RESUMEN
The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
Asunto(s)
ADN Mitocondrial , Proteínas Mitocondriales , Animales , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Dimerización , Humanos , Mamíferos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN/metabolismo , ARN Mitocondrial , ARN Citoplasmático Pequeño , Partícula de Reconocimiento de Señal , Transcripción GenéticaRESUMEN
The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.
Asunto(s)
Regulación de la Expresión Génica , Silenciador del Gen , Genes Mitocondriales , Transporte de Electrón , Complejo IV de Transporte de Electrones/genética , Células HEK293 , Humanos , Proteínas Mitocondriales/metabolismo , Oligonucleótidos/química , Fosforilación Oxidativa , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.
Asunto(s)
5-Metilcitosina , Citosol , Mitocondrias , Estabilidad del ARN , ARN Bicatenario , ARN Mitocondrial , Humanos , Citosol/metabolismo , 5-Metilcitosina/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Células HEK293 , Células HeLa , Metiltransferasas/metabolismo , Metiltransferasas/genética , Inmunidad Innata , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Sistemas CRISPR-CasRESUMEN
Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Ciclooxigenasa 1/biosíntesis , Ciclooxigenasa 1/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Células HEK293 , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mitocondrial , Ribosomas/metabolismoRESUMEN
Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.
Asunto(s)
Dieta Alta en Grasa , Epigénesis Genética , Mitocondrias , ARN Mitocondrial , Espermatozoides , Animales , Femenino , Humanos , Masculino , Ratones , Índice de Masa Corporal , Dieta Alta en Grasa/efectos adversos , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Epidídimo/citología , Epigénesis Genética/genética , Fertilización/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Obesidad/genética , Obesidad/metabolismo , Obesidad/etiología , Oocitos/metabolismo , Sobrepeso/genética , Sobrepeso/metabolismo , Herencia Paterna/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Espermatozoides/metabolismo , Testículo/citología , Transcripción GenéticaRESUMEN
Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.
Asunto(s)
Alelos , ADN Polimerasa gamma , Virus de la Encefalitis Transmitidos por Garrapatas , Herpesvirus Humano 1 , Tolerancia Inmunológica , SARS-CoV-2 , Animales , Femenino , Humanos , Masculino , Ratones , Edad de Inicio , COVID-19/inmunología , COVID-19/virología , COVID-19/genética , ADN Polimerasa gamma/genética , ADN Polimerasa gamma/inmunología , ADN Polimerasa gamma/metabolismo , ADN Mitocondrial/inmunología , ADN Mitocondrial/metabolismo , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/genética , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/virología , Efecto Fundador , Técnicas de Sustitución del Gen , Herpes Simple/genética , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/inmunología , Mutación , ARN Mitocondrial/inmunología , ARN Mitocondrial/metabolismo , SARS-CoV-2/inmunologíaRESUMEN
The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer-binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , ARN Helicasas DEAD-box/metabolismo , Ribonucleasa III/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Inmunoprecipitación de Cromatina , Humanos , MicroARNs/metabolismo , Fotoquímica , ARN/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , ARN Mitocondrial , Proteínas de Unión al ARN/metabolismo , Sitio de Iniciación de la Transcripción , TranscriptomaRESUMEN
Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.
Asunto(s)
Fumarato Hidratasa , Interferón beta , Macrófagos , Mitocondrias , ARN Mitocondrial , Humanos , Argininosuccinato Sintasa/metabolismo , Ácido Argininosuccínico/metabolismo , Ácido Aspártico/metabolismo , Respiración de la Célula , Citosol/metabolismo , Fumarato Hidratasa/antagonistas & inhibidores , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Fumaratos/metabolismo , Interferón beta/biosíntesis , Interferón beta/inmunología , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Lupus Eritematoso Sistémico/enzimología , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial , Metabolómica , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Mitocondrial/metabolismoRESUMEN
Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
Asunto(s)
ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , ARN Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Sitios de Unión , Microscopía por Crioelectrón , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mitocondrial/química , ARN Mitocondrial/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinámica , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Iniciación de la Transcripción GenéticaRESUMEN
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
Asunto(s)
Metiltransferasas/metabolismo , ARN Mitocondrial/química , ARN de Transferencia/química , Animales , Anticodón , Proliferación Celular , Codón , Citoplasma , ADN Mitocondrial/metabolismo , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales , Proteínas Mitocondriales/química , Consumo de Oxígeno , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Ribosomas/metabolismo , Regulación hacia ArribaRESUMEN
Mitochondrial double-stranded RNA (dsRNA) can form spontaneously in mitochondria, blocking mitochondrial gene expression and triggering an immune response. A recent study by Kim, Tan, et al. identified a safeguard mechanism in which NOP2/Sun RNA methyltransferase 4 (NSUN4)-mediated RNA methylation (m5C) recruits the RNA degradation machinery to prevent dsRNA formation.
