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Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m 1 G9 methylation, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving the structure and activity of the variant. These results have therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases., (© 2024. The Author(s).)

Brain functions are mediated via the complex interplay between several complex factors, and hence, identifying the underlying cause of an abnormality within a certain brain region can be challenging. In mitochondrial disease, abnormalities in brain function are thought to be attributed to accumulation of mitochondrial DNA (mtDNA) with pathogenic mutations; however, only few previous studies have directly demonstrated that accumulation of mutant mtDNA induced abnormalities in brain function. Herein, we examined the effects of mtDNA mutations on brain function via behavioral analyses using a mouse model with an A2748G point mutation in mtDNA tRNA Leu(UUR) . Our results revealed that mice with a high percentage of mutant mtDNA showed a characteristic trend toward reduced prepulse inhibition and memory-dependent test performance, similar to that observed in psychiatric disorders, such as schizophrenia; however, muscle strength and motor coordination were not markedly affected. Upon examining the hippocampus and frontal lobes of the brain, mitochondrial morphology was abnormal, and the brain weight was slightly reduced. These results indicate that the predominant accumulation of a point mutation in the tRNA Leu(UUR) gene may affect brain functions, particularly the coordination of sensory and motor functions and memory processes. These abnormalities probably caused by both direct effects of accumulation of the mutant mtDNA in neuronal cells and indirect effects via changes of systemic extracellular environments. Overall, these findings will lead to a better understanding of the pathogenic mechanism underlying this complex disease and facilitate the development of optimal treatment methods., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kaori Ishikawa reports financial support was provided by Japan Science and Technology Agency. Kaori Ishikawa, Kazuto Nakada reports financial support was provided by Japan Society for the Promotion of Science. Kazuto Nakada reports financial support was provided by Japan Agency for Medical Research and Development. Tsuyoshi Miyakawa reports financial support was provided by Government of Japan Ministry of Education Culture Sports Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This paper discusses the cases of siblings that were born healthy, then diagnosed in their neonatal periods with cardiomyopathy and/or severe metabolic acidosis, which ran progressive courses and contributed to death in infancy. Molecular testing of the children confirmed the presence of an m.3303C>T point mutation in the mitochondrial DNA in the MT-TL1 gene, which was also present in their oligosymptomatic mother and their mother's sister, an asymptomatic carrier.

In the yeast genera Saccharomycopsis and Ascoidea, which comprise the taxonomic order Ascoideales, nuclear genes use a nonstandard genetic code in which CUG codons are translated as serine instead of leucine, due to a tRNA-Ser with the unusual anticodon CAG. However, some species in this clade also retain an ancestral tRNA-Leu gene with the same anticodon. One of these species, Ascoidea asiatica, has been shown to have a stochastic proteome in which proteins contain ∼50% Ser and 50% Leu at CUG codon sites, whereas previously examined Saccharomycopsis species translate CUG only as Ser. Here, we investigated the presence, conservation, and possible functionality of the tRNA-Leu(CAG) gene in the genus Saccharomycopsis. We sequenced the genomes of 23 strains that, together with previously available data, include almost every known species of this genus. We found that most Saccharomycopsis species have genes for both tRNA-Leu(CAG) and tRNA-Ser(CAG). However, tRNA-Leu(CAG) has been lost in Saccharomycopsis synnaedendra and Saccharomycopsis microspora, and its predicted cloverleaf structure is aberrant in all the other Saccharomycopsis species. We deleted the tRNA-Leu(CAG) gene of Saccharomycopsis capsularis and found that it is not essential. Proteomic analyses in vegetative and sporulating cultures of S. capsularis and Saccharomycopsis fermentans showed only translation of CUG as Ser. Despite its unusual structure, the tRNA-Leu(CAG) gene shows evidence of sequence conservation among Saccharomycopsis species, particularly in its acceptor stem and leucine identity elements, which suggests that it may have been retained in order to carry out an unknown nontranslational function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

