Mitochondrial DNA mutations in rRNA and tRNA loci associated with maternally inherited deafness

Examination of the nucleotides following the ATG or GTG initiation codons of a file of 251 genes from Escherichia coli has shown that 247 (98.4%) of them contain a sequence of at least three and 168 (66.9%) of them a sequence of at least four consecutive nucleotides that is complementary to some part of the 16 nt at the 5' terminus of the bacterial 16S rRNA. It is proposed that this sequence, which falls within the first 24 nt coding for the genetic message, might be involved in mRNA recognition through a mechanism analogous to the well-established 'Shine--Dalgarno' interaction with the 3' terminus of the 16S rRNA. Comparison of these data with data derived from a file of 117 'false' gene starts that have a Shine--Dalgarno-like sequence followed by a suitably spaced ATG or GTG triplet but which are believed not to lie at the beginnings of genetic messages shows the association that we have found to be statistically significant at the 99.9% level.

Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases

In recent years, the roles of mitochondrial RNA and its associated human diseases have been reported to increase significantly. Treatments based on mtRNA metabolic processes and nuclear gene mutations are thus discussed. The mitochondrial oxidative phosphorylation process is affected by mtRNA metabolism, including mtRNA production, maturation, stabilization, and degradation, which leads to a variety of inherited human mitochondrial diseases. Moreover, mitochondrial diseases are caused by mitochondrial messenger RNA, mitochondrial transfer RNA, and mitochondrial ribosomal RNA gene mutations. This review presents the molecular mechanisms of human mtRNA metabolism and pathological mutations in mtRNA metabolism-related nuclear-encoded/nonencoded genes and mitochondrial DNA mutations to highlight the importance of mitochondrial RNA-related diseases and treatments.

The role of mitochondria in white adipose tissue has traditionally received little attention. This historic neglect is based on the assumption that the white adipocyte's poor mitochondrial equipment, being confined to the small cellular space adjacent to the fat droplet, could have little relevance to white adipose cell function. In contrast, the brown adipocyte, which specializes in thermogenesis, has a robust content of mitochondria; thus the role of mitochondrial function in brown adipose cells has been recognized as a worthwhile topic of study. Brown fat has been traditionally considered to play only a minor role in adult humans, but recent data from positron emission tomography and other tools of nuclear medicine have evidenced the presence of substantial amounts of brown adipose tissue in adult humans ((1),(2)). Hence, the role of brown adipose tissue in adult human metabolic pathologies, such as obesity, has not been elucidated but the discovery of previously uncovered brown adipose tissue in adult humans is expected to stimulate further research on the role of brown fat in adult human energy balance. On the other hand, in recent years, accumulating evidence from a number of research areas, including studies on obesity, type 2 diabetes and the lipodystrophies, has highlighted the previously unrecognized relevance of mitochondrial function for white adipose tissue biology and systemic metabolic regulation. Mitochondria in mammalian cells, including adipocytes, have a unique feature—the mitochondrial genome—that distinguishes them from other cellular organelles. This distinct 16-kb circular DNA-based genetic system encodes mitochondrial components that are important for mitochondrial function. In mammals, mitochondrial DNA (mtDNA) encodes proteins of the respiratory chain/oxidative phosphorylation system (OXPHOS) as well as other components of the mitochondrial translation system (i.e., mitochondrial transfer RNAs and ribosomal RNAs) ((3)). Coordinate regulation of mtDNA- and nuclear genome–mediated gene expression is required for the correct synthesis of a functional mitochondrial OXPHOS ((4)). In addition to its genetic variability in humans, mtDNA is particularly susceptible to somatic mutations that can affect mtDNA-dependent gene expression and mitochondrial function. This is likely due to the direct exposure of mtDNA to locally produced oxygen superoxides arising from the respiratory chain as well as to poor performance of repair systems in mtDNA replication ((5)). Tissues from patients bearing mtDNA mutations show a mixture of mutated and wild-type forms of mtDNA at a given proportion (heteroplasmy) and the