Background: Personal anguish, incapacity, and a decline in work and life quality are all associated with neck and low back pain, making it a significant socioeconomic burden for individuals and society. It is well known that engaging in regular physical exercise has considerable health benefits.Objective: The purpose of this research was to investigate the factors contributing to the high rates of musculoskeletal pain experienced by the Saudi Arabian population.Methods: This population-based, cross-sectional study was done in Saudi Arabia with 2,717 participants aged 18 to 60. A questionnaire was provided online to assess neck, shoulder, and lower back discomfort, time spent in general or aerobic physical activity, time spent sitting, sleep problems, general health, work satisfaction, and nutrition. Using logistic regression, we observed potential risk factors for musculoskeletal pain.Results: The prevalence of neck pain, shoulder pain, and lower back pain (LBP) were found to be 48.1%, 47.6%, and 63.8%, respectively. It was found that being a female , married , and having poor general health status , sleep disturbances  and poor job satisfaction  were independently associated with the prevalence of musculoskeletal pain. The diet of the individuals did not significantly influence the prevalence of MSPs.Conclusion: Good general health, good sleep, and good job satisfaction were associated with a reduced risk of experiencing neck or shoulder pain, but there was no association between physical activity and MSPs Longitudinal studies are required to acquire a better understanding of the relationship between MSP, aerobic activity, sleep, and diet. In many countries, neck and upper body musculoskeletal pain (MSP) is a leading source of morbidity and disability. Individuals' physical, social, and psychological well-being can be compromised, resulting in higher healthcare expenditures ,2. The pGovernments face enormous expenditures due to the recurrence and increasing incidence of MSP, which necessitates the implementation of effective new preventive reforms. Numerous studies on MPS have been performed, particularly in developed nations -12. HoweA population-based, cross-sectional study was an online survey conducted involving participants from different provinces in Saudi Arabia. The study population included adults aged between 18 years and 60 years (considering the age of retirement in Saudi Arabia). Any individual that is younger than 18 years or older than 60 years of age was excluded. A non-probability sampling technique that used a mixture of convenience and snowball sampling was used to collect responses from the participants based on the eligibility criteria. Twenty-two data collectors, who are medical students at Taif University, helped in distributing the questionnaires. An online pretested self-reported questionnaire was used to collect responses from the participants after obtaining their consent. The data were collected between the period of June to August 2022. On the basis of their relevance and prior identification in the literature as risk factors for neck and shoulder pain, several potentially important factors were assessed and identified.The questionnaire consisted of three parts (Appendix A). The first part included sociodemographic details . Part B, the second one, had items that measured the prevalence of neck and shoulder pain, general health status, general state of health, sleep disturbances, aerobic physical activity, time spent sitting, job satisfaction, and time spent working in a week). Responses to the question “Have you suffered any pain in your (body area) within the previous 12 months?” were used to determine the prevalence of lower back pain and neck and shoulder pain. Using a scale from 0 (no pain) to 9 (the greatest pain conceivable) as a reference point. We classified people with back pain into two groups: those with “pain” and “no pain.” In the third part, the dietary intake of each participant was recorded on a dietary chart. This chart had items that recorded the consumption of various food items such as beef, mutton/lamb, chicken, fish, vegetables, fruits, ice cream, chocolates, soft drinks, fruit juice, milk, tea, and coffee. The response options were: (a) Never, (b) 1-2 times/month, (c) once a week, (d) 2-4 times/week, (e) 5-6 times/week, (f) once daily, (g) 2-3 times/day, and (h) ≥ four times/day.Ethical considerationEthical approval for the study was obtained from the research ethics committee of Taif University, Saudi Arabia (IRB No.: 44-069).A pilot study was done on 20 participants to test the questionnaire for validity and reliability. Expert review and focused group discussion were utilized to evaluate the questionnaire's content and face validity. We did not include the items that had a correlation coefficient of more than 0.70. The test's internal consistency was evaluated (Chronbach's α= 0.714), but the test's test-retest reliability could not be determined due to a lack of available time. Online consent was taken after explaining the purpose and benefits of the study .Statistical analysis and data managementAll the collected information was tabulated on a Microsoft Excel sheet and then transferred to IBM Statistical Package for Social Sciences, Version 23  for data analysis. Descriptive statistics in the form of frequencies and percentages using suitable tables and figures were used to represent categorical data. Continuous variables were presented using mean and standard deviation. Comparison of continuous variables between categorical variables was evaluated using the Students' “t” test and/or Analysis of variance. Any possible association between categorical variables was measured using Pearson's chi-square test. Multivariate logistic regression was used to analyze key predictive factors for neck and shoulder pain. A p-value <0.05 was considered statistically significant.We received around 2,798 responses, of which 2,717 gave consent to participate and completed the total items in the survey. The socio-demographic analysis showed that 2,069 (76.2%) were females, 1,417 (52.2%) belonged to the age group of 18-25 years, 1,621 (59.7%) were single (unmarried), 989 (36.4%) were from the Western Province, 780 (28.7%) were employed, 1,716 (63.2%) had an executive, administrative or clerical duties type of job and 473 (17.4%) were obese , shoulder pain (SP), and lower back pain (LBP) were found to be 48.1%, 47.6%, and 63.8%, respectively (Figure The self-reported physical activities and personal habits are given in Table We evaluated the relationship of the prevalence of various MSPs with the participants' sociodemographic, physical activity, and other personal habits Table . It was The Diet Diversity chart of the participants is given in Table We compare the dietary diversity and the prevalence of three types of MSPs Table . There wMultivariate logistic regression was done to predict the risk factors and protective factors for MSPs Table . It was The findings of this study showed that female participants, being married, those with poor general health and sleep disturbances, and those with poor job satisfaction were independently associated with developing all three MSPs. Our findings are consistent with previous studies in different parts of the world. Rashid et al.  observedIt is widely acknowledged that a lack of exercise contributes to NP and poor health -22. StilThe present research found that all three types of MSPs were associated with disrupted sleep. Our findings support the previous pieces of evidence that reported an association between sleep disturbances and MSPs. It has been previously observed by Holm et al.  and FinaNumerous clinical and experimental studies have demonstrated that cigarette smoking negatively impacts the musculoskeletal system and negatively impacts the outcome of a variety of orthopedic illnesses and surgical interventions ,30. Our Our study findings did not show a significant impact of diet on MSPs. Fruits, vegetables, and whole grains are among the foods suggested to be effective for easing MSPs . Omega-3Due to the cross-sectional design of our research, we were unable to establish any causative relationships between the various factors and MSPs. This is the primary limitation of our study. Self-reported estimates of exercise activity levels, general health condition, and diet have also been questioned, which is another limitation. To test these characteristics objectively in every single individual who is a participant in a large population-based cohort with many participants would be incredibly expensive and possibly not even practicable.The current study found that female gender, married, poor general health status, poor sleep quality, and poor job satisfaction were associated with MSPs. However, no significant association was observed between different foods consumed and MSPs. More high-quality studies are needed to evaluate the relationship between MSP and diet. Insights into the relationship between upper body pain, physical activity, sleep, and health may be useful in developing effective ways to prevent and manage neck, shoulder, and lower back pain. Despite the negative effects of smoking on osteoarthritis, rheumatoid arthritis, intervertebral disc degeneration, reduced muscle mass and strength, muscular discomfort, tendons degeneration, and ruptures, smoking was not independently associated with MSP in our study. Some individuals may have quit smoking due to health concerns, but tobacco smoke may also have altered their pain perception threshold. Initiatives to encourage healthy habits should be an organizational policy priority to assist employees in staying healthy and coping with work, especially given the relatively high incidence of unhealthy lifestyles in the general working population. In order to fully comprehend the relationships between MSP and health, sleep difficulties, and aerobic activity, long-term longitudinal studies are warranted.


