RESUMEN
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 tRNALeu(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 tRNALeu(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.
Asunto(s)
Encéfalo , ADN Mitocondrial , Mutación Puntual , Animales , ADN Mitocondrial/genética , Masculino , Encéfalo/metabolismo , ARN de Transferencia de Leucina/genética , Ratones Endogámicos C57BL , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Inhibición Prepulso/genética , Memoria , Conducta AnimalRESUMEN
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 â¼50% 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.
Asunto(s)
Evolución Molecular , Código Genético , Saccharomycopsis/genética , Biosíntesis de Proteínas , Filogenia , ARN de Transferencia de Leucina/genética , Anticodón/genética , ARN de Transferencia de Serina/genéticaRESUMEN
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.
Asunto(s)
Sistema Libre de Células , Escherichia coli , Biosíntesis de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Serina/metabolismo , ARN de Transferencia de Serina/genética , Código Genético , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Ingeniería de Proteínas/métodos , Transcripción Genética , Anticodón/genética , Anticodón/metabolismoRESUMEN
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.
Asunto(s)
Aminoacil-ARNt Sintetasas , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/genética , Anticodón/genética , Leucina/genética , ARN de Transferencia de Leucina/genética , Código Genético , Codón , ARN de Transferencia/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Alanina/genética , Mamíferos/genéticaRESUMEN
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.
Asunto(s)
Células Madre Pluripotentes Inducidas , Mutación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Línea Celular , ARN de Transferencia de Leucina/genética , Diferenciación Celular , ADN Mitocondrial/genética , Fibroblastos/metabolismo , Fibroblastos/citologíaRESUMEN
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.
Asunto(s)
Activación de Linfocitos , Síndrome MELAS , Humanos , Síndrome MELAS/genética , Linfocitos T CD4-Positivos/inmunología , Heteroplasmia/genética , ARN de Transferencia de Leucina/genética , Masculino , Femenino , ADN Mitocondrial/genética , AdultoRESUMEN
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, BioRNAGly and BioRNALeu, 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 BioRNAGly and BioRNALeu 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 BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/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.
Asunto(s)
Antineoplásicos , Bioingeniería , MicroARNs , ARN de Transferencia de Glicerina , ARN de Transferencia de Leucina , ARN de Transferencia de Glicerina/química , ARN de Transferencia de Glicerina/genética , ARN de Transferencia de Glicerina/aislamiento & purificación , ARN de Transferencia de Glicerina/farmacología , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/aislamiento & purificación , ARN de Transferencia de Leucina/farmacología , MicroARNs/química , MicroARNs/genética , MicroARNs/aislamiento & purificación , MicroARNs/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Antineoplásicos/farmacología , Expresión Génica/efectos de los fármacos , Simulación por Computador , Línea Celular TumoralRESUMEN
The 3243A > G in mtDNA is a representative mutation in mitochondrial diseases. Mitochondrial protein synthesis is impaired due to decoding disorder caused by severe reduction of 5-taurinomethyluridine (τm5U) modification of the mutant mt-tRNALeu(UUR) bearing 3243A > G mutation. The 3243A > G heteroplasmy in peripheral blood reportedly decreases exponentially with age. Here, we found three cases with mild respiratory symptoms despite bearing high rate of 3243A > G mutation (>90%) in blood mtDNA. These patients had the 3290T > C haplotypic mutation in addition to 3243A > G pathogenic mutation in mt-tRNALeu(UUR) gene. We generated cybrid cells of these cases to examine the effects of the 3290T > C mutation on mitochondrial function and found that 3290T > C mutation improved mitochondrial translation, formation of respiratory chain complex, and oxygen consumption rate of pathogenic cells associated with 3243A > G mutation. We measured τm5U frequency of mt-tRNALeu(UUR) with 3243A > G mutation in the cybrids by a primer extension method assisted with chemical derivatization of τm5U, showing that hypomodification of τm5U was significantly restored by the 3290T > C haplotypic mutation. We concluded that the 3290T > C is a haplotypic mutation that suppresses respiratory deficiency of mitochondrial disease by restoring hypomodified τm5U in mt-tRNALeu(UUR) with 3243A > G mutation, implying a potential therapeutic measure for mitochondrial disease associated with pathogenic mutations in mt-tRNAs.