percentage of mutated mtDNA is usually associated with the severity of the pathology symptoms ((6)). Moreover, the mtDNA gene expression machinery is distinct from that which determines expression from nuclear genes. Pathogenic processes may alter components of the mtDNA-specific expression machinery, which is comprised mainly of nuclear-encoded proteins that are transported to the mitochondria after cytosolic synthesis ((7)). These pathologies may lead to alterations in overall mitochondrial function, altering energy balance and disturbing the respiratory chain, and leading to enhanced oxygen superoxide production or promotion of mitochondrially driven apoptosis—even in the absence of mtDNA mutations. Current research coming from the fields of genetics, endocrinology, and pharmacology indicate that mtDNA biology may be relevant to white adipose tissue function, and suggest that altered mtDNA in white fat may have local and systemic consequences. However, we are far from having developed a full mechanistic understanding of how mtDNA alterations cause these effects. The present review is intended to provide the first summary of our current knowledge of mtDNA biology in white adipose tissue and how it might be relevant to human pathologies, from obesity to lipodystrophy, that are related to energy balance. Human mtDNA was first sequenced in 1981. Since that time, extensive knowledge of the genetics and physiological processes that rely on mtDNA gene expression in human cells has become available ((3),(4)). Mitochondrial DNA in humans encodes two ribosomal RNAs, components of mitochondrial ribosomes; the 22 mitochondrial transfer RNAs; and 13 messenger RNAs that are translated into protein components of distinct complexes of the mitochondrial respiratory chain and OXPHOS. Thus, the energy-producing machinery in mammalian mitochondria relies on respiratory complexes built from nuclear and mtDNA-encoded polypeptides, and thus constitutes a unique cellular system that results from the coordinate expression of two distinct genomes. However, despite the fact that mtDNA encodes structural components of the OXPHOS system, most of the protein components of the mtDNA replication and expression machinery are encoded by the nuclear genome. The replication of mtDNA depends on mitochondrial DNA polymerase-γ, whereas mitochondrial RNA polymerase is responsible for mtDNA-driven transcription. Proteins, such as TFAM, TFB1M, and TFB2M, are involved in the control of these processes through interaction with the regulatory D-loop region of mtDNA. As shown in Figure 1, the control of mtDNA expression appears to be directed by nuclear gene–encoded transcription factors (e.g., nuclear respiratory factors-1 and -2), nuclear hormone receptors (e.g., thyroid hormone receptor, estrogen-related receptor-α), transcriptional coactivators (e.g., PGC-1s), and corepressors (e.g., RIP140). These factors control the expression of nuclear genes that encode structural components of the OXPHOS system and of the nuclear-encoded components of mtDNA replication and transcription (TFAM, TFB1M, and TFB2M) ((8)). The amounts of mtDNA in the mitochondria and in the whole cell, and the extent of mtDNA-dependent protein expression are therefore under complex control of nuclear-encoded components of the machinery. In contrast with nuclear gene expression, the expression of mtDNA genes can not only be controlled at the level of transcription but also at the level of mtDNA replication. Thus, mtDNA gene dosage may affect the extent of mtDNA-encoded gene expression, with consequences for mitochondrial function. Several physiological adaptive processes, such as chronic exercise training effects on muscle, mediate enhanced mtDNA expression by increasing mtDNA replication and thereby increasing mtDNA gene dosage ((9)). It is not known whether this mechanism of regulation of mtDNA expression through changes in mtDNA levels occurs in adipose tissue in response to physiological adaptations, although it takes place in association with brown and white adipocyte differentiation processes in vitro ((10),(11)), in pathological conditions, and under several pharmacological treatments (see below). Schematic representation of the factors involved in the control of mitochondrial DNA (mtDNA) expression. DNA-Polγ, DNA polymerase γ; ERRα, estrogen-related receptor-α; mtRPOL, mitochondrial RNA polymerase; TFAM, mitochondrial transcription factor A; TFB1M and TFB2M, mitochondrial transcription factors B1 and B2; NRF-1 and NRF-2, nuclear respiratory factors-1 and -2; OXPHOS, respiratory chain/oxidative phosphorylation system; PGC-1; peroxisome