In this research, the consequence of using rubber tire aggregates (RTA) on the durability and mechanical characteristics of polypropylene fibers (PF) reinforced concrete is evaluated. Fifteen concrete mixtures were produced and tested in the laboratory. RTA was utilized instead of fine natural aggregates (FNA) to the concrete at concentrations of 0%, 5%, 10%, 15%, and 20% by a volumetric fraction; also, the contents of PF in the concrete mixtures were 0%, 1%, and 2% by weight fraction. Finally, the following parameters were tested for all the mixtures: compressive and tensile resistances, fracture, changes in drying shrinkage, bulk electrical resistivity, elastic moduli, and resonance occurrences. The control sample was the one without RTA and PF. According to the results, by adding RTA to the mixtures, the shrinkage deformation amplified, but the PF addition caused a decrease in the shrinkage deformation. Furthermore, adding 0%, 5%, 10%, and 15% RTA, with 2% PF leads to an upsurge in the flexural resistance by 34%, 24%, 16%, and 6%, respectively, relative to the control sample without PF and RTA. Moreover, the fracture energy of mixtures increased by utilizing PF and RTA simultaneously. Compiling RTA around the world can cause a huge environmental risk if we cannot get rid of them properly. One efficient method, from both environmental and economic points of view, is to recycle these waste materials by using them as an aggregate for producing cement-based concrete ,2,3,4. TIn another study, Li et al.  assessedAccording to Feng et al. , strain The subject of another research by Farhad and Ronny  was the Literature reviews illustrate that a significant number of research studies have been performed on the evaluation of RTA-incorporated concrete. The application of RTA-based concrete has been limited to non-structural functions because of some of its significant weaknesses, such as early cracking, low strength, and stiffness, due to the inadequate bonding between RTA and concrete paste. Additionally, the previous finding showed that the incorporation of RTA to produce eco-friendly concrete led to reducing the mechanical characteristics of concrete. Therefore, using other improvement techniques is necessary to improve the performance of green concrete having RTA. In this regard, previous investigations showed that the incorporation of fibers significantly enhanced the mechanical characteristics of various types of concrete ,28,29,303 and 160 °C, correspondingly.To produce fiber-reinforced mixtures, 12 mm length PF were introduced at three volumetric fractions of 0%, 1% and 2%. The tensile resistance, density and melting point of used fibers are 360 MPa, 890 kg/mRTA comes from waste tires with particle sizes of 5–30 mm. RTA is used as a fractional substitution for FNA at five volumetric fractions- 0%, 5%, 10%, 15%, and 20%. Based on the Si et al.  study, ROrdinary Portland cement was utilized for concrete production in a drum-type mixer based on the ASTM C150/C150M . At firsSlum flow is applied to evaluate the fresh concrete behavior. The goal of this is to test the sample’s workability determination based on the ASTM C143 . All theP, D and L are the tensile resistance, the failure point, the samples’ diameter, and the cylinder height, respectively.After 28 days, tests were performed using the ASTM C496  to deterThe tensile resistance of RTA-based concretes that were modified with PF is illustrated in As shown in 2 greater than 92%, as provided in Equation (2). In this equation, New relationships that considered the effects of both RTA and PF were developed in accordance with the findings of the compressive and tensile resistance tests, as shown in L, F, h, and b stand for the span width, maximum load, broken ligament height, and broken ligament width (area above the notch), respectively. The findings are shown in According to the JCI-002-2003 standard, the fracture energy of the sample was calculated . Therefogand Therefore, the JCI-002-2003 standard’s proposed formula  was usedAs shown in Furthermore, the addition of 2% PF together with 0%, 5%, 10%, and 15% RTA caused an improvement in the flexural resistance of the concrete mixtures by 34%, 24%, 16%, and 6%, correspondingly. In contrast, when RTA and PF were utilized in the mixture together, rubber particles increased the mixture’s fracture energy . FollowiThe relations among flexural resistance and fracture energy, as well as among flexural and compressive resistances, are shown in As per the Guo et al. study , the ultAs per ASTM C157 , concretHamed et al.  developeThe quick alkali-silica response test to gauge concrete’s length expansion is described in ASTM C1260 . The speAs per ASTM C666-15 , concretMoreover, the specimens’ resonance frequency was evaluated. The resonant frequency of the samples during the freeze-thaw testing is demonstrated in As per the consequences of the dynamic elastic moduli utilizing the next equation, durability was taken into consideration:DF, P, N, and M, correspondingly. The durability of the samples is shown in The durability feature of the samples, the relation dynamic elastic moduli at N cycles, the number of cycles until P ranges the detailed lowest rate for stopping the examination, and the quantified number of cycles until the experience is to be stopped is represented by the letters At each exposure stage, the length variations of several samples were documented, as can be shown in Workability was reduced by adding more RTA, especially when a combination of 20% RA and 2% PF was utilized, which resulted in a slump reduction of nearly 49%. Conversely, when PF was not utilized in the concrete mixtures, workability was reduced less. So, this property was reduced by about 18% when only 20% RTA was used. Additionally, utilizing 1% PF and 20% rubber reduced slump by about 31%.Utilizing RTA reduced the concrete’s tensile resistance. While PF greatly enhanced this feature. Due to the minor value of RTA relative to FNA, the tensile strength decreased by 28% when 20% RTA was used. Though, incorporating 2% PF amplified the tensile resistance by 5% and prohibited the drop of tensile resistance because of the fiber-bridging character in preventing the quick cracks growth.Compressive resistance of concrete was decreased when RTA was used; however, this characteristic was significantly increased when PF was incorporated. In comparison to concrete produced with merely 0%, 5%, 10%, 15%, and 20% RTA combining 2% PF increased the concrete’s 7-day compressive resistance by about 24%, 29%, 24%, 25% and 22%, correspondingly.By employing RTA, the specimens’ flexural resistance was decreased while PF significantly enhances the flexural performance of the specimens, particularly when RTA was not utilized, due to its bridging function, which increases particle connection and elevates concrete’s tensile and compressive resistances.Incorporating SF and RTA significantly declined the ultrasonic pulse velocity due to the elastic modulus of RTA being lesser than that of the concrete paste and FNA, when 20% RTA was employed, the ultrasonic pulse velocity decreased in the weak zone.Due to the RTA’s lower stiffness than that of the FNA, and because of which RTA could easily deform under the internal drying shrinkage stress, the specimens’ shrinkage length changes increased with the RTA content; however, the addition of PF decreased these length changes. Therefore, using 5%, 10%, 15%, and 20% RTA as a substitute for FNA increased shrinkage length at 28 days