T cells have been shown to maintain a lower percentage (heteroplasmy) of the pathogenic m.3243A>G variant (MT-TL1, associated with maternally inherited diabetes and deafness [MIDD] and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes [MELAS]). The mechanism(s) underlying this purifying selection, however, remain unknown. Here we report that purified patient memory CD4+ T cells have lower bulk m.3243A>G heteroplasmy compared to naïve CD4+ T cells. In vitro activation of naïve CD4+ m.3243A>G patient T cells results in lower bulk m.3243A>G heteroplasmy after proliferation. Finally, m.3243A>G patient T cell receptor repertoire sequencing reveals relative oligoclonality compared to controls. These data support a role for T cell activation in peripheral, purifying selection against high m.3243A>G heteroplasmy T cells at the level of the cell, in a likely cell-autonomous fashion., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Mitochondrial diseases are disorders caused primarily by mutations in mitochondrial DNA, with the mitochondrial 3243A > G (m.3243A > G) mutation being one of the most common pathogenic mutations. Here, a pluripotent stem cell line with high m.3243A > G mutation load was generated by reprogramming the skin fibroblasts from a patient with mitochondrial disease. This cell line exhibited pluripotency, multilineage differentiation potential and normal karyotype, representing a valuable cell resource for studying the pathogenesis of mitochondrial diseases and screening drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNA Ser GAG and tRNA Leu GGA . This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors., (© 2024. The Author(s).)

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNA Gly and BioRNA Leu , respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNA Gly and BioRNA Leu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNA Gly /miR-7-5p exhibited greater efficiency to regulate a known target gene expression ( EGFR ) than BioRNA Leu /miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNA Gly /miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development., (© 2024 Traber et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNA Leu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNA Leu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNA Leu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNA Leu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNA Leu mutants with synonymous anticodons and for different tRNA Leu isoacceptors with the same anticodon. A comparison to tRNA Ala anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Background: Essential hypertension (EH) was associated with mitochondrial tRNA mutations., Aims: This study was designed to assess the association between EH and mitochondrial dysfunction., Methods: A total of 30 individuals from two different Chinese families exhibit maternally inherited EH were assessed for genetic, clinical, and biochemical phenotypes pertaining to EH and mitochondrial functionality. These analyses included assessments of tRNA Leu(UUR) 3261A > G mutation status, mitochondrial membrane permeability, mitochondria-associated ATP and reactive oxygen species (ROS) generation, and electron transport chain functionality., Results: EH was detected in 6 total analyzed members of the two families assessed in the present study, with its initial age of onset and presentation varying among patients. These patients with EH exhibited the tRNA Leu(UUR) 3261A > G mutation and were of the B5 and D4 Eastern Asian mitochondrial haplogroups. This 3261A > G mutation was predicted to result in disruption of normal tRNA Leu(UUR) activity owing to the destabilization of conserved base pairing (30A-40U). Consistent with this prediction, we found that cybrid cell lines exhibiting this 3261A > G mutation exhibited a ~49.05% decrease in baseline tRNA Leu(UUR) levels. These cells additionally exhibited ~44.81% reductions in rates of mitochondrial translation., Conclusions: To facilitate future molecular diagnosis, the 3261A > G mutation should be included in the list of hereditary risk factors. Our findings will aid in the counseling of EH families., (© 2022. The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland.)

tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at tRNA Leu and 5S rRNA genes in this breast cancer cell line. While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer., (Copyright © 2022. Published by Elsevier B.V.)

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line., (© 2021. The Author(s).)

Objective: 3' tRNA-derived fragments (3' tRFs) are important epigenetic regulators in normal and pathological conditions. In this study, we aimed to explore the potential value of a 3' tRF as a prognostic and/or screening biomarker for B-cell chronic lymphocytic leukemia (B-CLL)., Methods: Publicly available next-generation sequencing data from 20 B-CLL cases were analyzed, followed by prediction of targets of the most abundantly and ubiquitously expressed 3' tRFs, leading to selection of tRF-Leu AAG/TAG . PBMCs were isolated from blood samples of 91 B-CLL patients and 43 non-leukemic donors, followed by total RNA extraction, in-vitro polyadenylation, and first-strand cDNA synthesis. Next, a real-time quantitative PCR (qPCR) assay was developed for the accurate quantification of tRF-Leu AAG/TAG and applied in all samples, prior to biostatistical analysis., Results: High tRF-Leu AAG/TAG levels are associated with inferior overall survival (OS) of B-CLL patients. The unfavorable significance of tRF-Leu AAG/TAG was independent of established prognostic factors in B-CLL. Stratified Kaplan-Meier OS analysis uncovered the unfavorable prognostic role of high tRF-Leu AAG/TAG levels for patients in Binet A or Rai I stage, negative CD38 expression, mutated, or unmutated IGHV genomic locus., Conclusion: Our approach revealed the independent prognostic value of a particular 3' tRF, derived from tRNA LeuAAG and tRNA LeuTAG (tRF-Leu AAG/TAG ) in B-CLL., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mitochondrial diseases are clinically and genetically heterogeneous disorders, caused by pathogenic variants in either the nuclear or mitochondrial genome. This heterogeneity is particularly striking for disease caused by variants in mitochondrial DNA-encoded tRNA (mt-tRNA) genes, posing challenges for both the treatment of patients and understanding the molecular pathology. In this review, we consider disease caused by the two most common pathogenic mt-tRNA variants: m.3243A>G (within MT-TL1, encoding mt-tRNA Leu(UUR) ) and m.8344A>G (within MT-TK, encoding mt-tRNA Lys ), which together account for the vast majority of all mt-tRNA-related disease. We compare and contrast the clinical disease they are associated with, as well as their molecular pathologies, and consider what is known about the likely molecular mechanisms of disease. Finally, we discuss the role of mitochondrial-nuclear crosstalk in the manifestation of mt-tRNA-associated disease and how research in this area not only has the potential to uncover molecular mechanisms responsible for the vast clinical heterogeneity associated with these variants but also pave the way to develop treatment options for these devastating diseases., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The m.3243A >G mutation in the tRNA Leu (UUR) gene (MT-TL1) of the mitochondrial DNA is the most widely seen pathogenic mtDNA mutation which has major phenotypic variations. The clinical phenotype involves various organs such as the brain and nerves, skeletal muscles, heart, endocrine system, gastrointestinal tract, and skin. Some phenotypes conform to well established syndromes, while most of the symptoms appear individually or concomitant to other syndromes, making identification difficult. Furthermore, some progress has been made on cardiac manifestations as well as complications during pregnancy and perinatal period. This article provides a systematic review of the non-syndromic phenotypes and latest developments in m.3243A>G mutation., (Copyright: © Neurosciences.)

