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1.
Nat Commun ; 15(1): 4683, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824131

RESUMO

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.


Assuntos
Mitocôndrias , RNA de Transferência , Ribonuclease P , tRNA Metiltransferases , Humanos , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Mitocôndrias/metabolismo , Ribonuclease P/metabolismo , Ribonuclease P/genética , Ribonuclease P/química , tRNA Metiltransferases/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/química , Processamento Pós-Transcricional do RNA , Microscopia Crioeletrônica , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/química , Metilação , Conformação de Ácido Nucleico , Modelos Moleculares , Precursores de RNA/metabolismo , Precursores de RNA/genética
2.
Sci Rep ; 14(1): 12602, 2024 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824202

RESUMO

Mitochondrial RNA modification (MRM) plays a crucial role in regulating the expression of key mitochondrial genes and promoting tumor metastasis. Despite its significance, comprehensive studies on MRM in lower grade gliomas (LGGs) remain unknown. Single-cell RNA-seq data (GSE89567) was used to evaluate the distribution functional status, and correlation of MRM-related genes in different cell types of LGG microenvironment. We developed an MRM scoring system by selecting potential MRM-related genes using LASSO regression analysis and the Random Survival Forest algorithm, based on multiple bulk RNA-seq datasets from TCGA, CGGA, GSE16011, and E-MTAB-3892. Analysis was performed on prognostic and immunological features, signaling pathways, metabolism, somatic mutations and copy number variations (CNVs), treatment responses, and forecasting of potential small-molecule agents. A total of 35 MRM-related genes were selected from the literature. Differential expression analysis of 1120 normal brain tissues and 529 LGGs revealed that 22 and 10 genes were upregulated and downregulated, respectively. Most genes were associated with prognosis of LGG. METLL8, METLL2A, TRMT112, and METTL2B were extensively expressed in all cell types and different cell cycle of each cell type. Almost all cell types had clusters related to mitochondrial RNA processing, ribosome biogenesis, or oxidative phosphorylation. Cell-cell communication and Pearson correlation analyses indicated that MRM may promoting the development of microenvironment beneficial to malignant progression via modulating NCMA signaling pathway and ICP expression. A total of 11 and 9 MRM-related genes were observed by LASSO and the RSF algorithm, respectively, and finally 6 MRM-related genes were used to establish MRM scoring system (TRMT2B, TRMT11, METTL6, METTL8, TRMT6, and TRUB2). The six MRM-related genes were then validated by qPCR in glioma and normal tissues. MRM score can predict the malignant clinical characteristics, abundance of immune infiltration, gene variation, clinical outcome, the enrichment of signaling pathways and metabolism. In vitro experiments demonstrated that silencing METTL8 significantly curbs glioma cell proliferation and enhances apoptosis. Patients with a high MRM score showed a better response to immunotherapies and small-molecule agents such as arachidonyl trifluoromethyl ketone, MS.275, AH.6809, tacrolimus, and TTNPB. These novel insights into the biological impacts of MRM within the glioma microenvironment underscore its potential as a target for developing precise therapies, including immunotherapeutic approaches.


Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Glioma/genética , Glioma/patologia , Prognóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Regulação Neoplásica da Expressão Gênica , Microambiente Tumoral/genética , Processamento Pós-Transcricional do RNA , Gradação de Tumores , Mitocôndrias/genética , Mitocôndrias/metabolismo , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Multiômica
3.
Nat Commun ; 15(1): 4814, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862469

RESUMO

A detailed understanding of how spaceflight affects human health is essential for long-term space exploration. Liquid biopsies allow for minimally-invasive multi-omics assessments that can resolve the molecular heterogeneity of internal tissues. Here, we report initial results from the JAXA Cell-Free Epigenome Study, a liquid biopsy study with six astronauts who resided on the International Space Station (ISS) for more than 120 days. Analysis of plasma cell-free RNA (cfRNA) collected before, during, and after spaceflight confirms previously reported mitochondrial dysregulation in space. Screening with 361 cell surface marker antibodies identifies a mitochondrial DNA-enriched fraction associated with the scavenger receptor CD36. RNA-sequencing of the CD36 fraction reveals tissue-enriched RNA species, suggesting the plasma mitochondrial components originated from various tissues. We compare our plasma cfRNA data to mouse plasma cfRNA data from a previous JAXA mission, which had used on-board artificial gravity, and discover a link between microgravity and the observed mitochondrial responses.