proliferator–related receptors coactivators; RIP140, receptor interacting protein-140. Until recently, studies on mtDNA biology in adipose tissue have been limited to the analysis of mitochondrial biogenesis in brown adipose tissue—the adipose tissue that specializes in thermogenesis. In contrast to white adipocytes, brown adipocytes possess large amounts of oxidative active mitochondria. During brown adipose tissue development and differentiation, mitochondrial biogenesis is enhanced and is accompanied by increased levels of mtDNA and expression of the mtDNA-encoded components of the OXPHOS system ((10)). The transcriptional activators, PGC-1α and PGC-1β, play an important role in this process by coordinately enhancing the expression of nuclear factors that stimulate mtDNA replication and transcription, and regulating the expression of specific thermogenic genes ((12)). Recent studies have highlighted the importance of mitochondrial biogenesis in white adipose tissue and the potential for mitochondrial alterations to disturb white adipocyte development and function. Studies by Corvera and collaborators have shown that (i) mitochondrial biogenesis is directly associated with white adipocyte differentiation; (ii) obese ob/ob mice displayed impaired mitochondrial mass and function in white fat; and (iii) thiazolidinediones, peroxisome proliferator–activated receptor-γ activators that favor adipocyte differentiation, ameliorated these alterations ((13),(14)). It has been also shown that white adipocyte differentiation is associated with increases in the relative abundance of mtDNA, and upregulation of components of the mtDNA replication and transcription machinery, such as TFAM ((11)), and components of deoxynucleotide metabolism required for mtDNA replication ((15)). Agents that promote white adipocytes differentiation in vitro, such as glitazones, also increase mtDNA levels in human adipocytes in vitro ((16)). Studies of potential alterations in white adipose tissue mtDNA as they relate to obesity have focused on two facets: changes in mtDNA levels that underlie obese phenotypes; and the occurrence of mutated, polymorphic, forms of mtDNA that are specifically associated with obesity. In experimental models of obesity, such as ob/ob or db/db mice, abnormally low levels of mtDNA have been reported ((17),(18)). As noted above for in vitro studies, treatment of obese mice with glitazones increases mtDNA levels in white fat ((18)). A study in which mice were treated with a diet enriched in polyunsaturated fatty acids identified mtDNA-encoded transcripts and proteins among the most upregulated genes in white adipose tissue; this upregulation was associated with an enhancement of fatty acid oxidation in white adipose tissue ((19)). In humans, the scenario appears to be more complex. It has been reported that mtDNA levels in adipose tissue are lowered in type 2 diabetic patients ((20)), and studies by Arner and collaborators have confirmed that mtDNA levels are not associated with obesity per se, but rather with type 2 diabetes phenotypes ((21)). Moreover, mtDNA levels were found to be strongly related to lipogenesis in white adipose tissue ((22)), rather than to BMI. The mechanism by which mtDNA copy number in white adipose tissue could affect lipogenesis rate remains to be established, but it stands in contrast to the expected relationship between mtDNA level variations and energy expenditure and fat oxidation. Moreover, in humans, as in rodents, pioglitazone treatment causes an increase in mtDNA levels in white adipose tissue of type 2 diabetes patients ((20)) but not in nondiabetic obese individuals ((23)). Probably, one of the more outstanding pieces of evidence in support of the potential relationship between mtDNA levels in white adipose tissue and human obesity comes from a recent study of a series of rare monozygotic twins discordant for obesity ((24)). Sequence analyses of mtDNA in subcutaneous adipose tissue revealed no aberrant heteroplasmy between the co-twins. However, mtDNA copy number was reduced by 47% in the obese co-twin's fat. These findings highlight the potential role of mtDNA levels in white adipose tissue mass. Further research will be needed to clarify the role of mtDNA levels in influencing biological processes in white adipose tissue beyond the expected consequences on the extent of metabolic energy consumption through oxidative pathways. However, recent data indicate that mitochondrial oxidative capacity may not be a major factor determining events such the extent of free fatty acid release by white adipose cells ((25)). Another important line of research is the