by 10%, 14%, 21%, and 27%, correspondingly.A total of 10% of RTA was the optimal amount to maximize bulk resistivity. With the addition of 2% PF and 10% RTA, this characteristic was improved by roughly 12%. It is worth noting that the ideal rate to achieve the highest possible bulk resistivity was established based on the amounts of PF and RTA employed in this investigation. Additionally, SF significantly improved bulk resistivity, particularly when more than 10% RTA was utilized;By adding RTA, the length growth under alkali-silica reaction assault was significantly reduced. Additionally, by adding more RTA, the impact of PF on the alkali-silica response development decreased, and there is no discernible alteration between the length growth of the sample with 2% PF reinforcement and concrete with 1% PF and 20% RTA.According to the materials utilized in this investigation, 10% is the ideal amount of RTA to maximize the durability factor before it begins to significantly decline. Additionally, the durability factor increased by 13%, 31%, and 37%, respectively, when 2% PF was added together with 0%, 5%, and 10% RTA compared to the control mix.Up to 50 freeze-thaw cycles resulted in a momentous change in the specimens’ length. Due to ongoing cement hydration, autogenous shrinkage happened when the specimens were kept in the freeze-thaw chamber. At low cycle counts, the length changes of the specimens abruptly decreased and then gradually increased. The specimens’ length reduction also got worse as PF content increased. Alternatively, it declined as the RTA content increased, and the length changes recovered less as the RTA increased.The current investigation evaluated the characteristics of concrete having PF and RTA. Fifteen mixes were created with this objective in mind. Five ratios of RTA  were employed instead of FNA. The concrete mixes also included PF at three different volume contents: 0%, 1%, and 2%. Then, measurements were made of the specimens’ workability, compressive and tensile and flexural resistances, ultrasonic pulse velocity, shrinkage, electrical resistance, dynamic elastic moduli, and resonance frequency. The subsequent conclusions could be made based on the achieved findings:


Saccharomyces cerevisiae to confirming the pathogenicity of novel mutations and, in some cases, for defining the molecular defects.In eukaryotes, mitochondrial RNAs (mt-tRNAs and mt-rRNAs) are subject to specific nucleotide modifications, which are critical for distinct functions linked to the synthesis of mitochondrial proteins encoded by mitochondrial genes, and thus for oxidative phosphorylation. In recent years, mutations in genes encoding for mt-RNAs modifying enzymes have been identified as being causative of primary mitochondrial diseases, which have been called modopathies. These latter pathologies can be caused by mutations in genes involved in the modification either of tRNAs or of rRNAs, resulting in the absence of/decrease in a specific nucleotide modification and thus on the impairment of the efficiency or the accuracy of the mitochondrial protein synthesis. Most of these mutations are sporadic or private, thus it is fundamental that their pathogenicity is confirmed through the use of a model system. This review will focus on the activity of genes that, when mutated, are associated with modopathies, on the molecular mechanisms through which the enzymes introduce the nucleotide modifications, on the pathological phenotypes associated with mutations in these genes and on the contribution of the yeast  In most eukaryotes, mitochondria are the organelles in which most of the ATP is synthesized. Mitochondria contain their own genome in multiple copies, which is called mitochondrial DNA, or mtDNA. Although in different organisms the length of the mtDNA and the number of mitochondrial genes is different, in most cases mtDNA encodes for all the mitochondrial tRNAs (mt-tRNAs), the mitochondrial rRNAs (mt-rRNAs) and for a few proteins which make part of the respiratory complexes. Most of the mitochondrial proteins, including those involved in oxidative phosphorylation (OXPHOS), are encoded by nuclear genes and transported into mitochondria. However, the few protein-encoding genes present in mtDNA are transcribed and translated inside the organelle thanks to its transcription and translation machinery.In mammals, mtDNA contains 22 genes for mt-tRNA, two genes for mt-rRNA and 13 genes for subunits of the respiratory complexes. Since all these subunits are crucial for the OXPHOS activity, mutations in mtDNA which affect mitochondrial translation are associated with mitochondrial diseases (MDs), which have a maternal inheritance, since the mtDNA is inherited from the mother. To date, more than 300 pathological mutations have been identified in genes encoding for mt-tRNAs and mt-rRNAs, which account for approximately 50% of the mitochondrial pathological mutations .MDs are caused also by mutations in nuclear genes encoding, in most cases, mitochondrial proteins. Approximately 300 genes have been associated with several MDs, which are inherited according to Mendelian laws, as autosomal recessive (AR), autosomal dominant (AD), X-linked recessive (XR), X-linked dominant (XD) or are de novo mutations . In the Most modopathies are caused by a lack of modification of one or more tRNAs. All tRNA molecules have a highly conserved secondary structure similar to a cloverleaf, which consists of stems and loops: acceptor stem, dihydrouridine (D) loop, anticodon loop, variable loop and thymidine-pseudouridine-cytidine, or TΨC (T), loop. Thanks to the base pairing in loops, tRNA form an L-shaped tertiary structure via multiple hydrogen bonds between loops and helices, characterized by an anticodon arm/D arm and a T arm/acceptor stem. All tRNAs possess several and specific modifications, which are post-transcriptionally added to tRNA by specific modifying enzymes. To date, 43 types of tRNA modifications have been identified in humans, which are introduced by 73 enzymes and accessory proteins [Overall, tRNA modifications have several functions. Most of the tRNA modifications regulate the local structure and stabilize the global structure of each tRNA, allowing the recognition of their interactors, in particular the specific aminoacyl-tRNA synthetase and the mRNA-ribosome complex. Some modifications avoid tRNAs being degraded by specific endonucleases, increasing their stability. Modifications in nucleotides of the anticodon are critical in order to recognize multiple synonymous codons for the specific amino acid and to avoid mismatch pairing with wrong codons. Most of the single human mt-tRNAs must recognize all the synonymous codons, up to four for some mt-tRNAs. Most synonymous codons diverge for the third base, which is called the wobble position, which is recognized by the base at position 34, the first base of the anticodon, forming the so-called wobble pair. Modifications at position 34 have the function to relax the recognition of the wobble position, with a limit. If an amino acid is encoded by four synonymous codons in which the wobble position is occupied by each of the four bases , the anticodon must pair with each of the codons, that is the first base of the anticodon should pair with each of the four bases. On the contrary, if an amino acid is encoded by two synonymous codons in which the wobble position is occupied by just a pyrimidine , the first base of the anticodon must pair with U and C, but not with purines, and vice-versa. For such reasons, position 34 in the tRNAs is the most modified base considering the whole group of tRNAs. Furthermore, position 37, adjacent to the third base of the anticodon, is strongly modified in most tRNA, especially with bulky groups. Modifications in such a position can have several functions: stabilization of codon–anticodon pairing, maintenance of the reading frame in order to avoid frameshift and/or preventing unwanted base pairing within the anticodon loop. Other modifications have specific roles: for example, some modifications serve as recognition for specific modifying enzymes which introduce the second modification in adjacent bases , which has the role of the 5S rRNA in cytoplasmic ribosomes [A second, minor group of modopathies are caused by mutations in genes encoding for enzymes involved in the modification of mitochondrial rRNAs (mt-rRNAs) ,59. Humaibosomes . Howeveribosomes ,97,98,99ibosomes ,101,102.Modifications of mt-rRNAs have several functions, including rRNA maturation and stability, the assembly of ribosomal subunits, the optimization of translation and, concerning the modifications of ribose, the stabilization of the structure of the ribosomal RNA-loop as well as the optimization