Assuntos
Antígenos CD36 , Ácidos Nucleicos Livres , DNA Mitocondrial , Voo Espacial , Ausência de Peso , DNA Mitocondrial/genética , DNA Mitocondrial/sangue , Humanos , Ácidos Nucleicos Livres/sangue , Animais , Camundongos , Antígenos CD36/metabolismo , Antígenos CD36/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Masculino , Astronautas , RNA/metabolismo , RNA/genética , Biópsia Líquida/métodos , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Feminino , Pessoa de Meia-Idade , Adulto
4.
Mitochondrion ; 77: 101907, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38777221

RESUMO

Mitochondrial mutations have been linked to changes in phenotypes such as fertility or longevity, however, these changes have been often inconsistent across populations for unknown reasons. A hypothesis that could explain this inconsistency is that some still uncharacterized mitochondrial products are mediating the phenotypic changes across populations. It has been hypothesized that one such product could be the small RNAs encoded in the mitochondrial genome, thus this work will provide new evidence for their existence and function. By using data from the 1000 genome project and knowledge from previously characterized nuclear small RNAs, this study found that 10 small RNAs encoded in tRNA fragments are consistently expressed in 450 individuals from five different populations. Furthermore, this study demonstrated that the expression of some small mitochondrial RNAs is different in individuals of African ancestry, similar to what was observed before in nuclear and mitochondria mRNAs. Lastly, we investigate the causes behind these differences in expression, showing that at least one of the mt-tRFs might be regulated by TRMT10B. The analyses presented in this work further support the small mitochondrial RNAs as functional molecules, and their population-specific expression supports the hypothesis that they act as a mediator between the nucleus and mitochondria differently across populations.


Assuntos
Linfócitos , RNA Mitocondrial , Humanos , RNA Mitocondrial/genética , Linfócitos/metabolismo , Transcrição Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo
5.
Hum Mol Genet ; 33(R1): R26-R33, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38779774

RESUMO

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.


Assuntos
Mitocôndrias , Polirribonucleotídeo Nucleotidiltransferase , Estabilidade de RNA , RNA Mitocondrial , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Estabilidade de RNA/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , RNA Helicases/metabolismo , RNA Helicases/genética , RNA/metabolismo , RNA/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Endorribonucleases , Exorribonucleases , Complexos Multienzimáticos
6.
Nat Commun ; 15(1): 4422, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38789440

RESUMO

The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduces their respective read counts by more than 85%, resulting in an increase in sequencing depth of 47% on average for long non-coding RNAs. We also apply RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as an increase in non-globin reads by 16% on average. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER's modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser .


Assuntos
RNA Mensageiro , Análise de Sequência de RNA , Análise de Sequência de RNA/métodos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Longo não Codificante/genética , RNA/genética , Software , Globinas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Aprendizado Profundo , Transcriptoma , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo
7.
Hum Mol Genet ; 33(R1): R19-R25, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38779769

RESUMO

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.


Assuntos
DNA Mitocondrial , Mitocôndrias , Processamento Pós-Transcricional do RNA , RNA Mitocondrial , Humanos , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Transcrição Gênica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo
8.
Mol Metab ; 84: 101955, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38704026

RESUMO

OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina , Mitocôndrias , Animais , Ratos , Humanos , Mitocôndrias/metabolismo , Células Secretoras de Insulina/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Masculino , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Camundongos , Ratos Wistar , Transporte de Elétrons
9.
RNA ; 30(7): 839-853, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38609156

RESUMO

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.


Assuntos
Ciclo do Ácido Cítrico , Citosol , Malato Desidrogenase , Mitocôndrias , Ligação Proteica , Malato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Citosol/metabolismo , Citosol/enzimologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , RNA/metabolismo , RNA/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , NAD/metabolismo , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
10.
Nature ; 628(8009): 844-853, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38570685

RESUMO

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.


Assuntos
Alelos , DNA Polimerase gama , Vírus da Encefalite Transmitidos por Carrapatos , Herpesvirus Humano 1 , Tolerância Imunológica , SARS-CoV-2 , Animais , Feminino , Humanos , Masculino , Camundongos , Idade de Início , COVID-19/imunologia , COVID-19/virologia , COVID-19/genética , DNA Polimerase gama/genética , DNA Polimerase gama/imunologia , DNA Polimerase gama/metabolismo , DNA Mitocondrial/imunologia , DNA Mitocondrial/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/genética , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Herpes Simples/genética , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/imunologia , Mutação , RNA Mitocondrial/imunologia , RNA Mitocondrial/metabolismo , SARS-CoV-2/imunologia
11.
RNA ; 30(6): 597-608, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38448244

RESUMO

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.


Assuntos
Mitocôndrias , Membranas Mitocondriais , RNA Mitocondrial , Mitocôndrias/metabolismo , Mitocôndrias/genética , Humanos , Animais , Membranas Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA/metabolismo , RNA/genética , Transporte de RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Biossíntese de Proteínas , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética
12.
Mol Cell Proteomics ; 23(4): 100746, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38447791

RESUMO

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive deficits, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo-Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded oxidative phosphorylation mRNAs (mt-Nd1, mt-Nd2, mt-Nd4, mt-Nd4l, mt-Nd5, mt-Nd6, mt-Co1, mt-Cytb, and mt-ATP8). We also applied tandem mass tag-based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein abundance of mitochondria-related genes in HD.