analysis of mtDNA mutations for which there is no direct evidence for pathogenic consequences, but may constitute mtDNA polymorphic forms related to obesity. Several reports indicate such association, but no clear-cut conclusions have been reached to date. The single-nucleotide polymorphism, 15497 G/A, which leads to a glycine-to-serine amino acid replacement at residue 251 (Gly251Ser) in the mtDNA-encoded cytochrome b, has been associated with obesity in a Japanese population ((26)). In fact, individuals bearing the A allele presented an increased waist-to-hip ratio and other obesity-related variables including elevated triglyceride levels relative to individuals bearing the G allele. However, the functional consequences of this polymorphism for adipose tissue biology, and whether they affect primarily white adipose tissue function or overall energy balance, and thus adipose tissue expansion, are unknown. Studies of white populations found that this mutation contributes to severe obesity only in rare instances ((27)), and similarly, a polymorphism in the mtDNA D-loop noncoding region was not found to be associated with obesity ((28)). Recently, single-nucleotide polymorphisms in the mtDNA-encoded reduced nicotinamide adenine dinucleotide dehydrogenase subunit I gene and in the 12S ribosomal RNA gene in mtDNA have also been associated with obesity in a Japanese population ((29)) whereas a common mtDNA variant has been associated with low BMI in white women ((30)). Since the 1980s, deletions and point mutations in mtDNA have been known to cause human diseases. The common pathogenic features of diseases attributed to genetic mtDNA alterations involve defects in neural and muscular tissues, but not adipose tissues. Nevertheless, there is a remarkable exception: mutations in the mtDNA-encoded tRNA-Lys cause lipomatosis. Most patients bearing pathogenic mutations in the mtDNA-encoded tRNA-Lys (A8344G, T8356C, G8361A, G8363A) show the myoclonic epilepsy associated with ragged-red fibers syndrome, characterized by neuropathy and myopathy symptoms. However, other patients show an abnormal enlargement of adipose tissue in the dorsal area, similar to the Madelung syndrome ((31),(32),(33),(34)). The lipomatous tissues in the dorso-cervical area of these patients do not show changes in mtDNA levels or in the expression of mtDNA genes. Nuclear DNA–encoded components of the mitochondria are similarly unchanged, despite altered expression of master regulators of adipogenesis. A distorted pattern of brown vs. white adipocyte gene expression, characterized by the expression of the brown adipose tissue–specific uncoupling protein-1 gene but without the acquisition of a complete brown fat phenotype, occurs in the lipomatous tissue ((35),(36)). How such a specific mutation in an mtDNA gene causes adipose tissue hypertrophy remains a mystery. However, it highlights the importance of mtDNA for the normal development of adipose tissue. Certain pathogenic mutations in mtDNA are strongly associated with diabetes caused by progressive insulinopenia, particularly the A3243G mutation in the mtDNA-encoded tRNA-Leu gene. This mutation has been associated with a low BMI ((37),(38)), and lipomatosis has been reported in some patients, despite good glycemic control ((39),(40)). However, it is not known to what extent the adipose disturbances are caused by direct alterations of mtDNA function in white adipose tissue, or are caused indirectly by insulinopenia. Finally, one additional piece of evidence that support the involvement of mtDNA-related pathologies in altering white adipose tissue is provided by mitochondrial diseases caused by mutations in nuclear genes involved in the mtDNA replication/transcription machinery. Obesity has been reported to be a feature of Finnish patients with ataxia due to mutations in mitochondrial DNA polymerase-γ ((41)). In a parallel experimental model, transgenic mice bearing a proof-reading-deficient version of the mitochondrial DNA polymerase-γ showed reduced subcutaneous white adipose tissue in association with the accumulation of somatic mtDNA mutations during ageing ((42)). Research interest in the role of mtDNA in adipose tissue biology has received a dramatic boost in the last few years from studies of human immunodeficiency virus (HIV) lipodystrophy. This pathology occurs in a substantial subset of HIV-infected patients under antiretroviral treatment; its main features include lipoatrophy of subcutaneous fat, visceral obesity and, occasionally, lipomatosis. A major hypothesis proposed to account for the etiopathogenesis of this lipodystrophy is based on the action of