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNA Leu(UUR) ) necessary for mitochondrial translation. Pathogenic variants in the MT-TL1 gene result in mitochondriopathy in humans. The m.3250T>C variant in the MT-TL1 gene has been previously associated with exercise intolerance and mitochondrial myopathy, yet disease classification for this variant has not been consistently reported. Molecular studies suggest the m.3250T>C variant does not alter tRNA Leu(UUR) structure but may have a modest impact on aminoacylation capacity. However, functional studies are limited. Our study aimed to further define the clinical presentation, inheritance pattern, and molecular pathology of the m.3250T>C variant. Families with the m.3250T>C variant were recruited from the Mitochondrial Disease Clinic at Cincinnati Children's Hospital Medical Center and GeneDx laboratory database. Affected individuals most frequently presented with cardiac findings, exercise intolerance, and muscle weakness. Hypertrophic cardiomyopathy was the most frequent cardiac finding. Many asymptomatic individuals had homoplasmic or near homoplasmic levels of the m.3250T>C variant, suggesting the penetrance is incomplete. Patient-derived fibroblasts demonstrated lowered ATP production and increased levels of reactive oxygen species. Our results demonstrate that the m.3250T>C variant exhibits incomplete penetrance and may be a possible cause of cardiomyopathy by impacting cellular respiration in mitochondria., (© 2020 Wiley Periodicals LLC.)

Mitochondrial diseases are rare, often go undiagnosed and can lead to devastating cascades of multisystem organ dysfunction. This report of a young woman with hearing loss and gestational diabetes illustrates a novel presentation of a cardiomyopathy caused by a previously described mutation in a mitochondrial gene, MT-TL1. She initially had biventricular heart dysfunction and ventricular arrhythmia that ultimately recovered with beta blockade and time. She continues to participate in sport without decline. It is important to keep mitochondrial diseases in the differential diagnosis and understand the testing and management strategies in order to provide the best patient care., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2 ∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2 ∆, of which kap123 ∆ and tl(gag)g ∆ appear the most specific. We show that kap123 ∆ and tl(gag)g ∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2 , provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement., (Copyright © 2020 by the Genetics Society of America.)

Background: Up to one third of patients on renal replacement programmes have an unknown cause of kidney disease, and the diagnosis may only be established following renal transplantation when the disease recurs or if new extra-renal symptoms develop., Case Presentation: We present two patients who presented with progressive chronic kidney disease of unknown cause. Both patients underwent successful renal transplantation but subsequently developed multisystem abnormalities, and were ultimately diagnosed with mitochondrial cytopathy 10-15 years following transplantation., Conclusions: Mitochondrial cytopathies are rare inborn errors of metabolism that should be considered in adults with renal impairment, especially in those with a family history of kidney or other multisystem disease. The widespread availability of genetic testing provides the potential for earlier diagnoses, thereby enhancing management decisions, anticipation of complications, avoidance of mitotoxic drugs, and informed prognosis prediction.