Assuntos
Doença de Huntington , Mitocôndrias , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Mitocôndrias/metabolismo , Humanos , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação Oxidativa , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Linhagem Celular , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , Espectrometria de Massas , Perfil de Ribossomos
13.
Biochim Biophys Acta Mol Basis Dis ; 1870(4): 167043, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38320662

RESUMO

Mitochondrial encephalopathy is a neurological disorder caused by impaired mitochondrial function and energy production. One of the genetic causes of this condition is the mutation of MT-TN, a gene that encodes the mitochondrial transfer RNA (tRNA) for asparagine. MT-TN mutations affect the stability and structure of the tRNA, resulting in reduced protein synthesis and complex enzymatic deficiency of the mitochondrial respiratory chain. Our patient cohort manifests with epileptic encephalopathy, ataxia, hypotonia, and bilateral basal ganglia calcification, which differs from previously reported cases. MT-TN mutation deficiency leads to decreased basal and maximal oxygen consumption rates, disrupted spare respiratory capacity, declined mitochondrial membrane potential, and impaired ATP production. Moreover, MT-TN mutations promote mitophagy, a process of selective degradation of damaged mitochondria by autophagy. Excessive mitophagy further leads to mitochondrial biogensis as a compensatory mechanism. In this study, we provided evidence of pathogenicity for two MT-TN mutations, m.5688 T > C and m.G5691A, explored the molecular mechanisms, and summarized the clinical manifestations of MT-TN mutations. Our study expanded the genotype and phenotypic spectrum and provided new insight into mt-tRNA (Asn)-associated mitochondrial encephalopathy.


Assuntos
Encefalopatias , Encefalomiopatias Mitocondriais , Mitofagia , Humanos , Mitofagia/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Encefalopatias/genética , Encefalopatias/metabolismo , RNA de Transferência/genética , RNA Mitocondrial/metabolismo
14.
Elife ; 132024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38251974

RESUMO

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.


Assuntos
Células Endoteliais , RNA , Humanos , RNA Mitocondrial/genética , Cromatina , Bioensaio
16.
Yeast ; 41(4): 256-278, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37642136

RESUMO

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.


Assuntos
RNA , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA/metabolismo , Íntrons , Transcriptoma , RNA Mitocondrial/metabolismo , Splicing de RNA , Mitocôndrias/genética , Mitocôndrias/metabolismo , Análise de Sequência de RNA , Glucose/metabolismo
18.
Plant Physiol ; 194(3): 1593-1610, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-37956067

RESUMO

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.


Assuntos
Proteínas de Plantas , Zea mays , Zea mays/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mitocondrial/metabolismo , Sementes/metabolismo , Endosperma/metabolismo
19.
Nucleic Acids Res ; 52(D1): D229-D238, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-37843123

RESUMO

We describe the Mitochondrial and Nuclear rRNA fragment database (MINRbase), a knowledge repository aimed at facilitating the study of ribosomal RNA-derived fragments (rRFs). MINRbase provides interactive access to the profiles of 130 238 expressed rRFs arising from the four human nuclear rRNAs (18S, 5.8S, 28S, 5S), two mitochondrial rRNAs (12S, 16S) or four spacers of 45S pre-rRNA. We compiled these profiles by analyzing 11 632 datasets, including the GEUVADIS and The Cancer Genome Atlas (TCGA) repositories. MINRbase offers a user-friendly interface that lets researchers issue complex queries based on one or more criteria, such as parental rRNA identity, nucleotide sequence, rRF minimum abundance and metadata keywords (e.g. tissue type, disease). A 'summary' page for each rRF provides a granular breakdown of its expression by tissue type, disease, sex, ancestry and other variables; it also allows users to create publication-ready plots at the click of a button. MINRbase has already allowed us to generate support for three novel observations: the internal spacers of 45S are prolific producers of abundant rRFs; many abundant rRFs straddle the known boundaries of rRNAs; rRF production is regimented and depends on 'personal attributes' (sex, ancestry) and 'context' (tissue type, tissue state, disease). MINRbase is available at https://cm.jefferson.edu/MINRbase/.


Assuntos
Bases de Dados de Ácidos Nucleicos , RNA Mitocondrial , RNA Ribossômico , Humanos , Sequência de Bases , Mitocôndrias/genética , Ribossomos , RNA Mitocondrial/genética , RNA Ribossômico/genética
20.
Nucleic Acids Res ; 52(3): 1341-1358, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38113276

RESUMO

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.


Assuntos
Mitocôndrias , Proteínas Mitocondriais , Peptídeo Hidrolases , tRNA Metiltransferases , Humanos , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Peptídeo Hidrolases/genética , Proteólise , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , Proteínas Mitocondriais/metabolismo
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