganglioside GM3 Synthase Deficiency in Mouse Models and Human Patients.

Gangliosides (glycosphingolipids containing one or more sialic acids) are highly expressed in neural tissues in vertebrates, and four species (GM1a, GD1a, GD1b, GT1b) are predominant in mammalian brains. GM3 is the precursor of each of these four species and is the major ganglioside in many nonneural tissues. GM3 synthase (GM3S), encoded by ST3GAL5 gene in humans, is a sialyltransferase responsible for synthesis of GM3 from its precursor, lactosylceramide. ST3GAL5 mutations cause an autosomal recessive form of severe infantile-onset neurological disease characterized by progressive microcephaly, intellectual disability, dyskinetic movements, blindness, deafness, intractable seizures, and pigment changes. Some of these clinical features are consistently present in patients with ST3GAL5 mutations, whereas others have variable expression. GM3S knockout (KO) mice have deafness and enhanced insulin sensitivity, but otherwise do not display the above-described neurological defects reported in ST3GAL5 patients. The authors present an overview of physiological functions and pathological aspects of gangliosides based on findings from studies of GM3S KO mice and discuss differential phenotypes of GM3S KO mice versus human GM3S-deficiency patients.

Structural analyses of a human lysyl-tRNA synthetase mutant associated with autosomal recessive nonsyndromic hearing impairment.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Elmod3 knockout leads to progressive hearing loss and abnormalities in cochlear hair cell stereocilia.

ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment.

Mutations in KARS cause early-onset hearing loss and leukoencephalopathy: Potential pathogenic mechanism.

Leukoencephalopathies are a broad class of common neurologic deterioration for which the etiology remains unsolved in many cases. In a Chinese Han family segregated with sensorineural hearing loss and leukoencephalopathy, candidate pathogenic variants were identified by targeted next-generation sequencing of 144 genes associated with deafness and 108 genes with leukoencephalopathy. Novel compound heterozygous mutations p.R477H and p.P505S were identified in KARS, which encodes lysyl-tRNA synthetase (LysRS), as the only candidate causative variants. These two mutations were functionally characterized by enzymatic assays, immunofluorescence, circular dichroism analysis, and gel filtration chromatography. Despite no alteration in the dimer-tetramer oligomerization and cellular distribution by either mutation, the protein structure was notably influenced by the R477H mutation, which subsequently released the protein from the multiple-synthetase complex (MSC). Mutant LysRSs with the R477H and P505S mutations had decreased tRNALys aminoacylation and displayed a cumulative effect when introduced simultaneously. Our studies showed that mutations in KARS lead to a newly defined subtype of leukoencephalopathy associated with sensorineural hearing impairment. The combined effect of reduced aminoacylation and release of LysRS from the MSC likely underlies the pathogenesis of the KARS mutations identified in this study.

A novel mutation MT-COIII m.9267G>C and MT-COI m.5913G>A mutation in mitochondrial genes in a Tunisian family with maternally inherited diabetes and deafness (MIDD) associated with severe nephropathy.

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia, maternal transmission and its association with a bilateral hearing impairment. Several studies reported mutations in mitochondrial genes as potentially pathogenic for diabetes, since mitochondrial oxidative phosphorylation plays an important role in glucose-stimulated insulin secretion from beta cells. In the present report, we studied a Tunisian family with mitochondrial diabetes (MD) and deafness associated with nephropathy. The mutational analysis screening revealed the presence of a novel heteroplasmic mutation m.9276G>C in the mitochondrial COIII gene, detected in mtDNA extracted from leukocytes of a mother and her two daughters indicating that this mutation is maternally transmitted and suggest its implication in the observed phenotype. Bioinformatic tools showed that m.9267G>C mutation (p.A21P) is « deleterious ¼ and it can modify the function and the stability of the MT-COIII protein by affecting the assembly of mitochondrial COX subunits and the translocation of protons then reducing the activity of the respective OXPHOS complexes of ATP synthesis. The nonsynonymous mutation (p.A21P) has not been reported before, it is the first mutation described in the COXIII gene which is related to insulin dependent mitochondrial diabetes and deafness and could be specific to the Tunisian population. The m.9267G>C mutation was present with a nonsynonymous inherited mitochondrial homoplasmic variation MT-COI m.5913 G>A (D4N) responsible of high blood pressure, a clinical feature detected in all explored patients.

Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection.

BACKGROUND: Mutations in either Aß Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. RESULTS: Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDD(KI), which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDD(KI) mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. CONCLUSION: These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

Mitochondrial diabetes in children: seek and you will find it.