Mitochondria play various important roles in energy production, metabolism, and apoptosis. Mitochondrial dysfunction caused by alterations in mitochondrial DNA (mtDNA) can lead to the initiation and progression of cancers and other diseases. These alterations include mutations and copy number variations. Especially, the mutations in D-loop, MT-ND1 , and MT-ND5 affect mitochondrial functions and are widely detected in various cancers. Meanwhile, several other mutations have been correlated with muscular and neuronal diseases, especially MT-TL1 is deeply related. These pieces of evidence indicated mtDNA alterations in diseases show potential as a novel therapeutic target. mtDNA repair enzymes are the target for delaying or stalling the mtDNA damage-induced cancer progression and metastasis. Moreover, some mutations reveal a prognosis ability of the drug resistance. Current efforts aim to develop mitochondrial transplantation technique as a direct cure for deregulated mitochondria-associated diseases. This review summarizes the implications of mitochondrial dysfunction in cancers and other pathologies; and discusses the relevance of mitochondria-targeted therapies, along with their contribution as potential biomarkers.

Aim: Pathogenic variants within mitochondrial tRNA and rRNA genes negatively affect protein synthesis function and cause oxidative phosphorylation defects. The majority of mitochondrial cytopathies are caused by pathogenic point variants within the mitochondrial tRNA gene for leucine ( MT-TL1 ). This study was designed to evaluate a novel amplification-refractory mutation system (ARMS)-PCR based assay to screen patient samples with a clinical diagnosis of mitochondrial cytopathies. Methods: Tissue DNA samples from 219 affected individuals were screened for the pathogenic variants m.3271T>C, m.3291Ty >C, m.3303C>T, m.3256C>T, and m.3260A>G along with the most frequent m.3243A>G mutation in the MT-TL1 gene. The assay included a "High Resolution Melt curve analysis" to enhance detection limits. The precision of the assay was verified using synthetic controls with variant heteroplasmy ratios. Results: The screening identified the second reported m.3303C>T case as well as two patients with m.3243A>G variants and a rare variant exhibiting m.3290T>C. Conclusion: ARMS-PCR is superior to Sanger sequencing for the detection of variations exhibiting low heteroplasmy. These results provide "proof of concepts" for the implementation of this application for future screening of rare mtDNA variations in sample repositories.

The m.3243A > G mutation in the mitochondrial tRNA Leu (UUR) gene is associated with a variety of phenotypic heterogeneity. The clinical spectrum and phenotypic-genotypic correlations in the Chinese patients are poorly understood. In the present study, we reported the clinical and genetic characterization, as well as haplogroups of seven Han Chinese families carrying the m.3243A > G mutation. Of the 39 matrilineal individuals, five suffered from mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), two had life-threatening mitochondrial myopathy (LTMM), and one patient had neuropathy, ataxia, and retinitis pigmentosa (NARP)-like syndrome. The LTMM and NARP like syndromes enriched the phenotypic profile of the m.3243A > G mutation. The heteroplasmy of the m.3243A > G mutation ranged from 16% to 59% in MELAS, 29% to 79% in LTMM, and 57% in a NARP-like syndrome patient. The levels ranged from 0% to 14% in patients that manifested with pure diabetes and pure hearing loss, and 0% to 5% in 13 normal family members. However, we particularly noticed heteroplasmy in four asymptomatic individuals in one LTMM family carried the heteroplasmy mutation ranged from 22% to 78%, implying that there were other modifying factors in this family. The modulation of the phenotype of mtDNA mutations requires further investigation., (Copyright © 2019. Published by Elsevier B.V.)

We describe a patient with chronic progressive external ophthalmoplegia (CPEO) due to a rare mitochondrial genetic variant. Muscle biopsy revealed numerous cytochrome c oxidase (COX)-deficient fibres, prompting sequencing of the entire mitochondrial genome in muscle which revealed a rare m.12334G>A variant in the mitochondrial (mt-) tRNALeu(CUN)(MT-TL2) gene. Analysis of several tissues showed this to be a de novo mutational event. Single fibre studies confirmed the segregation of high m.12334G>A heteroplasmy levels with the COX histochemical defect, confirming pathogenicity of the m.12334G>A MT-TL2 variant. This case illustrates the importance of pursuing molecular genetic analysis in clinically-affected tissues when mitochondrial disease is suspected.