Asunto(s)
Síndrome MELAS , Enfermedades Mitocondriales , Humanos , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , ARN de Transferencia de Leucina/metabolismo , Taurina , Haplotipos , Mutación , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genéticaRESUMEN
Certain mutations of the model cyanobacterium Synechococcus elongatus PCC 7942 during laboratory storage have resulted in some divergent phenotypes. One laboratory-stored strain (H1) shows a temperature-sensitive (ts) growth phenotype at 40 °C. Here, we investigated the reason for this temperature sensitivity. Whole genome sequencing of H1 identified a single nucleotide mutation in synpcc7942_R0040 encoding tRNA-Leu(CAA). The mutation decreases the length of the tRNA-Leu t-arm from 5 to 4 base pairs, and this explains the ts phenotype. Secondary mutations suppressing the ts phenotype were identified in synpcc7942_1640, which putatively encodes a NYN domain-containing protein (nynA). The NYN domain is thought to be involved in tRNA/rRNA degradation. Thus, the structural stability of tRNA-Leu is critical for growth at 40 °C in Synechococcus elongatus PCC 7942.
Asunto(s)
ARN de Transferencia de Leucina , Synechococcus , Temperatura , ARN de Transferencia de Leucina/metabolismo , Mutación , Proteínas Bacterianas/genéticaRESUMEN
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.
Asunto(s)
Mitocondrias , ARN de Transferencia de Leucina , Humanos , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/metabolismo , Linaje , Mutación , Mitocondrias/metabolismo , Hipertensión Esencial/genéticaRESUMEN
Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A-SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation.
Asunto(s)
Neoplasias , ARN de Transferencia de Leucina , Línea Celular Tumoral , Muerte Celular , Apoptosis , Biosíntesis de ProteínasRESUMEN
Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.
Asunto(s)
Metiltransferasas , ARNt Metiltransferasas , Metiltransferasas/genética , Mutación , ARN de Transferencia/metabolismo , ARN de Transferencia de Leucina , ARNt Metiltransferasas/química , Bacillaceae/genética , Bacillaceae/metabolismoRESUMEN
Urbanization can change biodiversity in both directions, positive and negative, and despite the rising global trend of urban beekeeping, little is known about the impact of urbanization on the genetic diversity of honey bees. We investigate how urbanization affects the genetic variability of feral and managed honey bee colonies that are spread throughout the entire city, even in highly urban areas, through genetic analysis of 82 worker bees. We found convincing evidence of high genetic differentiation between these two groups. Additionally, by comparing city samples with 241 samples from 46 apiaries in rural parts of the country, variations in mitochondrial tRNAleu-cox2 intergenic region and microsatellite loci indicated that feral colonies have distinct patterns of genetic diversity. These results, with evidence that feral honey bees find niches within highly modified and human-dominated urban landscapes, lead us to conclude that urbanization is a driver of the genetic diversity of feral honey bees in the city.
Asunto(s)
Ecosistema , ARN de Transferencia de Leucina , Humanos , Abejas/genética , Animales , Ciclooxigenasa 2 , Variación Genética , ADN IntergénicoRESUMEN
To correctly aminoacylate tRNALeu, leucyl-tRNA synthetase (LeuRS) catalyzes three reactions: activation of leucine by ATP to form leucyl-adenylate (Leu-AMP), transfer of this amino acid to tRNALeu and post-transfer editing of any mischarged product. Although LeuRS has been well characterized biochemically, detailed structural information is currently only available for the latter two stages of catalysis. We have solved crystal structures for all enzymatic states of Neisseria gonorrhoeae LeuRS during Leu-AMP formation. These show a cycle of dramatic conformational changes, involving multiple domains, and correlate with an energetically unfavorable peptide-plane flip observed in the active site of the pre-transition state structure. Biochemical analyses, combined with mutant structural studies, reveal that this backbone distortion acts as a trigger, temporally compartmentalizing the first two catalytic steps. These results unveil the remarkable effect of this small structural alteration on the global dynamics and activity of the enzyme.
Asunto(s)
Leucina-ARNt Ligasa , ARN de Transferencia de Leucina , Catálisis , Dominio Catalítico , Leucina-ARNt Ligasa/química , Leucina-ARNt Ligasa/genética , Leucina-ARNt Ligasa/metabolismo , Péptidos , ARN de Transferencia de Leucina/metabolismoRESUMEN
Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.