Maternally Inherited Diabetes and Deafness (MIDD) is a rare form of diabetes due to defects in mitochondrial DNA (mtDNA). 3243 A>G is the mutation most frequently associated with this condition, but other mtDNA variants have been linked with a diabetic phenotype suggestive of MIDD. From 1989 to 2009, we clinically diagnosed mitochondrial diabetes in 11 diabetic children. Diagnosis was based on the presence of one or more of the following criteria: 1) maculopathy; 2) hearing impairment; 3) maternal heritability of diabetes/impaired fasting glucose and/or hearing impairment and/or maculopathy in three consecutive generations (or in two generations if 2 or 3 members of a family were affected). We sequenced the mtDNA in the 11 probands, in their mothers and in 80 controls. We identified 33 diabetes-suspected mutations, 1/33 was 3243A>G. Most patients (91%) and their mothers had mutations in complex I and/or IV of the respiratory chain. We measured the activity of these two enzymes and found that they were less active in mutated patients and their mothers than in the healthy control pool. The prevalence of hearing loss (36% vs 75-98%) and macular dystrophy (54% vs 86%) was lower in our mitochondrial diabetic adolescents than reported in adults. Moreover, we found a hitherto unknown association between mitochondrial diabetes and celiac disease. In conclusion, mitochondrial diabetes should be considered a complex syndrome with several phenotypic variants. Moreover, deafness is not an essential component of the disease in children. The whole mtDNA should be screened because the 3243A>G variant is not as frequent in children as in adults. In fact, 91% of our patients were mutated in the complex I and/or IV genes. The enzymatic assay may be a useful tool with which to confirm the pathogenic significance of detected variants.

Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia.

BACKGROUND: A mutation in the BRI2/ITM2b gene causes familial Danish dementia (FDD). BRI2 is an inhibitor of amyloid-ß precursor protein (APP) processing, which is genetically linked to Alzheimer's disease (AD) pathogenesis. The FDD mutation leads to a loss of BRI2 protein and to increased APP processing. APP haplodeficiency and inhibition of APP cleavage by ß-secretase rescue synaptic/memory deficits of a genetically congruous mouse model of FDD (FDDKI). ß-cleavage of APP yields the ß-carboxyl-terminal (ß-CTF) and the amino-terminal-soluble APPß (sAPPß) fragments. γ-secretase processing of ß-CTF generates Aß, which is considered the main cause of AD. However, inhibiting Aß production did not rescue the deficits of FDDKI mice, suggesting that sAPPß/ß-CTF, and not Aß, are the toxic species causing memory loss. RESULTS: Here, we have further analyzed the effect of γ-secretase inhibition. We show that treatment with a γ-secretase inhibitor (GSI) results in a worsening of the memory deficits of FDDKI mice. This deleterious effect on memory correlates with increased levels of the ß/α-CTFs APP fragments in synaptic fractions isolated from hippocampi of FDDKI mice, which is consistent with inhibition of γ-secretase activity. CONCLUSION: This harmful effect of the GSI is in sharp contrast with a pathogenic role for Aß, and suggests that the worsening of memory deficits may be due to accumulation of synaptic-toxic ß/α-CTFs caused by GSI treatment. However, γ-secretase cleaves more than 40 proteins; thus, the noxious effect of GSI on memory may be dependent on inhibition of cleavage of one or more of these other γ-secretase substrates. These two possibilities do not need to be mutually exclusive. Our results are consistent with the outcome of a clinical trial with the GSI Semagacestat, which caused a worsening of cognition, and advise against targeting γ-secretase in the therapy of AD. Overall, the data also indicate that FDDKI is a valuable mouse model to study AD pathogenesis and predict the clinical outcome of therapeutic agents for AD.

ß- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.

A mutation in the BRI2/ITM2b gene causes loss of BRI2 protein leading to familial Danish dementia (FDD). BRI2 deficiency of FDD provokes an increase in amyloid-ß precursor protein (APP) processing since BRI2 is an inhibitor of APP proteolysis, and APP mediates the synaptic/memory deficits in FDD. APP processing is linked to Alzheimer disease (AD) pathogenesis, which is consistent with a common mechanism involving toxic APP metabolites in both dementias. We show that inhibition of APP cleavage by ß-secretase rescues synaptic/memory deficits in a mouse model of FDD. ß-cleavage of APP yields amino-terminal-soluble APPß (sAPPß) and ß-carboxyl-terminal fragments (ß-CTF). Processing of ß-CTF by γ-secretase releases amyloid-ß (Aß), which is assumed to cause AD. However, inhibition of γ-secretase did not ameliorate synaptic/memory deficits of FDD mice. These results suggest that sAPPß and/or ß-CTF, rather than Aß, are the toxic species causing dementia, and indicate that reducing ß-cleavage of APP is an appropriate therapeutic approach to treating human dementias. Our data and the failures of anti-Aß therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aß.