of the peptidyl transferase reaction ,112,113.METTL15 is associated with obesity [TFB1M is associated with type 2 diabetes [MRM2 gene involved in mt-rRNA modification: mitochondrial DNA depletion syndrome 17 (OMIM #618567) with infancy onset [Whereas a polymorphism in  obesity  and a podiabetes , only a cy onset .It must be underlined that, besides covalent modifications of mtRNAs catalyzed by modifying enzymes, mtRNAs undergo non-covalent interactions with other proteins or molecules such as iron-containing groups and polyamines which are critical for their function or the function of their interactors, such as heme bioavailability ,118,119.Saccharomyces cerevisiae has been intensively used as a model system to confirm the pathogenicity and the molecular and phenotypic consequences of mutations in genes associated with MDs, either mitochondrial or nuclear (reviewed in [S. cerevisiae is able to grow either on fermentable or non-fermentable (or oxidative) carbon sources. If mitochondrial oxidative phosphorylation (OXPHOS) is impaired, yeast has a limited capacity or is unable to perform respiration and to grow on non-fermentable carbon sources. Since most MDs result in an OXPHOS deficiency, the pathogenicity of novel putative mutations, in genes already known to be associated with an MD or in novel genes, can be assessed primarily by measuring the growth on non-fermentable carbon sources and the oxygen consumption rate. Several yeast models have been constructed to validate such mutations. These models are mainly based on the gene disruption of the yeast ortholog of the human gene and on complementation studies with wild type and mutant alleles. Depending on specific genes, these assays are performed through homologous complementation with the yeast gene carrying a mutation in the equivalent position of the human ortholog, heterologous complementation with the human gene carrying the mutation or complementation with a mutant chimeric construct harboring a fragment of the yeast gene and a fragment of the human gene [The yeast iewed in ,123,124.COB for complex III, COX1, COX2, COX3 for complex IV, ATP6, ATP8 and ATP9 for complex V), one gene, VAR1, encoding for a subunit of the mitochondrial ribosome; one gene, RPM1, encoding an RNA subunit of the RNase P, which is involved in the processing of the mitochondrial pre-tRNAs; 24 genes for tRNAs; and two genes for the 15S rRNA and for the 21S rRNA. In addition, some genes encoding for maturases, endonucleases and a reverse transcriptase are present inside the introns of the COX1, COB and 21S rRNA genes.Yeast mtDNA, which is longer compared with its mammalian counterpart and can be 68 Kbp (in short strains) to 86 Kbp (in long strains), contains seven genes encoding for subunits of the respiratory complexes  [R mutant yeast strain 15S mt-rRNA. However, mutations in these positions, namely 1555AàG, which pairs with C1494, or 1494CàU, which pairs with A1555, lead to a stabilized hairpin structure that allows the binding of paromomycin and other aminoglycosides, conferring aminoglycosides sensitivity [In ynthesis . If a C esis [R) ,131,132.sitivity . These msitivity ,135.S) strain, but inhibit OXPHOS activity in the mutant, paromomycin resistant strain. Due to the similarity between yeast PR 15S mt-rRNA and human 12S rRNA A-sites, it is possible to exploit yeast to perform complementation analyses of such genes in a paromomycin-resistant background to mimic the normal conditions found in humans  straiR strains. First, point mutations and deletion in MSS1 were shown to determine a respiratory impairment, caused by the block in the splicing of introns of COX1 mRNA due to the inhibition of the synthesis of maturases encoded by mtDNA genes, only in a PR background [MTO1 or MSS1 showed a significant growth defect just in PR strains [MTO2, when studied in a PR background, showed an OXPHOS defect due to a defective expression of some mitochondrial genes, among which COB and COX1 [TRM5 gene were studied both in paromomycin-sensitive and paromomycin-resistant backgrounds; in the latter case, a worsening of the phenotype was visible [Different mutations have been studied in Pckground . Second, strains , probabl strains . Third, and COX1 . Last, m visible , as descMTO1  and GTPBP3 (GTP binding protein 3) are nuclear genes encoding the enzymes that catalyze the formation of 5-taurinomethyluridine (τm5U) (Gln(UUG), tRNAGlu(UUC), tRNALys(UUU) and, putatively, of tRNALeu(UAA) and tRNATrp(UCA) [5s2U) (TRMU (or MTU1 or MTO2) in tRNAGln(UUG), tRNAGlu(UUC) and tRNALys(UUU) [e (τm5U)  in the fTrp(UCA) ,138. Thi) [5s2U) , performLys(UUU) .MTO1 was isolated and characterized starting from the yeast S. cerevisiae orthologous gene MTO1, whose protein sequence was subjected to a BLAST search to identify human homolog. The predicted human MTO1 protein contains 692 amino acids, a typical mitochondrial target sequence, and a predicted molecular mass of 77 kDa. The protein is well conserved through evolution; in fact, the sequence identity is also higher than 50% between S. cerevisiae and E. coli [MTO1 gene maps to chromosome 6q13, and contains 12 exons: five transcriptional variants were identified, resulting from the alternative splicing of exons 4, 6A and 12A or intron 5. Transfection experiments showed that MTO1 localizes exclusively to mitochondria. Northern blot analysis detected a 2.6-kb MTO1 transcript in all the tissues examined, with the highest expression in tissues with high metabolic rates. An additional transcript was also identified in the liver and skeletal muscle [Post-transcriptional modifications of U34 increase the fidelity and the efficiency of mtDNA translation through impact on the tRNA structure, interaction with the ribosomal complex, the stabilization of the codon-anticodon pairing, and tRNA recognition by the correct aminoacyl-transferase (summarized in ). Human  E. coli . MTO1 gel muscle .GTPBP3, containing a typical mitochondrial targeting pre-sequence, were identified starting from the yeast MSS1 gene. The variants are due to alternative splicing and the use of different polyadenylation signals. The GTPBP3 gene maps to chromosome 19p13.11 and contains nine exons. Two variants differ in their 3′-UTRs and encode an identical 492-amino acid protein with a predicted molecular mass of 52 kDa. Two other variants encode an identical 471-amino acid protein, and another variant encodes a longer 524-amino acid protein. [Five variants of protein. . The exoprotein. . The proprotein. .TRMU was identified by searching for sequences similar to the yeast SLM3/MTO2 gene . The cDNA ORF spans 1266bp and encodes for a 421 amino acids long protein with a predicted molecular mass of 48 kDa [TRMU gene maps on chromosome 22q13 and contains 11 exons. Three TRMU splicing variants differing for 99 amino acids were identified; all variants lack exon 3, and one also lacks exon 10, resulting in a truncated protein. Through Northern blot analyses, expression levels were assessed, identifying a 1.9-kb transcript characterized by ubiquitous expression, with the highest levels identified in tissues with high metabolic rates [f 48 kDa . TRMU geic rates .E. coli, GidA  and MnmE are the orthologs of MTO1 and GTPBP3, respectively, and constitute a functional hetero-tetrameric complex and perform the flavin adenine dinucleotide (FAD) and the GTP-dependent incorporation of the carboxymethylaminomethyl (cmnm) group at position five of the first anticodon nucleotide (U34) of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAArg and tRNAGly [Lys, tRNAGlu and tRNAGln, MnmA, the ortholog of TRMU, performs the thiolation at position two of U34 [E. coli and humans, GidA/MTO1, and not MnmE/GTPBP3, seem to be involved in the interaction with the cognate tRNA. However, while in E. coli and yeast GidA/Mto1 and MnmE/Mss1 catalyze the 5-carboxymethyl-aminomethylation of uridine, resulting in cmnm5U, in humans, MTO1 and GTPBP3 catalyze the 5-taurinomethylation of uridine, resulting in τm5U. In bacteria and yeast, 5,10-methylenetetrahydrofolate  is used as the methyl donor and glycine as the substrate which is added to the methyl moiety. In humans, the τm5U modification is synthesized starting from 5,10-CH2-THF and taurine instead of glycine. However, in conditions of taurine starvation, the human MTO1/GTPBP3 complex can use glycine as substrate, resulting in cmnm5U modification. Potassium, GTP and FAD act as cofactors in all cases [In  tRNAGly ,141,142.o of U34 . While to of U34 . GidA sto of U34 . The Mnmo of U34 . The α/βo of U34 . The G do of U34 . Both inll cases ,28,60.2U modification. In yeast and humans, the two cysteines are conserved in TRMU/Mto2 proteins, suggesting a similar mechanism.MnmA presents a cradle-like shape