the nucleoside-analog inhibitors of the viral reverse transcriptase, a class of antiretroviral drugs used in HIV-1 infected patients. A side effect of these drugs is inhibition of DNA polymerase-γ, the enzyme responsible for mtDNA replication. Accordingly, antiretroviral-treated patients show abnormal reductions (depletion) in mtDNA abundance ((43)). A systematic analysis of published reports based on cross-sectional studies indicates that most such studies consistently identify an association between mtDNA depletion in subcutaneous adipose tissue and lipoatrophy ((44)). However, other tissues besides white adipose tissue also exhibit a reduction in mtDNA abundance; thus, if mtDNA depletion is the cause of the syndrome, it remains unknown why it so specifically affects adipose tissue. Perhaps the positive association between mtDNA levels and lipogenesis reported elsewhere ((22)) may underlie the association between mtDNA depletion in adipose tissue and lowered fat mass accretion in subcutaneous white adipose tissue from patients. The reduction in mtDNA levels occurs in conjunction with impaired expression of genes related to adipocyte differentiation (i.e., peroxisome proliferator–activated receptor-γ), lipid accretion (e.g., lipoprotein lipase, GLUT4), and adipokines (i.e., adiponectin) ((45),(46),(47),(48)). In fact, thiazolidinedione treatment–mediated activation of peroxisome proliferator–activated receptor-γ in humans leads to increased body weight through the upregulation of genes facilitating adipocyte lipid storage ((49)) and, therefore, the impaired peroxisome proliferator–activated receptor-γ expression in adipose tissue from patients may contribute to lipoatrophy. However, studies in which antiretroviral treatment of HIV-1 patients with lipoatrophy has been interrupted ((50)) or shifted to drugs with a lesser effect on DNA polymerase-γ have indicated a substantial reversion of mtDNA depletion in white adipose tissue but a milder amelioration of lipoatrophy ((51),(52),(53),(54)). Thus, cause-and-effect relationships remain unclear. Exposure of brown or white adipocytes in culture to nucleoside-analog reverse transcriptase inhibitors leads to a reduction in mtDNA as well as to an impairment in adipocyte differentiation ((55),(56),(57)). However, other antiretroviral drugs that do not inhibit DNA polymerase-γ nor cause changes in mtDNA levels, such as protease inhibitors, are also powerful blockers of white adipocyte differentiation in vitro ((58)). Ultimately, the potential impact of mtDNA depletion on cell and tissue function can be expected to depend on the extent to which the depletion reduces the levels of mtDNA-encoded gene products. Although mtDNA depletion is usually associated with a reduction in mtDNA-encoded transcripts ((47)), mtDNA-encoded protein levels may be unaltered ((48),(59)), suggesting the action of compensatory mechanisms that upregulate the synthesis of mtDNA-encoded proteins. It is worth noting that mitochondrial mass, as measured directly through mitochondrial protein quantification after mitochondria isolation ((45),(59)) or indirectly by assessment of citrate synthase activity ((48)), may even be increased in white adipose tissue from patients with lowered mtDNA levels. This indicates that, at least in pathogenic conditions, mtDNA levels may be dissociated from mitochondrial mass in adipose tissue. However, the enhancement of mitochondrial biogenesis in mtDNA-depleted adipose tissue does not necessarily lead to enhancement of mitochondria functional activity. The scenario may be similar to the proliferation of abnormal mitochondria occurring in skeletal muscle from patients with mtDNA pathogenic mutations and leading to the appearance of the ragged-red structures in muscle fibers from these patients ((60)). The consequences on bioenergetic mitochondrial function of the enhanced mitochondrial mass observed in mtDNA-depleted white adipose tissue from patients with lipodystrophy have not been extensively studied, but a recent report indicates no increase in cytochrome c oxidase activity in association with the increased mitochondrial mass ((48)). On the other hand, even if mtDNA depletion causes some impairment in respiratory chain, it has been proposed that its consequences may result not only from lowered ATP production but also from a reduced synthesis of uridine. An active respiratory chain is required for the function of dihydroorotate dehydrogenase, a key enzyme in the de novo synthesis of all intracellular pyrimidines. Treatment of white adipocytes with uridine ameliorates the disturbances elicited by drugs that cause mtDNA depletion, thus