Asunto(s)
Enfermedades Mitocondriales , ARN de Transferencia de Leucina , Humanos , Animales , Ratones , ARN de Transferencia de Leucina/metabolismo , Enfermedades Mitocondriales/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mutación , Procesamiento Postranscripcional del ARNRESUMEN
Pancreatic cancer (PC) is a life-threatening cancer with increasing incidence in developed countries. Reports indicate that tRNA-derived fragments (tRFs) are possible therapeutic targets and biomarkers for cancer treatment. Nonetheless, the effect of tRF-Leu-AAG on PC is unclear. This study aims to explore the role of tRF-Leu-AAG and upstream frameshift mutant 1 (UPF1) in the development of PC and its potential underlying mechanisms. High-throughput second-generation sequencing techniques were used to detect the expression of tRFs in cancerous and adjacent normal tissues from PC patients. The role of tRF-Leu-AAG proliferation in PC cells was investigated via the Cell Counting Kit-8 (CCK8) assay. The effect of tRF-Leu-AAG on the invasion and migration ability of PC cells was also determined by the transwell assay. Thereafter, the downstream target genes of tRF-Leu-AAG were comprehensively predicted using bioinformatics analysis databases. We also used the Dual-Luciferase Reporter assay to assess the nexus between tRF-Leu-AAG and UPF1. Eventually, Western Blot was used to validate the expression of UPF1 in PC cells. A total of 33 tRF expressions significantly varied from PC patients. RT-qPCR confirmed that the expression of tRF-Leu-AAG was observably up-regulated in PC cells as compared to the control cells. Importantly, knockdown of tRF-Leu-AAG observably inhibited cell proliferation, migration, and invasion. Furthermore, according to the predicted frameshift database results, the UPF1 acted as downstream target genes for tRF-Leu-AAG and significantly down-regulated UPF1 expression.
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Neoplasias Pancreáticas , ARN de Transferencia de Leucina , Proliferación Celular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pancreáticas/genética , ARN Helicasas , Reacción en Cadena en Tiempo Real de la Polimerasa , TransactivadoresRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , ARN Largo no Codificante/genética , ARN Citoplasmático Pequeño/genética , ARN de Transferencia/genética , Partícula de Reconocimiento de Señal/genética , Neoplasias de la Mama/genética , Ciclo Celular , Femenino , Humanos , Células MCF-7 , Metástasis de la Neoplasia , ARN Ribosómico 5S/genética , ARN de Transferencia de Leucina/genéticaRESUMEN
BACKGROUND: Sequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt-tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined. METHODS: A maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree. RESULTS: Four of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNAAla and m.12311T>C in tRNALeu(CUN) , were identified via PCR-Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNAAla (position 59), while the T-to-C substitution at position 12311 located at position 48 in the variable stem of tRNALeu(CUN) and was believed to alter the aminoacylation and the steady-state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes. CONCLUSION: Our data indicated that mt-tRNAAla m.5601C>T and tRNALeu(CUN) 12311T>C mutations were associated with NSHL.
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ARN de Transferencia de Alanina , ARN de Transferencia de Leucina , Humanos , Adenosina Trifosfato , Sordera , ADN Mitocondrial/genética , Mutación/genética , Especies Reactivas de OxígenoRESUMEN
Streptomycetes are characterized by their ability to produce structurally diverse compounds as secondary metabolites and by their complex developmental life cycle, which includes aerial mycelium formation and sporulation. The production of secondary metabolites is growth-stage dependent, and generally coincides with morphological development on a solid culture. Streptomyces sp. BB47 produces several types of bioactive compounds and displays a bald phenotype that is devoid of an aerial mycelium and spores. Here, we demonstrated by genome analysis and gene complementation experiments that the bald phenotype arises from the bldA gene, which is predicted to encode the Leu-tRNAUUA molecule. Unlike the wild-type strain producing jomthonic acid A (1) and antarlide A (2), the strain complemented with a functional bldA gene newly produced milbemycin (3). The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as milbemycin A4, which is an insecticidal/acaricidal antibiotic. These results indicate that genetic manipulation of genes involved in morphological development in streptomycetes is a valuable way to activate cryptic biosynthetic pathways.
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Streptomyces , Vías Biosintéticas , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Macrólidos , ARN de Transferencia de Leucina/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
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.