structure consisting of three domains: the N-terminal catalytic domain, the central domain and the C-terminal domain. The catalytic domain is characterized by a mixed α/β fold, contains a P-loop motif and shares similarities with the N-type ATP pyrophosphatases ,148,149.MTO1 are responsible for combined oxidative phosphorylation deficiency 10 , characterized by autosomal recessive inheritance, and resulting in variable defects of the mitochondrial oxidative respiration. The pathology is usually characterized by infancy onset and patients present hypertrophic cardiomyopathy and lactic acidosis. Several patients have been identified as being affected by hypertrophic cardiomyopathy and lactic acidosis due to mutations in the MTO1 gene. Ghezzi and coauthors [MTO1 variants were identified: 17 missenses, five frameshifts, two deletions and one duplication. Two variants, Ala428Thr and Thr411IIe, were frequent in the 16 patients who reported death, suggesting that these mutations could lead to more severe impairment of the mitochondrial translation. The main clinical features are lactic acidosis, cardiovascular, combined complex deficiency, developmental delay, hypotonia, intellectual disability, delayed psychomotor development and abdominal disease/feeding difficulties. Encephalopathy, optic atrophy, ataxia, cognitive impairment, poor speech and spasticity are also present in a fewer number of patients [Mutations in oauthors  reportedoauthors . In the patients . Complexpatients .GTPBP3 are responsible for combined oxidative phosphorylation deficiency 23 , characterized by autosomal recessive inheritance. The pathology is usually characterized by the early childhood onset of hypertrophic cardiomyopathy and/or neurologic symptoms, including delayed psychomotor development and hypotonia [GTPBP3 were reported through the years. Kopajtich and coauthors reported 11 patients from nine families affected by combined oxidative phosphorylation deficiency [Mutations in ypotonia . Fifteenficiency . All patficiency . Three oficiency . All theTRMU are related to two distinct groups of pathologies. Homozygous and compound heterozygous variants are the cause of transient infantile liver failure . Mutations in this gene impair mitochondrial protein synthesis and oxidative phosphorylation, determining the disease. A total of sixty-two patients carrying mutations in TRMU have been reported. Zeharia and coauthors first described 13 individuals with mutations in this gene; four of them died and the others showed no further neurologic or hepatological issues over the years of follow-up [TRMU acts as a nuclear modifier to modulate the clinical manifestation of deafness-associated 12S mt-rRNA mutations (OMIM #580000); aminoglycoside-induced deafness is a maternally inherited pathology that determines sensorineural deafness, ranging from hearing loss to moderate hearing impairment, with no myopathy or neurological symptoms [TRMU was identified as a nuclear modifier gene for the phenotypic expression of 12S mt-rRNA-related deafness; in particular, TRMU carrying the mutation p.Ala10Ser, leading to a mitochondrial translation impairment, and the A1555G mutation in 12S mt-rRNA modulates the manifestation of the pathology [TRMU alleles worsens the translation impairment caused by the A1555G variant, determining impaired respiratory activity and, subsequently, elevated ROS production and reduced ATP synthesis [Mutations in ollow-up . Subsequollow-up . Forty-eollow-up . Furthersymptoms ,152. Thesymptoms  and resuathology . Impaireynthesis .5U34) group (Lys(UUU), mt-tRNAGlu(UUC) and mt-tRNAGln(UUG), and the impairment of Mss1 and Mto1 activity can determine the loss of this modification [5s2U34 (Lys(UUU), tRNAGlu(UUC), and tRNAGln(UUG) [MTO2 determines the instability of mt-tRNAs and the subsequent impairment in the aminoacylation of the tRNAs [MTO2 and of MTO1 or MSS1, but not of the single gene, results in the inability of growing on oxidative carbon sources, as a consequence of reduced levels of the three mt-tRNA substrates of the enzymes, low levels of COX1 and COB mRNA and the complete loss of mitochondrial protein synthesis.Yeast Mto1 and Mss1 are responsible for the biogenesis of the 5-carboxymethylaminomethyl (cmnm4) group  of the wfication ,60. Mto2 [5s2U34  of mitocGln(UUG) . The delhe tRNAs . The delR yeast strain harboring mutation C1477G in the 15S mt-rRNA showed resistance to paromomycin but hypersensitivity to neomycin, another aminoglycoside; in fact, transcription levels of mtDNA, respiratory activity and membrane potential were severely decreased after neomycin treatment. The mto2 null mutant strain reduces hypersensitivity to this antibiotic, suggesting an interaction between the 2-thiolic group introduced by Mto2 and the A-site of 15S mt-rRNA. These findings show the involvement of MTO2 in the modulation of aminoglycoside sensitivity in the PR background in S. cerevisiae, as in humans harboring mutations in the corresponding region of the 12S mt-tRNA [The P mt-tRNA .MTO1 in yeast, it was possible to exploit this model organism to study the alleged pathological mutations. Human mutations p.Arg620Lysfs*8 and p.Ala428Thr were studied using the strains carrying the respective mutations in yeast MTO2, P622* and A431T [S strain), the mto1 null mutant strain showed a slight decrease in the oxygen consumption rate, and expression of the P622*mto1 allele does not recover this phenotype, indicating that the variant truncated at amino acid 620 is functionally inactive. The decreased oxygen consumption rate was only partially rescued by the A431Tmto1 allele compared with the full rescue by the MTO1 wild type allele, also showing that this mutation determines pathological defects. However, no oxidative growth defect was visible for all the strains. To better understand the role of these mutations in human disease, mutations under analysis were studied also in the PR background, mimicking the wild type human condition of the small subunit mt-rRNA, as reported in mto1 null strain was not able to grow on oxidative carbon sources such as glycerol. No oxidative growth was visible for the P622* mutant, while a leaky growth defect was visible for the strain carrying the A431T variant compared with the fully restored growth obtained with the expression of the wild type allele. The respiratory activity results parallelized oxidative growth analyses, supporting the pathogenicity of these mutations. To confirm the pathogenicity of a second group of variants identified in five patients, PR mto1 null strain carrying T414Imto1 and R481Hmto1 mutant alleles, equivalent to human mutations p.Thr411Ile and p.Arg477His, respectively, were studied [Thanks to the presence of nd A431T . In a ge studied . No oxidT414Imto1 gene product is fully impaired [Structural analysis showed that substitution with isoleucine of Thr414 may alter the position of serine 371, involved in the catalytic process through binding or stabilization of FAD; therefore, in an attempt to rescue the phenotype with a small molecule, the effect of riboflavin supplementation was evaluated on the T414I mutant. No effect was visible on oxidative growth and respiratory activity, suggesting that the catalytic activity of the impaired .R background. No defects in the de novo mitochondrial protein synthesis profile were observed in the strain harboring the A431T or R481H variants. On the contrary, the strain without mto1 or harboring the P622* or the T414I variants showed normal levels of newly synthetized Var1, Cox3, Atp6 and Atp8/9, whereas Cox1, Cox2 and Cob were absent, indicating that complex III and IV should be impaired. Indeed, complex IV activity was abolished in the strains with P622* and T414I variants, whereas a significant reduction of the activity was observed for the strain carrying the A431T variant compared with the strain carrying the MTO1 wild type allele, and no impairment in CIV activity was observed for the strain carrying the R481H variant. Functional analyses in yeast allowed the validation of the human variants identified in patients and the severity of the phenotype are in accordance with the clinical manifestation in patients, supporting the utility of yeast as a model for the validation of alleged pathological mutations. In agreement with this hypothesis, the most deleterious mutations are associated with a strong defect in protein synthesis, whereas the mild mutations are associated with normal levels of protein synthesis; the latter observation leads to the hypothesis that the mitochondrial protein are still synthesized but their sequence may be altered due to incorporation of the wrong amino acids due to defect in the codon–anticodon pairing when the cmnm5 modification does not occur [Molecular consequences were assessed by performing mitochondrial protein synthesis in the Pot occur .MSS1 and TRMU allegedly associated with primary mitochondrial diseases have been modeled in yeast, although yeast has been used, as reported previously, for studying a possible effect of mutations in the genes reported in this section with aminoglycoside-induced ototoxicity and deafness.To date, no mutations in TRMT5, also known as TRM5, is a nuclear gene encoding the tRNA methyltransferase 5. TRMT5 methylates the N1 position of guanosine 37 (m1G) (Leu(GAC) and the mitochondrial tRNAPro(UGG) using S-adenosyl methionine (AdoMet) as substrate [TRMT5 was first discovered by sequencing clones deriving from a size-fractionated cDNA library from the human brain and called KIAA1393 [TRM5 sequence, KIAA1393 was found as a homologous human sequence, encoding a protein made up of 500 amino acids, and was renamed TRMT5. TRMT5, which maps to chromosome 14q23.1, contains five exons with an additional upstream exon with an importation signal that could enable the protein localization in the mitochondria, to allow the methylation of mitochondrial tRNAs [1G37 in bacteria, has a size of 28 kDa and functions as a homodimer [Methanocaldococcus jannaschii protein. D3, the C-terminal domain, defining the class I methyltransferases, is characterized by the Rossmann fold and binds the substrate AdoMet. D2 is the central domain associated with D3, and these two domains play the leading role in tRNA methylation. Moreover, D1, the N-terminal domain, not conserved through evolution, is structurally independent of D2-D3 [+.37 (m1G) , the nucubstrate . The metubstrate ,156. TRMKIAA1393 . Throughal tRNAs . TrmD, tomodimer : the intomodimer ,160. Arcomodimer ,161,162.omodimer , while Tomodimer ,164. Theof D2-D3 . This enMutations in this gene are responsible for peripheral neuropathy with variable spasticity, exercise intolerance and developmental delay , characterized by autosomal recessive inheritance. This pathology is a multisystemic disorder with variable manifestations, within the same family too. Some patients show symptoms with infantile/childhood onset. Among the symptoms, there are hypotonia and global developmental delay with poor or absent motor skill acquisition and poor growth. Other patients present symptoms as young adults with exercise intolerance and muscle weakness. All patients have peripheral neuropathy, usually demyelinating, with distal muscle weakness, atrophy and distal sensory impairment. Spasticity, extensor plantar responses, contractures, cerebellar signs, seizures, short stature and the rare involvement of other organ systems are additional features of this pathology. Deficiencies in mitochondrial respiratory complex enzyme activities in patient tissues are sometimes identified through biochemical analyses, despite the fact that they are not always apparent. As a mitochondrial dysfunction hallmark, lactate is frequently increased ,90.Eight patients have been identified with PNSED; two unrelated patients with a high variable phenotype, caused by a defective mitochondrial respiratory chain activity, were reported . One patTRM5, the yeast ortholog of human TRMT5, encodes for a protein that is responsible for the methylation of both cytoplasmatic and mitochondrial tRNA. In cytoplasm, Trm5 methylates cytoplasmic tRNALeu(UAR), tRNAPro(NGG), tRNAArg(CGG), tRNAHis(GUG), tRNAAsp(GUC) [Ala(IGC) that presents, after modification catalyzed by the adenosine deaminase Tad1 [1I37). In addition, it participates, catalyzing the methylation at the N1 position, to the formation at position G37 of tRNAPhe(GAA) of the deeply modified nucleoside wybutosine, an unusual nucleoside deriving from guanosine containing three rings, whose aim it is to restrict the flexibility of the anticodon and whose synthesis requires at least five genes [fMet(CAU) and tRNAPhe(GAA) [His(GUG), tRNALeu(UAA), tRNAPhe(GAA), tRNAPro(UGG), tRNASer(UGA) and tRNAThr(UAG) [Asp(GUC)  and tRNAase Tad1 , inosineve genes ,171,172.Phe(GAA) , and putThr(UAG) .Leu(UAG). With regard to the structural aspect, Arg291 amino acid is in close proximity to Glu288, a conserved amino acid involved in the catalytic domain of the protein, so that a substitution of this could affect the interaction with residue 288, leading to a destabilization of the active site. Met386 does not have a direct catalytic role, but a small aliphatic amino acid, such as valine, may alter the position of Asn387, an amino acid of the catalytic pocket. Although the structural analysis suggested a pathogenic role of these mutations, an ad hoc functional analysis was necessary to correlate the role of these mutations with the defects in the respiratory complexes observed in patients. In the study, yeast was exploited to validate the pathogenicity of these two mutations, constructing a strain expressing mitochondrial TRM5 wild type or mutant alleles harboring the equivalent mutation, R270H and M396V, respectively. Since Trm5 acts both on cytoplasmatic and mitochondrial tRNAs, the trm5 null mutant is lethal. To bypass the lethality and study the effect only on mt-tRNAs, a strain lacking the gene fragment which encodes for mitochondrial targeting sequence was created (trm5Δ1-33). This strain showed only a leaky respiratory deficiency, suggesting that methylation of 37G in mt-tRNAs is not critical in yeast for proper mitochondrial protein synthesis. However, as in the case of MTO1/MSS1 and MTO2, the respiratory deficiency was exacerbated in the PR strain described in TRM5, whereas strains carrying the mutations R270H or M396V led just to a partial rescue of the respiratory activity, suggesting that they are the cause of the pathogenicity in patients when in compound with a null allele.Thanks to the presence of the human ortholog in yeast, the pathogenicity of two variants, p.Arg291His and p.Met386Val, found in different patients in compound heterozygosis with the frameshift mutation p.Ile105Serfs∗4, was confirmed . Both paTRIT1 encodes for the human tRNA isopentenyl transferase (IPTase), an enzyme of 50 kDa responsible for the addition of an isopentenyl group at position N6 of adenosine 37 (i6A) , tRNASer(CGA), tRNASer(UGA) and tRNASec(UCA), which inserts selenocysteine in specific proteins, and the mitochondrial tRNACys(GCA), tRNATrp(UCA), tRNATyr(GUA), tRNASer(UGA) and tRNAPhe(GAA) [37 (i6A) , in seve library . TRIT1, Phe(GAA) . All thePhe(GAA) ,177.S. cerevisiae IPTases bound to their substrates have shown a direct recognition of the anticodon loops (ACLs) of their target tRNAs, and the i6A37 formation using dimethylallyl-pyrophosphate (DMAPP) as an isopentenyl donor. The eukaryotic dimethylallyl-pyrophosphate transferase structure is composed of a core domain, remarkably similar to that of the bacterial enzyme, and of an insertion domain with a five-helix bundle and connected to the core domain through two loops. It is also composed of the C-terminal extension that can be divided into two parts and this is unique to the eukaryotic enzyme. The C-terminus is a zinc finger connected to the core domain trough a linker region of 30 amino acids and composed of two short helices and loops traversing along the core domain. Interaction between tRNA and DMAPP transferase is extensive and is mainly concentrated at the anticodon stem-loop [The homologous tRNA isopentenyl transferases have been well conserved during the evolution from bacteria to humans; however, several changes have occurred in order to also target the proteins to mitochondria. Biochemical studies and the structures of bacterial and tem-loop ,181,182.TRIT1 on chromosome 1p34. To date, only eight cases and seven allelic variants have been reported of this disorder. The affected patients are characterized by variable deficiencies in mitochondrial respiratory enzyme complexes and the most frequent symptoms observed are intellectual disability, hypotonia, microcephaly, global development delay and central nervous system involvement. These symptoms can occur in early infancy and the severity can be variable. The mutations identified in these patients are p.Arg323Gln, p.His419Pro, p.Ile283Ser, p.Arg402Ter, p.Lys286Glu, p.Arg8Ter and p.Glu327Lys. The p.Arg323Gln mutation was identified by whole-exome sequencing in two siblings born from consanguineous parents of Pakistani origin [Mutations in this gene lead to a pathology called combined oxidative phosphorylation deficiency 35 , a rare autosomal recessive disease caused by homozygous or compound heterozygous mutations in the tRNA isopentenyl transferase gene i origin . The p.Gi origin .MOD5 is the yeast homolog of the human TRIT1 and the similarity between the two proteins is 53%. Besides its enzymatic activity, the yeast