suggesting that unaltered mtDNA levels are required for correct rates of uridine synthesis and that uridine homeostasis is closely related to the maintenance of white adipocyte differentiation and function ((56)). It is likely that a reduction in the levels of mtDNA to a certain extent is capable of maintaining normal function; at less than permissive levels, a threshold is crossed below which white adipocyte homeostatic mechanisms cannot ensure unaltered levels of mtDNA-encoded gene products. Currently, a precise understanding of these mtDNA expression regulatory mechanisms, which are likely to involve the mtDNA transcription and translation machinery, is lacking. This is a promising area of research for pharmacological or nutritional intervention designed to enhance the rates of mtDNA gene expression, and thereby ameliorate the pathogenic consequences of suprathreshold mtDNA depletion in white adipose tissue. Most studies have focused on relating mtDNA depletion in fat to white adipose tissue atrophy in HIV-1 patients. This reflects the ease of access to the atrophic subcutaneous adipose tissue for biopsies and subsequent analysis. Other signs of HIV lipodystrophy, such as visceral obesity or lipomatosis are not associated with any loss of adipose tissue mass. A study of biopsies from “buffalo hump,” an enlargement of the adipose mass in the dorso-cervical area that occurs in some HIV-1 patients with lipodystrophy, revealed a depletion of mtDNA similar to that found in subcutaneous lipoatrophic adipose tissue ((47)). Recently, the availability of samples from HIV-1 patient visceral adipose tissue, a site prone to enlargement in patients with HIV lipodystrophy, revealed also a similar reduction in mtDNA levels with respect to subcutaneous white fat ((61)). These observations question the presumption that abnormally low mtDNA levels could influence the outcome—atrophy vs. hypertrophy—for adipose tissue in a simple manner. On the other hand, some reports indicate that the first signs of mtDNA depletion in white adipose tissue occur in the absence of treatment, simply due to HIV infection ((62)) although most reports attribute mtDNA depletion exclusively to antiretroviral treatment ((63)). In experimental rodent models in which there is no HIV-1 infection, it has been very difficult to induce mtDNA depletion and lipodystrophy, even using large doses of antiretroviral drugs ((64)), suggesting that HIV-1 events contribute to the full of the mtDNA depletion Recently, two models of mtDNA have been through or of forms of the gene for mitochondrial the enzyme responsible for for DNA synthesis the mitochondria In there was a depletion of mtDNA levels in white and brown adipose tissue. In addition to a pathology of human in patients with adipose tissues were also strongly and mice showed signs of lipodystrophy including loss of subcutaneous white fat and abnormal brown adipose tissue Another recent report mtDNA depletion in fat in a a of the nuclear estrogen-related This receptor appears to play a major role in the expression of nuclear genes involved in mitochondrial among these are related to the mtDNA replication machinery. show amounts of white adipose tissue, abnormal accumulation of brown fat, and to obesity Although no data are available for white fat, the brown fat from these mice also a reduction of mtDNA levels and of mtDNA-encoded transcripts This summary of the available data evidence for a role for mtDNA in white adipose tissue biology, and indicates that changes in mtDNA levels and expression of mtDNA-encoded genes in white adipose tissue may have important consequences in pathogenic conditions, including obesity. However, we are far from having a full understanding of the mechanistic that relate mtDNA biology and white adipose tissue The relationship between mtDNA and adipose tissue mass is not and our present knowledge does not a mechanistic that our current knowledge of the role of mtDNA in cellular with the of white adipose tissue disturbances associated with mtDNA Studies of genetic mtDNA diseases in several tissues and have provided evidence to support the relevance of threshold effects that relate mtDNA altered levels and function with cellular and there are that such threshold effects may be do not what threshold level of white adipocyte mtDNA is with the of white adipose cell However, certain observations suggest that changes in mtDNA levels are not involved in determining intracellular oxidative in white glitazones increase mtDNA levels in white adipocytes, with increased differentiation and enhancement of fat in adipocytes, and mtDNA levels are associated