Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set.

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 tRNASerGAG and tRNALeuGGA. 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.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

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 tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu 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 tRNALeu 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 tRNALeu 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 tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla 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.

T cell activation contributes to purifying selection against the MELAS-associated m.3243A>G pathogenic variant in blood.

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.

Generation of a human induced pluripotent stem cell line harboring heteroplasmic m.3243A > G mutation in MT-TL1 gene.

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.

Novel mitochondrial tRNALeu(UUR) 3261A > g mutation in two pedigrees with essential hypertension.

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 tRNALeu(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 tRNALeu(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 tRNALeu(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 tRNALeu(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.

Widespread association of ERα with RMRP and tRNA genes in MCF-7 cells and breast cancers.

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 tRNALeu 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.

Sonlicromanol improves neuronal network dysfunction and transcriptome changes linked to m.3243A>G heteroplasmy in iPSC-derived neurons.

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.

A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis.

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.

The non-syndromic clinical spectrums of mtDNA 3243A>G mutation.

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.

A 3' tRNA-derived fragment produced by tRNALeuAAG and tRNALeuTAG is associated with poor prognosis in B-cell chronic lymphocytic leukemia, independently of classical prognostic factors.

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-LeuAAG/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-LeuAAG/TAG and applied in all samples, prior to biostatistical analysis. RESULTS: High tRF-LeuAAG/TAG levels are associated with inferior overall survival (OS) of B-CLL patients. The unfavorable significance of tRF-LeuAAG/TAG was independent of established prognostic factors in B-CLL. Stratified Kaplan-Meier OS analysis uncovered the unfavorable prognostic role of high tRF-LeuAAG/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 tRNALeuAAG and tRNALeuTAG (tRF-LeuAAG/TAG ) in B-CLL.

Kickboxing a cardiomyopathy: mitochondrial sequencing provides answer for young athlete and her family.

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.

The molecular pathology of pathogenic mitochondrial tRNA variants.

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-tRNALeu(UUR) ) and m.8344A>G (within MT-TK, encoding mt-tRNALys ), 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.

Mitochondrial genome variant m.3250T>C as a possible risk factor for mitochondrial cardiomyopathy.

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNALeu(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 tRNALeu(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.

Suppressors of mRNA Decapping Defects Restore Growth Without Major Effects on mRNA Decay Rates or Abundance.

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.

Delayed diagnoses of mitochondrial cytopathies in patients presenting with end stage kidney disease: two case reports.

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.

Chronic Progressive External Ophthalmoplegia due to a Rare de novo m.12334G>A MT-TL2 Mitochondrial DNA Variant1.

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.

A Novel Amplification-Refractory Mutation System-PCR Strategy to Screen MT-TL1 Pathogenic Variants in Patient Repositories.

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.

Deregulated Mitochondrial DNA in Diseases.

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.