Asunto(s)
ARN Mitocondrial , ARN Mitocondrial/metabolismo , Humanos , Metilación , Citosina/metabolismo , Mitocondrias/metabolismo , Estabilidad del ARN , ARN/metabolismo , ARN Bicatenario/metabolismo , Metiltransferasas/metabolismo , AnimalesRESUMEN
Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
Asunto(s)
5-Metilcitosina , Citosina/análogos & derivados , Glucólisis , Mitocondrias , Metástasis de la Neoplasia , Fosforilación Oxidativa , ARN Mitocondrial , 5-Metilcitosina/biosíntesis , 5-Metilcitosina/metabolismo , Antígenos CD36 , Supervivencia Celular , Citosina/metabolismo , Progresión de la Enfermedad , Glucólisis/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Fosforilación Oxidativa/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismoRESUMEN
Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial-encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes.
Asunto(s)
MicroARNs/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Biosíntesis de Proteínas/fisiología , ARN/metabolismo , Animales , Transporte Biológico Activo/fisiología , Transporte de Electrón/fisiología , Humanos , MicroARNs/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , ARN/genética , ARN MitocondrialRESUMEN
Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
Asunto(s)
Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/metabolismo , Exorribonucleasas/metabolismo , Mitocondrias/enzimología , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mitocondrial/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Biomarcadores de Tumor/genética , Exorribonucleasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mitocondrial/genética , Células Sf9 , SpodopteraRESUMEN
Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.
Asunto(s)
Codón de Terminación , Proteínas de Drosophila/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Deficiencias en la Proteostasis/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HeLa , Humanos , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Proteínas Mitocondriales/genética , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismoRESUMEN
Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.
Asunto(s)
ADN Mitocondrial , Mitocondrias , Procesamiento Postranscripcional del ARN , ARN Mitocondrial , Humanos , ADN Mitocondrial/genética , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Transcripción Genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.
Asunto(s)
Mitocondrias , Polirribonucleótido Nucleotidiltransferasa , Estabilidad del ARN , ARN Mitocondrial , Humanos , Mitocondrias/metabolismo , Mitocondrias/genética , Estabilidad del ARN/genética , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Polirribonucleótido Nucleotidiltransferasa/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN/metabolismo , ARN/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Endorribonucleasas , Exorribonucleasas , Complejos MultienzimáticosRESUMEN
The mammalian mitochondrial proteome comprises over 1000 proteins, with the majority translated from nuclear-encoded messenger RNAs (mRNAs). Mounting evidence suggests many of these mRNAs are localized to the outer mitochondrial membrane (OMM) in a pre- or cotranslational state. Upon reaching the mitochondrial surface, these mRNAs are locally translated to produce proteins that are cotranslationally imported into mitochondria. Here, we summarize various mechanisms cells use to localize RNAs, including transfer RNAs (tRNAs), to the OMM and recent technological advancements in the field to study these processes. While most early studies in the field were carried out in yeast, recent studies reveal RNA localization to the OMM and their regulation in higher organisms. Various factors regulate this localization process, including RNA sequence elements, RNA-binding proteins (RBPs), cytoskeletal motors, and translation machinery. In this review, we also highlight the role of RNA structures and modifications in mitochondrial RNA localization and discuss how these features can alter the binding properties of RNAs. Finally, in addition to RNAs related to mitochondrial function, RNAs involved in other cellular processes can also localize to the OMM, including those implicated in the innate immune response and piRNA biogenesis. As impairment of messenger RNA (mRNA) localization and regulation compromise mitochondrial function, future studies will undoubtedly expand our understanding of how RNAs localize to the OMM and investigate the consequences of their mislocalization in disorders, particularly neurodegenerative diseases, muscular dystrophies, and cancers.
Asunto(s)
Mitocondrias , Membranas Mitocondriales , ARN Mitocondrial , Mitocondrias/metabolismo , Mitocondrias/genética , Humanos , Animales , Membranas Mitocondriales/metabolismo , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN/metabolismo , ARN/genética , Transporte de ARN , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Biosíntesis de Proteínas , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.
Asunto(s)
Ciclo del Ácido Cítrico , Citosol , Malato Deshidrogenasa , Mitocondrias , Unión Proteica , Malato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Citosol/metabolismo , Citosol/enzimología , Humanos , Mitocondrias/metabolismo , Mitocondrias/genética , Mitocondrias/enzimología , ARN/metabolismo , ARN/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , NAD/metabolismo , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.