with lipogenesis in Thus, the impact of in white adipocyte mtDNA not be as the simple of the potential changes in mitochondrial and cellular ATP synthesis elicited by altered mtDNA function. In this a recent study has reported that reductions in mtDNA levels and respiratory chain activity in adipocytes, elicited by of TFAM, caused impaired and through unknown mechanisms It is likely that the mtDNA levels or mtDNA expression in the white adipocyte mitochondria may influence nuclear gene expression through (e.g., through intracellular oxygen or changes in and thus could affect overall adipocyte in a complex manner. Moreover, the consequences of in mtDNA levels and expression of mtDNA-encoded genes on the expression of nuclear genes in the white adipocyte may involve the synthesis and release of adipokines and therefore may have systemic consequences that underlie the of In evidence a role for mtDNA in white adipose tissue and indicates that disturbances in mtDNA function may be involved in adipose tissue pathologies, including obesity. Further research will be required to the precise mechanisms by which mtDNA functional changes result in of adipose tissue mass, and identify pharmacological nutritional for mtDNA that This was by de and de de The no of

Mitochondrial transcription produces polycistronic transcripts, in which ribosomal RNA (rRNA) and messenger RNA (mRNA) regions are interspersed with transfer RNA (tRNA) sequences. Endonucleolytic cleavages at the 5' and 3' ends of tRNA sequences in polycistronic transcripts are the first critical post-transcriptional steps in mitochondrial gene expression, essential for the release of all functional rRNA, mRNA, and tRNA species. Mitochondrial tRNA 5’ processing is catalyzed by the mitochondrial RNase P (mt-RNase P) complex composed of an endonuclease subunit, PRORP, and two auxiliary subunits forming a tRNA methyltransferase subcomplex, TRMT10C and SDR5C1. As a multi-subunit protein-only RNase P, the human mt-RNase P complex is distinct from the ubiquitous ribonucleoprotein RNase Ps or single-subunit protein-only RNase Ps. The 3’ processing of both mitochondrial and nuclear tRNAs is catalyzed by a common RNase Z enzyme, ELAC2. Recent evidence indicates that ELAC2 may also depend on TRMT10C and SDR5C1 specifically for mitochondrial tRNA processing. Thus, mitochondrial RNases P and Z appear to be compositionally atypical multimeric enzymes, and the molecular underpinnings of their function are not well understood. This dissertation focuses on structural and mechanistic characterization of human mt-RNase P and Z complexes. First, to understand how mt-RNase P complex carries out tRNA 5’ processing and what role TRMT10C and SDR5C1 play therein, we determined a cryo-electron microscopy structure of human mt-RNase P bound to precursor tRNA. The structure reveals that the subunits TRMT10C and SDR5C1 form a subcomplex that recognizes tRNAs via a sequence-independent mechanism, enabling the recognition of structurally diverse mt-tRNAs. Binding to TRMT10C–SDR5C1 subcomplex distorts the tRNA structure and positions the 9th nucleobase in the methyltransferase active site. The endonuclease PRORP is recruited via interactions with TRMT10C, which stabilize PRORP in an active conformation and ensure precise cleavage at the 5' end of the pre-tRNA. Subsequently, we investigated the mechanism of tRNA 3’ processing and the role of TRMT10C and SDR5C1 in this process through a combination of structural and biochemical studies. We found that TRMT10C and SDR5C1 are specifically required for processing of mt-tRNAs lacking the canonical elbow regions. ELAC2 recognizes nuclear and mitochondrial tRNAs with canonical elbows via direct interactions with the conserved elbow elements. By contrast, most mt-tRNAs, which lack a canonical elbow, depend on compensatory protein-protein interactions between ELAC2 and TRMT10C for processing. Together, these results elucidate the structural and mechanistic basis of the mitochondrial tRNA 5’ and 3’ processing, provide catalytic insights into tRNA-R9 methylation and explain the role of TRMT10C and SDR5C1 in mt-tRNA processing. Furthermore, they highlight the role of TRMT10C–SDR5C1 complex as a general mt-tRNA maturation platform that functions as a mitochondrial tRNA ‘prosthesis’, stabilizing the tertiary fold of mt-tRNAs and compensating for the absence of conserved structural features otherwise necessary for their recognition. This work represents an important milestone towards a complete structural understanding of mitochondrial gene expression and provides a framework for future studies aimed at achieving this goal.