In the present work, a pilot creation of four cybrid cultures with high heteroplasmy level was performed using mitochondrial genome mutations m.12315G>A and m.1555G>A. According to data of our preliminary studies, the threshold heteroplasmy level of mutation m.12315G>A is associated with atherosclerosis. At the same time, for a mutation m.1555G>A, such a heteroplasmy level is associated with the absence of atherosclerosis. Cybrid cultures were created by fusion of rho0-cells and mitochondria from platelets with a high heteroplasmy level of the investigated mutations. To create rho0-cells, THP-1 culture of monocytic origin was taken. According to the results of the study, two cybrid cell lines containing mutation m.12315G>A with the heteroplasmy level above the threshold value (25% and 44%, respectively) were obtained. In addition, two cybrid cell lines containing mutation m.1555G>A with a high heteroplasmy level (24%) were obtained. Cybrid cultures with mtDNA mutation m.12315G>A can be used to model both the occurrence and development of atherosclerosis in cells and the titration of drug therapy for patients with atherosclerosis. With the help of cybrid cultures containing single nucleotide replacement of mitochondrial genome m.1555G>A, it is possible to develop approaches to the gene therapy of atherosclerosis.

Recent evidences highlight the importance of mitochondria-nucleus communication for the clinical phenotype of oxidative phosphorylation (OXPHOS) diseases. However, the participation of small non-coding RNAs (sncRNAs) in this communication has been poorly explored. We asked whether OXPHOS dysfunction alters the production of a new class of sncRNAs, mitochondrial tRNA fragments (mt tRFs), and, if so, whether mt tRFs play a physiological role and their accumulation is controlled by the action of mt tRNA modification enzymes. To address these questions, we used a cybrid model of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), an OXPHOS disease mostly caused by mutation m.3243A>G in the mitochondrial tRNA Leu(UUR) gene. High-throughput analysis of small-RNA-Seq data indicated that m.3243A>G significantly changed the expression pattern of mt tRFs. A functional analysis of potential mt tRFs targets (performed under the assumption that these tRFs act as miRNAs) indicated an association with processes that involve the most common affected tissues in MELAS. We present evidences that mt tRFs may be biologically relevant, as one of them (mt i-tRF GluUUC), likely produced by the action of the nuclease Dicer and whose levels are Ago2 dependent, down-regulates the expression of mitochondrial pyruvate carrier 1 (MPC1), promoting the build-up of extracellular lactate. Therefore, our study underpins the idea that retrograde signaling from mitochondria is also mediated by mt tRFs. Finally, we show that accumulation of mt i-tRF GluUUC depends on the modification status of mt tRNAs, which is regulated by the action of stress-responsive miRNAs on mt tRNA modification enzymes., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA Leu UAA (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms 2 i 6 A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular., (© 2019 John Wiley & Sons Ltd.)

A hallmark of translation in human immunodeficiency virus type 1 (HIV-1) is a -1 programmed ribosome frameshifting event that produces the Gag-Pol fusion polyprotein. The constant Gag to Gag-Pol ratio is essential for the virion structure and infectivity. Here we show that the frameshifting efficiency is modulated by Leu-tRNALeu that reads the UUA codon at the mRNA slippery site. This tRNALeu isoacceptor is particularly rare in human cell lines derived from T-lymphocytes, the cells that are targeted by HIV-1. When UUA decoding is delayed, the frameshifting follows an alternative route, which maintains the Gag to Gag-Pol ratio constant. A second potential slippery site downstream of the first one is normally inefficient but can also support -1-frameshifting when altered by a compensatory resistance mutation in response to current antiviral drug therapy. Together these different regimes allow the virus to maintain a constant -1-frameshifting efficiency to ensure successful virus propagation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a multisystemic mitochondrial disorder that usually presents in childhood. Patients can have a wide array of neurological symptoms when presenting with stroke-like episodes, and imaging characteristics during the episodes can overlap with different neurological disorders., Case Report: A 61-year-old woman presented with communication difficulties consistent with auditory agnosia and was found to have bitemporal abnormalities on imaging that first raised the concern for herpes simplex virus encephalitis. Further work-up, in conjunction with the patient's past medical and family history, suggested a mitochondrial disorder. Mitochondrial full genome analysis revealed m.3243A>G variant in the MT-TL1 gene, with 6% heteroplasmy in blood leading to a diagnosis of MELAS., Conclusions: MELAS is a disorder with clinical variability. Neuroimaging studies during stroke-like episodes in MELAS can provide significant clues to the underlying disorder. Although patients typically present in childhood, the first stroke-like episode can occur later in life in some patients, potentially related to a lower heteroplasmy level.