counterpart furthermore binds the RNA polymerase III transcription complexes on nuclear tRNA genes helping the “silence” of the transcription of RNA polymerase II [Ser(AGA), tRNASer(CGA) and tRNASer(UGA), as in humans, and the specific tRNATyr(GUA) and tRNACys(GCA)), and three mitochondrial tRNAs (tRNATrp(UCA), tRNATyr(GUA) and tRNACys(GCA)) instead of five as found in humans [Schizosaccharomyces pombe, it was shown that i6A37 increases the translational efficiency and the fidelity in a codon-specific manner cognate with tRNAs containing the i6A37 [erase II . Unlike n humans . In the he i6A37 .6A37 modification. To evaluate whether these defects correlated with a defective oxidative phenotype, an S. cerevisiae strain deleted in MOD5 was generated and transformed with a mod5 allele harboring arginine instead of the naturally occurring lysine at position 294, equivalent to p.Arg323 in TRIT1 (humanized MOD5) or a mod5 allele harboring glutamine at position 294 as found in patients . The yeast strain transformed with the mutant allele showed reduced growth in a medium supplemented with ethanol, a non-fermentative carbon source, compared with the strain with the humanized MOD5. The respiratory activity was reduced in the mutant strain compared with the humanized strain as well, indicating that the growth defect on oxidative carbon sources was due to impaired respiratory activity. The correlation between enzymatic defect found in patients’ cells and a respiratory phenotype was confirmed also in another yeast model, the fission yeast Schizosaccharomyces pombe deleted in the orthologous gene TIT1 and transformed through heterologous complementation with a human TRIT1 wild type and the mutant allele . The wild type allele, but not the mutant one, rescued the oxidative growth defect on a medium supplemented with the non-fermentable carbon source glycerol. In addition, a defect in the enzymatic activity was confirmed by the red–white assay in S. pombe. The red-colored phenotype in the presence of low adenine in the medium was due to nonsense mutation in ADE6. The ADE6 mutant allele contains a premature UGA stop codon, which can be suppressed by the suppressor tRNASer(UCA), an isoform derived from the gene for the tRNASer(UGA) after a G→C transversion provided that it, as does its human counterpart, contains i6A37, which is critical for the pairing of the anticodon to UGA and thus for the translational activity of the suppressor-tRNA [Using the yeast model has been critical to confirm the pathogenicity of a homozygous variant harboring mutation p.Arg323Gln found in two siblings with encephalopathy and myoclonic epilepsy due to multiple OXPHOS deficiencies in skeletal muscle, born from consanguineous parents of Pakistani origin as reported above. Studies on patients’ cells showed a decrease in the respiratory complex activity, in the in vivo mitochondrial protein synthesis, in the steady state levels of several subunits of respiratory complexes, and in the levels of cytosolic and mitochondrial tRNA harboring isor-tRNA . Coloniesor-tRNA .TRNT1 encodes for the tRNA-nucleotidyltransferase, an enzyme involved in the addition of the CCA trinucleotide to the 3′ end of all mitochondrial and cytoplasmic tRNAs. Although, strictly speaking, the addition of the CCA stretch is not a nucleotide modification, here we discuss also the effects of TRNT1 mutations since the addition of CCA is considered an ad hoc modification that occurs after transcription and processing of the pre-tRNAs [re-tRNAs . Indeed,re-tRNAs ,188. In re-tRNAs ,191,192.TRNT1, which maps to chromosome 3p26.2, contains seven exons and encodes for at least one cytoplasmic isoform and two mitochondrial isoforms. TRNT1 is an essential gene: siRNA knockdown of TRNT1 in human fibroblasts is responsible for cytotoxicity and apoptosis [CCA1, is lethal. Although the catalyzed reaction is identical, there are important differences found in the three kingdoms of life both in the overall structure and in the organization of the catalytic core [Archaeoglobus fulgidus, a 51 kDa protein containing 437 amino acids and its nucleotide complexes with ATP, CTP and UTP [poptosis , while ttic core ,193. The and UTP . The str and UTP .TRNT1 gene cause a pathology called congenital sideroblastic anemias , which include a group of autosomal recessive syndromic and nonsyndromic inherited diseases [TRNT1 gene. The main mutations include substitutions p.Thr154Ile, p.Leu166Ser, p.Arg190Ile, p.Ile233Thr and p.Glu43 in-frame deletion, and several intron mutations which result in altered splicing or frameshift mutations which result in a truncated protein.Mutations in the diseases ,197. Thediseases ,198. TheSaccharomyces cerevisiae, a single gene CCA1 encodes for tRNA CCA nucleotidyltransferase. Aebi and coauthors demonstrated its role after the isolation of a temperature-sensitive (ts) allele, ts352 [In the yeast e, ts352 . Cells be, ts352 . Using me, ts352 .Saccharomyces cerevisiae was exploited by examining at first the ability of human TRNT1 variants, cloned under the ADH1 promoter, to complement the lethality at high temperatures of the mutant strain harboring the ts352 mutant allele. At first, it was observed that the heterologous expression of the wild type human TRNT1 allele restored the growth at the non-permissive temperature in a similar way as the wild type yeast CCA1 allele, indicating that the human enzyme is also capable of modifying yeast tRNAs. TRNT1 mutant alleles harboring the mutations p.Thr154Ile, p.Met158Val, p.Leu166Ser, p.Arg190Ile, p.Ile223Thr or p.Ile326Val, found in different patients, are able to partially complement the temperature-sensitive growth defect, but at a lower extent compared with wild type CCA1 or TRNT1 and, at a different extent, among them, indicating that all the mutations affect the modification of tRNAs. Comparable results were reported by expressing wild type and mutant TRNT1 alleles, under the ADH1 promoter or the native CCA1 promoter, in a strain deleted in CCA1. Transformation through plasmid shuffling showed that the wild type TRNT1 allele restored the viability and the full growth of the strain. The same results were observed for TRNT1 variants harboring p.Ile326Val and p.Lys416Glu mutations, which are located far from the active site, suggesting that these mutant alleles are milder. On the contrary, TRNT1 variants harboring the remaining mutations, all close to the active site, restored just partially and at different extents the growth, being the mutations p.Leu166Ser, p.Arg190Ile and p.Ile223Thr the most deleterious. In addition, the latter two mutant strains showed an extremely poor growth on a medium supplemented with a non-fermentable carbon source such as glycerol, suggesting a defect in oxidative phosphorylation likely due to an impairment of the mitochondrial translation. Moreover, the protein levels of all TRNT1 variants were decreased compared to wild type TRNT1, though at different extents and with the exception of the p.Ile326Val variant, whose levels seem to be increased, indicating that the mutation affects the stability of TRNT1, as found also in patients’ fibroblasts [The yeast roblasts .MRM2  encodes for the mitochondrial rRNA methyltransferase 2, an enzyme of 27 kDa responsible for the methylation of the 2′-O-ribose of U1369 of the 16S mt-rRNA A-loop, resulting in Um  binding domain. It was also observed that MRM2 is essential for the biogenesis of the large subunit of the mitochondrial ribosome (mtLSU) due to its capacity to remodel the conformation of 16S rRNA in the late stages of the process: this activity is independent from the methyltransferase activity, since mutations which abolish the latter did not affect the biogenesis [MRM2. These defects are caused by the presence of mtLSUs trapped in immature assembly states with unstructured IV and V domains of the mt-rRNA which prevent the initial engagement of the immature particles in translation [7Gppp-cap [2+ ion seems to be important for the substrate binding and the placement of the 2′-hydroxyl group of the ribose near to the methyl group of AdoMet. 