Comparison of cytoplasmic and mitochondrial 4 s RNA from cultured hamster cells: Physical and metabolic properties

Mitochondria are found in all nucleated human cells and perform various essential functions, including the generation of cellular energy. Mitochondria are under dual genome control. Only a small fraction of their proteins are encoded by mitochondrial DNA (mtDNA), whereas more than 99% of them are encoded by nuclear DNA (nDNA). Mutations in mtDNA or mitochondria-related nDNA genes result in mitochondrial dysfunction leading to insufficient energy production required to meet the needs for various organs, particularly those with high energy requirements, including the central nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system. Because cardiac muscles are one of the high energy demanding tissues, cardiac involvement occurs in mitochondrial diseases with cardiomyopathies being one of the most frequent cardiac manifestations found in these disorders. Cardiomyopathy is estimated to occur in 20–40% of children with mitochondrial diseases. Mitochondrial cardiomyopathies can vary in severity from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. Hypertrophic cardiomyopathy is the most common type; however, mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Cardiomyopathies are frequent manifestations of mitochondrial diseases associated with defects in electron transport chain complexes subunits and their assembly factors, mitochondrial transfer RNAs, ribosomal RNAs, ribosomal proteins, translation factors, mtDNA maintenance, and coenzyme Q10 synthesis. Other mitochondrial diseases with cardiomyopathies include Barth syndrome, Sengers syndrome, TMEM70-related mitochondrial complex V deficiency, and Friedreich ataxia.

Mitochondrial disorders are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial energy metabolism, and they represent one of the largest groups of inborn errors of metabolism. Mitochondrial disorders can be caused by mutation of genes encoded by either nuclear DNA or mitochondrial DNA (mtDNA), with more than 300 disease-associated genes identified to date. Among these genes, around 100 have so far been associated with cardiac manifestations. Cardiomyopathy is estimated to occur in 20–40% of children with mitochondrial disorders. Genetic defects can affect a vast range of different mitochondrial functions including electron transport chain complex subunits and their assembly factors, mitochondrial transfer or ribosomal RNAs, factors involved in translation or mtDNA maintenance, and cofactor metabolism such as coenzyme Q10 synthesis. With collectively more than 1000 described cases, the most frequent mitochondrial cardiac diseases include Barth syndrome, Sengers syndrome, ACAD9- or TMEM70-related mitochondrial complex I or V deficiency, and Friedreich ataxia. Hypertrophic cardiomyopathy is the most common type of cardiomyopathy, but mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Mitochondrial cardiomyopathy can vary in severity from asymptomatic to severe manifestations including heart failure and sudden cardiac death. Congenital arrhythmias and congenital heart defects (CHDs) are also part of the clinical spectrum of mitochondrial disorders. In this chapter, we provide an overview of the constantly growing number of mitochondrial cardiac disorders and comment on the current practice in the diagnosis and treatment of patients with mitochondrial cardiomyopathy, including optimal therapeutic management and long-term monitoring.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.

Biosynthesis of ribonucleic acid in yeast: Some properties of mitochondrial ribosomal ribonucleic acid in Saccharomyces carlsbergensis

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are “hotspots” for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

In silico model of mtDNA mutations effect on secondary and 3D structure of mitochondrial rRNA and tRNA in Leber’s hereditary optic neuropathy

15] Physical studies of the interaction of Escherichia coli translational initiation factor 3 protein with ribosomal RNA

Pathogenic mutations of the human mitochondrial genome are associated with well-characterized, progressive neurological syndromes, with mutations in the transfer RNA genes being particularly prominent. To describe a novel mitochondrial transfer RNA(Pro) gene mutation in a woman with a myoclonic epilepsy with ragged-red fibers-like disease. Design, Setting, and Patient Case report of a 49-year-old woman presenting with a myoclonic epilepsy with ragged-red fibers-like disease comprising myoclonic jerks, cerebellar ataxia, and proximal muscle weakness. Histochemical analysis of a muscle biopsy revealed numerous cytochrome-c oxidase-deficient, ragged-red fibers, while biochemical studies indicated decreased activity of respiratory chain complex I. Molecular investigation of mitochondrial DNA revealed a new heteroplasmic mutation in the TpsiC stem of the mitochondrial transfer RNA(Pro) gene that segregated with cytochrome-c oxidase deficiency in single muscle fibers. Our case serves to illustrate the ever-evolving phenotypic spectrum of mitochondrial DNA disease and the importance of performing comprehensive mitochondrial genetic studies in the absence of common mitochondrial DNA mutations.

Mitochondrial DNA and disease