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D., (© 2019 Sanz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Inherited mitochondrial DNA (mtDNA) mutations cause mitochondrial disease, but mtDNA mutations also occur somatically and accumulate during ageing. Studies have shown that the mutation load of some inherited mtDNA mutations decreases over time in blood, suggesting selection against the mutation. However, it is unknown whether such selection occurs in other mitotic tissues, and where it occurs within the tissue. Gastrointestinal epithelium is a canonical mitotic tissue rapidly renewed by stem cells. Intestinal crypts (epithelium) undergo monoclonal conversion with a single stem cell taking over the niche and producing progeny. We show: (1) that there is a significantly lower mtDNA mutation load in the mitotic epithelium of the gastrointestinal tract when compared to the smooth muscle in the same tissue in patients with the pathogenic m.3243A>G and m.8344A>G mutations; (2) that there is considerable variation seen in individual crypts, suggesting changes in the stem cell population; (3) that this lower mutation load is reflected in the absence of a defect in oxidative phosphorylation in the epithelium. This suggests that there is selection against inherited mtDNA mutations in the gastrointestinal stem cells that is in marked contrast to the somatic mtDNA mutations that accumulate with age in epithelial stem cells leading to a biochemical defect. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Transcriptome analysis reveals a strong positive correlation between human Schlafen family member 11 (SLFN11) expression and the sensitivity of tumor cells to DNA-damaging agents (DDAs). Here, we show that SLFN11 preferentially inhibits translation of the serine/threonine kinases ATR and ATM upon DDA treatment based on distinct codon usage without disrupting early DNA damage response signaling. Type II transfer RNAs (tRNAs), which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. Messenger RNAs encoded by genes with high TTA (Leu) codon usage, such as ATR, display utmost susceptibility to translational suppression by SLFN11. Specific attenuation of tRNA-Leu-TAA sufficed to ablate ATR protein expression and restore the DDA sensitivity of SLFN11-deficient cells. Our study uncovered a novel mechanism of codon-specific translational inhibition via SLFN11-dependent tRNA cleavage in the DNA damage response and supports the notion that SLFN11-deficient tumor cells can be resensitized to DDAs by targeting ATR or tRNA-Leu-TAA.

The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria-nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A>G in mitochondrial tRNA Leu(UUR) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A>G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A>G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A>G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-β superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Although the "universal" genetic code is now known not to be universal, and stop codons can have multiple meanings, one regularity remains, namely that for a given sense codon there is a unique translation. Examining CUG usage in yeasts that have transferred CUG away from leucine, we here report the first example of dual coding: Ascoidea asiatica stochastically encodes CUG as both serine and leucine in approximately equal proportions. This is deleterious, as evidenced by CUG codons being rare, never at conserved serine or leucine residues, and predominantly in lowly expressed genes. Related yeasts solve the problem by loss of function of one of the two tRNAs. This dual coding is consistent with the tRNA-loss-driven codon reassignment hypothesis, and provides a unique example of a proteome that cannot be deterministically predicted. VIDEO ABSTRACT., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The genetic code used in nuclear genes is almost universal, but here we report that it changed three times in parallel during the evolution of budding yeasts. All three changes were reassignments of the codon CUG, which is translated as serine (in 2 yeast clades), alanine (1 clade), or the 'universal' leucine (2 clades). The newly discovered Ser2 clade is in the final stages of a genetic code transition. Most species in this clade have genes for both a novel tRNA Ser (CAG) and an ancestral tRNA Leu (CAG) to read CUG, but only tRNA Ser (CAG) is used in standard growth conditions. The coexistence of these alloacceptor tRNA genes indicates that the genetic code transition occurred via an ambiguous translation phase. We propose that the three parallel reassignments of CUG were not driven by natural selection in favor of their effects on the proteome, but by selection to eliminate the ancestral tRNA Leu (CAG).