Functional null mutations of MSRB3 encoding methionine sulfoxide reductase are associated with human deafness DFNB74.

The DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53-67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing.

c-Ret-mediated hearing loss in mice with Hirschsprung disease.

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c-RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c-Ret and c-RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c-Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-kappaB but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c-Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c-RET-mediated severe HSCR, our results indicate that c-Ret and c-RET are a deafness-related molecule in mice and humans.

Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into maternally inherited deafness.

Mitochondrial biogenesis is controlled by signaling networks that relay information to and from the organelles. However, key mitochondrial factors that mediate such pathways and how they contribute to human disease are not understood fully. Here we demonstrate that the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 are key downstream effectors of mitochondrial biogenesis that perform unique, yet cooperative functions. The primary function of h-mtTFB2 is mtDNA transcription and maintenance, which is independent of its rRNA methyltransferase activity, while that of h-mtTFB1 is mitochondrial 12S rRNA methylation needed for normal mitochondrial translation, metabolism and cell growth. Over-expression of h-mtTFB1 causes 12S rRNA hypermethylation, aberrant mitochondrial biogenesis and increased sorbitol-induced cell death. These phenotypes are recapitulated in cells harboring the pathogenic A1555G mtDNA mutation, implicating a deleterious rRNA methylation-dependent retrograde signal in maternally inherited deafness pathology and shedding significant insight into how h-mtTFB1 acts as a nuclear modifier of this disease.

Spermine synthase deficiency leads to deafness and a profound sensitivity to alpha-difluoromethylornithine.

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken beta-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO). Within 2-3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.

A catechol-O-methyltransferase that is essential for auditory function in mice and humans.

We have identified a previously unannotated catechol-O-methyltranferase (COMT), here designated COMT2, through positional cloning of a chemically induced mutation responsible for a neurobehavioral phenotype. Mice homozygous for a missense mutation in Comt2 show vestibular impairment, profound sensorineuronal deafness, and progressive degeneration of the organ of Corti. Consistent with this phenotype, COMT2 is highly expressed in sensory hair cells of the inner ear. COMT2 enzymatic activity is significantly reduced by the missense mutation, suggesting that a defect in catecholamine catabolism underlies the auditory and vestibular phenotypes. Based on the studies in mice, we have screened DNA from human families and identified a nonsense mutation in the human ortholog of the murine Comt2 gene that causes nonsyndromic deafness. Defects in catecholamine modification by COMT have been previously implicated in the development of schizophrenia. Our studies identify a previously undescribed COMT gene and indicate an unexpected role for catecholamines in the function of auditory and vestibular sense organs.

Preparation, functional characterization, and NMR studies of human KCNE1, a voltage-gated potassium channel accessory subunit associated with deafness and long QT syndrome.

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.

Lesions of an avian forebrain nucleus prevent changes in protein kinase C levels associated with deafening-induced vocal plasticity in adult songbirds.

We investigated the participation of protein kinase C (PKC) in the regulation of vocal plasticity in songbirds. Deafening of adult Bengalese finches causes initial song alteration, followed by stabilization. In parallel, the expression of PKC beta1 increases transiently 2 weeks after deafening, and then decreases gradually in the robust nucleus of the arcopallium (RA) of Bengalese finches, similar to the pattern observed during developmental song learning. First, we showed that in adult zebra finches, whose songs change more gradually after auditory deprivation than those of Bengalese finches, PKC in RA also increased to an equal degree 2 weeks after deafening, despite the species difference. Second, double-labeling with an anterograde tracer and PKC immunofluorescence revealed that PKC immunoreactivity in RA was detected on the synaptic terminals from a high premotor vocal nucleus (HVC), but not from the lateral magnocellular nucleus of the anterior nidopallium (LMAN). To determine what causes deafening-induced PKC increases, we blocked signals from LMAN, the final output nucleus to RA in the anterior forebrain pathway (AFP), by a unilateral LMAN lesion prior to auditory deprivation of adult Bengalese finches. The PKC immunoreactivity increased in RA of the intact hemisphere; however, in RA on the lesioned side, it was less intense than that of the unlesioned side. Thus, the deafening-induced PKC expression was suppressed by lesioning of LMAN. These results suggest that an output signal from the AFP via LMAN induces the increase in PKC activity on HVC-RA synapses that may regulate song plasticity.

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations.

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.