2′-O-ribose methylation on the RNA changes the local properties of the RNA, which can be easily detected [f 27 KDa . MRM2 be members . The humogenesis . For thinslation . AlthougGppp-cap . From thdetected ,209,210.detected ,207.Mutations in this gene lead to a pathology called mitochondrial DNA depletion syndrome 17 (OMIM #606906), an autosomal recessive pathology characterized by development delay, generalized dyskinesia and ballismus, also involving cervical and oropharyngeal muscles. To date, only one mutation, the p.Gly189Arg, has been reported in homozygosis in a boy with a MELAS-like mitochondrial depletion syndrome, and is absent in the ExAC database. Glu189 is located near the AdoMet binding site and the catalytic site .S. cerevisiae there are three proteins similar to the E.coli 2-O-methyltransferase FtsJ/RrmJ [MRM2 [MRM2 gene did not show any difference in growth at 30 °C compared with the wild type isogenic strain regardless of whether the carbon source is fermentable or non-fermentable. However, growth was severely reduced on a medium supplemented with glycerol at 37 °C, suggesting that the methylation catalyzed by Mrm2 is critical for a proper mitochondrial translation at high temperatures [In the yeast tsJ/RrmJ : Spb1, atsJ/RrmJ ; Trm7, atsJ/RrmJ ; and MrmmJ [MRM2 . The deleratures .Saccharomyces cerevisiae was exploited [MRM2 wild type allele and the G259Rmrm2 mutant allele, harboring the mutation corresponding to the human one, were cloned in a centromeric vector under its promoter and inserted in a strain deleted in the endogenous MRM2 gene, which showed a strong decrease in the oxygen consumption rate. Contrarywise to the wild type allele, the mutant allele failed to restore the oxygen consumption rate, indicating that the mutant protein is unable to complement the respiratory phenotype. It was also studied to examine if the mutant allele determines a reduction in Um2791 modification of the yeast 21S mt-rRNA, which corresponds to Um1369 in human 16S mt-rRNA [mrm2 null strain demonstrated extremely low 2′-O-methyl pausing at U2791, consistent with the lack of modification at this site due to the lack of Mrm2. The expression of a wild type MRM2 allele restored the Um2791 methylation, whereas the expression of the G259Rmrm2 mutant allele resulted in a 30% decrease in the methylated 21S mt-RNA levels compared with the former strain. Analysis in yeast thus confirmed the pathogenicity of the mutant variant, which is associated with a reduction of the methylation activity of the mt-rRNA and a consequent impairment of the oxidative phosphorylation [To study the effects of the p.Gly189Arg mutation identified in humans, the yeast xploited . The MRM mt-rRNA . The abirylation .Primary mitochondrial disorders (MDs) are a group of clinically heterogeneous defects caused directly or indirectly by an energy impairment. MDs can be caused by mutations in either the nuclear or mitochondrial genome, with a collective incidence of 1.6 in 5000; however, given their high genetic heterogeneity, each single gene defect is extremely rare ,215.Among MDs, a particular class is represented by modopathies , caused MRM2 [A large variety of modifications can occur on tRNAs and at least 18 types have been reported for human mt-tRNA . tRNA moMRM2 .Most of the mutations in genes coding for mt-tRNA or mt-rRNA modifiers known to date have been identified thanks to next generation sequencing techniques (NGS), encompassing panel sequencing, whole exome sequencing (WES) and whole genome sequencing (WGS). The use of NGS has been fundamental for achieving a genetic diagnosis of several mitochondrial diseases because MDs are amongst the most genetically and phenotypically diverse groups of inherited diseases and suffer from the lack of a single reliable biomarker . The oveSaccharomyces cerevisiae has been extensively used to study mitochondrial diseases and has proved its usefulness in validating the pathogenicity of newly identified variants in several genes and in assessing the mechanisms involved in mitochondrial dysfunction. Yeast, therefore, represents a powerful tool to deepen the knowledge about mitochondrial pathways both in health and disease. More generally, the success of this experimental single-cell organism relies on its efficient homologous recombination, which easily and quickly allows the creation of genetic knockouts, and on the easiness of its manipulation thanks to well-known genetic tools, including transformation and plasmid shuffling. Another particularly useful feature is that yeast exists in both haploid and diploid forms. The study of a mutant variant in a haploid genetic background allows it to be immediately validated even if it is recessive in humans. In addition, several intracellular pathways are highly conserved between yeast and humans, including mitochondrial ones. Remarkably, more than 50% of the 1000 proteins estimated to be in yeast mitochondria have a human homolog, and 70% of the nuclear genes involved in human mitochondrial diseases are conserved in yeast [Saccharomyces cerevisiae without the need for a patient-derived tissue or cell line. One of the most valuable features of using yeast to study the effect of putative pathogenic variants is that it is possible to introduce every allele under investigation in a genetically identical background thus avoiding the possible confounding effects due to the genetic heterogenicity of the patients that could influence the severity and the progression of the disease. It is also possible to study separately two alleles found in compound in a patient in order to determine the contribution of each of them to the phenotype. In addition, the severity of the mitochondrial function impairment observed in yeast often correlates with the severity of the clinical phenotype of the patients.Thanks to its ability to survive and grow when the mitochondrial metabolism is impaired, the yeast in yeast . NotablySaccharomyces cerevisiae will continue to prove to be an invaluable model system both to prove the pathogenicity of new variants and to dissect the molecular mechanisms underpinning these diseases. Although yeast, as a unicellular organism, cannot help to elucidate tissue-specific defects or phenotypes related to cell-to-cell communication or to immune response, nor for studying the effect of alleged pathological mutations at the level of tissues or organs, it can be exploited to point out the intracellular pathways involved in the disease.Although in the last few years the genetic diagnosis of MDs has been improved thanks to the extensive use of NGS, in a large fraction of patients the genetic basis is still unknown. Considering that the human mitochondrial proteome consists of approximately 1500 proteins, it is expected that many more disease genes will be discovered in the coming years. Some functions fulfilled by human mitochondria do not exist in yeast; however, Despite some differences in mitochondrial DNA length and codon usage, a high degree of similarity is shared between yeast and human mtDNA transcription and translation. Several of the mt-RNA modifying enzymes found in humans are present and conserved in yeast, especially the enzymes which modify the tRNAs inside or near the anticodon, thus allowing the use of yeast as a model system. For the non-conserved genes/pathways, other models must be used.Val are not present in yeast [Saccharomyces cerevisiae [To date, few mutations associated with modopathies were modeled in yeast and the studies designed until now are mostly limited to the validation based on oxidative growth defect and the reduced oxygen consumption rate of the mutant carrying the alleged pathological mutation. In some cases, the molecular consequences were assessed by performing mitochondrial protein synthesis or measuring the levels of the modified RNA. However, it is expected that the number of novel mutations, the number of novel genes and the number of novel modopathies will increase, and the role of yeast in determining a genotype–phenotype correlation and the defects at the molecular level will be likely fundamental. However, it must be underlined that the mitochondrial translation machinery is partially different between humans and yeast, and this could limit, in some cases, the usefulness of yeast for validation. First of all, the 5S rRNA is not imported in yeast mitochondria, so the effects of mutations in such molecules or in proteins involved in its import cannot be studied in yeast. In addition, the number of the proteins of the mitoribosomes and the structures of some of them are different between the two organisms, both in the mtSSU and in the mtLSU, where, for example, the proteins which bind to the tRNAin yeast ,220. Thiin yeast . Finallyrevisiae ,222.Despite the tremendous progress made in MD’s diagnosis over the last years, therapy is still insufficient and mostly limited to relieving symptoms. Therefore, there is an urgent need to develop new drugs to treat these diseases, especially pathologies recently discovered, such as most modopathies. In order to identify drugs able to rescue the detrimental effects of pathological mutations, a high-throughput phenotype-based approach called “drug drop test” was set up in yeast allowing to quickly screen a large number of molecules taking advantage of the oxidative defect of the strains defective in mitochondrial functions . In the