Polycystic ovary syndrome (PCOS) is a very prevalent endocrine disease affecting reproductive women. Clinically, patients with this disorder are more vulnerable to develop type 2 diabetes mellitus (T2DM), cardiovascular events, as well as metabolic syndrome (MetS). To date, the molecular mechanism underlying PCOS remains largely unknown. Previously, we showed that mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) mutation was an important cause for PCOS. In the current study, we described the clinical and biochemical features of a three-generation pedigree with maternally transmitted MetS, combined with PCOS. A total of three matrilineal relatives exhibited MetS including obesity, high triglyceride (TG) and Hemoglobin A1c (HbA1c) levels, and hypertension. Whereas one patient from the third generation manifestated PCOS. Mutational analysis of the whole mitochondrial genes from the affected individuals identified a set of genetic variations belonging to East Asia haplogroup B4b1c. Among these variants, the homoplasmic C3275T mutation disrupted a highly evolutionary conserved base-pairing (28A-46C) on the variable region of tRNA Leu(UUR) , whereas the T4363C mutation created a new base-pairing (31T-37A) in the anticodon stem of tRNA Gln , furthermore, the A8343G mutation occurred at the very conserved position of tRNA Lys and may result the failure in mitochondrial tRNAs (mt-tRNAs) metabolism. Biochemical analysis revealed the deficiency in mitochondrial functions including lower levels of mitochondrial membrane potential (MMP), ATP production and mtDNA copy number, while a significantly increased reactive oxygen species (ROS) generation was observed in polymononuclear leukocytes (PMNs) from the individuals carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for the clinical phenotypes. Taken together, our data indicated that mt-tRNA mutations were associated with MetS and PCOS in this family, which shaded additional light into the pathophysiology of PCOS that were manifestated by mitochondrial dysfunction., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Focal segmental glomerulosclerosis (FSGS) is a pathological lesion rather than a disease, with a diverse etiology. FSGS may result from genetic and non‑genetic factors. FSGS is considered a podocyte disease due to the fact that in the majority of patients with proven‑FSGS, the lesion results from defects in the podocyte structure or function. However, FSGS does not result exclusively from podocyte‑associated genes, however also from other genes including collagen IV‑associated genes. Patients who carry the collagen type IVA3 chain (COL4A3) or COL4A4 mutations usually exhibit Alport Syndrome (AS), thin basement membrane neuropathy or familial hematuria (FH). Previous studies revealed that long‑time persistent microscopic hematuria may lead to FSGS. A case of a family is presented here where affected individuals exhibited FH with FSGS‑proven, or chronic kidney disease. Renal biopsies were unhelpful and failed to demonstrate glomerular or basement membrane defects consistent with an inherited glomerulopathy, and therefore a possible underlying genetic cause for a unifying diagnosis was pursued. Genomic DNA of the siblings affected by FH with biopsy‑proven FSGS was analyzed, and their father was screened for 18 gene mutations associated with FSGS [nephrin, podocin, CD2 associated protein, phospholipase C ε, actinin α 4, transient receptor potential cation channel subfamily C member 6, inverted formin, FH2 and WH2 domain containing, Wilms tumor 1, LIM homeobox transcription factor 1 β, laminin subunit β 2, laminin subunit β 3, galactosida α, integrin subunit β 4, scavenger receptor class B member 2, coenzyme Q2, decaprenyl diphosphate synthase subunit 2, mitochondrially encoded tRNA leucine 1 (UUA/G; TRNL1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a like 1] using matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry technology. Then whole exome sequencing (WES) was performed in the two probands to ascertain whether there were other known or unknown gene mutations that segregated with the disease. Using mass array technology, a TRNL1 missense homozygous mutation (m. 3290T>C) was identified in the probands diagnosed with FH and manifested as FSGS on biopsy. In addition, a COL4A4 missense mutation c. 4195A>T (p. M1399L) in heterozygous pattern was identified using WES. None of these variants were detected in their father. In the present study, a mutation in TRNL1 (m. 3290T>C) was identified, which was the first reported variant associated with FSGS. The COL4A4 (c. 4195A>T) may co‑segregate with FSGS. Screening for COL4A mutations in familial FSGS patients is suggested in the present study. Genetic investigations of families with similar clinical phenotypes should be a priority for nephrologists. The combination of mass array technology and WES may improve the detection rate of genetic mutation with a high level of accuracy.

While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNA Leu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNA Leu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNA Leu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNA Leu -binding protein. We suspected that the overexpression of free tRNA Leu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNA Leu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNA Leu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Streptomyces albus J1074 is one of the most popular and convenient hosts for heterologous expression of gene clusters directing the biosynthesis of various natural metabolic products, such as antibiotics. This fuels interest in elucidation of genetic mechanisms that may limit secondary metabolism in J1074. Here, we report the generation and initial study of J1074 mutant, deficient in gene bldA for tRNA Leu UAA , the only tRNA capable of decoding rare leucyl TTA codon in Streptomyces. The bldA deletion in J1074 resulted in a highly conditional Bld phenotype, with depleted formation of aerial hyphae on certain solid media. In addition, bldA mutant of J1074 was unable to produce endogenous antibacterial compounds and two heterologous antibiotics, moenomycin and aranciamycin, whose biosynthesis is directed by TTA-containing genes. We have employed a new TTA codon-specific β-galactosidase reporter system to provide genetic evidence that J1074 bldA mutant is impaired in translation of TTA. In addition, we have discussed the possible reasons for differences in the phenotypes of bldA mutants described here and in previous studies, providing knowledge to study bldA-based regulation of antibiotic biosynthesis.

The m.3243A > G mitochondrial DNA mutation was originally described in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The phenotypic spectrum of the m.3243A > G mutation has since expanded to include a spectrum of neuromuscular and ocular manifestations, including reduced vision with retinal degeneration, the underlying mechanism of which remains unclear. We used dermal fibroblasts, from patients with retinal pathology secondary to the m.3243A > G mutation to generate heteroplasmic induced pluripotent stem cell (hiPSC) clones. RPE cells differentiated from these hiPSCs contained morphologically abnormal mitochondria and melanosomes, and exhibited marked functional defects in phagocytosis of photoreceptor outer segments. These findings have striking similarities to the pathological abnormalities reported in RPE cells studied from post-mortem tissues of affected m.3243A > G mutation carriers. Overall, our results indicate that RPE cells carrying the m.3243A > G mutation have a reduced ability to perform the critical physiological function of phagocytosis. Aberrant melanosomal morphology may potentially have consequences on the ability of the cells to perform another important protective function, namely absorption of stray light. Our in vitro cell model could prove a powerful tool to further dissect the complex pathophysiological mechanisms that underlie the tissue specificity of the m.3243A > G mutation, and importantly, allow the future testing of novel therapeutic agents.

We reported a 53-year-old with late-onset mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) accompanied by aneurysm and large vessel dilations. Most studies have focused on microangiopathy causing stroke-like episodes. We report a case to describe large vessel involvement in clinical considerations, and possible mechanisms of aneurysm formation. We recommended regular angiographic examination for patients with MELAS.

Background: The presence of giant ring mitochondria on endomyocardial biopsy is rarely reported and does not have a well-defined differential diagnosis., Methods: We report the case of a 54-year-old man with heart failure and preserved ejection fraction and left ventricular hypertrophy, initially thought to have an infiltrative cardiomyopathy., Results: The patient was found to have extensive vacuolization caused by giant ring mitochondria on endomyocardial biopsy. Mitochondrial genetic testing revealed an A3243G mutation in the MT-TL1 gene, which is a mitochondrial encoded transfer RNA-leucine molecule., Conclusions: Mitochondrial disease should be considered in patients presenting with unexplained cardiomyopathy and skeletal muscle, cerebral, or metabolic abnormalities. In this case, the presence of unexpected extensive cardiomyocyte vacuolization and giant, ring-shaped mitochondria on endomyocardial biopsy prompted mitochondrial genetic testing, which ultimately resulted in the correct diagnosis and treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ultrastructural changes in skeletal muscle biopsy in a 24-year-old female patient with clinically suspected mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) syndrome are presented. We observed proliferation and/or pleomorphism of mitochondria in skeletal muscle and smooth muscle cells of arterioles, as well as in pericytes of capillaries. Paracrystalline inclusions were found only in damaged mitochondria of skeletal muscle. Genetic testing revealed a point mutation in A3243G tRNALeu(UUR) typical for MELAS syndrome. We conclude that differentiated pathological changes of mitochondria in the studied types of cells may be associated with the different energy requirements of these cells.

Objective: In this study, we aimed to identify mt3243A > G mutation carriers in a group of Chinese elderly type 2 diabetic patients by a rapid and noninvasive diagnostic system., Methods: DNA was extracted from blood, saliva, and urine sediment samples. The mutation screening and quantitation of heteroplasmy were performed by high-resolution melting (HRM) curve and pyrosequencing, respectively. Patients with mt3243A > G mutation underwent a detailed audiometric, ophthalmologic, neurological, and cardiac examination., Results: Two patients (2/1041) carrying the mt3243A > G mutation were detected among all type 2 diabetic patients. In patient 1, the heteroplasmy was 0.8%, 2.8%, and 14.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. In patient 2, the heteroplasmy was 5.3%, 8.4%, and 37.7% in peripheral blood leukocytes, saliva, and urine sediment, respectively. Both of the two patients showed hearing impairment. Abnormal ophthalmologic conditions and hyperintensity on T2-weighted magnetic resonance images were showed in patient 1., Conclusion: The occurrence of mt3243 A > G mutation was 0.2% in Chinese elderly type 2 diabetic patients. Moreover, detection of mt3243 A > G mutation in urine sediment with high-resolution melting (HRM) curve and pyrosequencing is feasible in molecular genetic diagnosis.