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1.
Am J Hum Genet ; 108(11): 2195-2204, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34715011

RESUMO

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.


Assuntos
Alelos , Pleiotropia Genética , Mitocôndrias/enzimologia , RNA Mitocondrial/genética , RNA de Transferência/genética , Ribonuclease P/genética , Adulto , Feminino , Humanos , Masculino , Linhagem
2.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639116

RESUMO

Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those operating in the host cell, and among eukaryotes. In recent decades, studies have revealed several plant-specific features of mitochondrial gene regulation. The polyadenylation status of mRNA is critical for its stability and translation in mitochondria. In this short review, I focus on recent advances in understanding the mechanisms regulating mRNA polyadenylation in plant mitochondria, including the role of poly(A)-specific ribonuclease-like proteins (PARNs). Accumulating evidence suggests that plant mitochondria have unique regulatory systems for mRNA poly(A) status and that PARNs play pivotal roles in these systems.


Assuntos
Embriófitas/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Poli A/genética , RNA Mensageiro/genética , RNA Mitocondrial/genética , Embriófitas/crescimento & desenvolvimento , Embriófitas/metabolismo , Exorribonucleases/genética , Poliadenilação , Estabilidade de RNA , RNA Mensageiro/química
3.
Biol Res ; 54(1): 33, 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34666824

RESUMO

BACKGROUND: The antisense noncoding mitochondrial RNAs (ASncmtRNAs) derive from the mitochondrial 16S gene. Knockdown of these transcripts with chemically-modified antisense oligonucleotides induces proliferative arrest, apoptosis and invasiveness reduction in tumor but not normal cells. One of these transcripts, ASncmtRNA-2, contains the complete and identical sequence of hsa-miR-4485-3p and, upon knockdown of this transcript, there is a strong increase in levels of this miRNA, suggesting ASncmtRNA-2 as a source for miR-4485-3p, which is supported by several evidences from our group and others, in the ex vivo setting. RESULTS: Here we show that incubation of in vitro-transcribed ASncmtRNA-2 with recombinant Dicer produces RNA fragments corresponding to hsa-miR-4485-3p, showing that Dicer binds to and processes ASncmtRNA-2, strongly supporting the hypothesis that ASncmtRNA-2 acts as a precursor for miR-4485-3p. CONCLUSION: The in vitro results presented here strengthen the hypothesis that miR-4485-3p is derived from ASncmtRNA-2 by Dicer processing. Since miR-4485-3p is classified as a tumor suppressor miRNA, this evidence strengthens the application of ASncmtRNA knockdown for cancer therapy.


Assuntos
MicroRNAs , RNA Longo não Codificante , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Antissenso/genética , RNA Mitocondrial/genética
4.
PLoS Genet ; 17(10): e1009808, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34665800

RESUMO

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that some regions of rho+ mtDNA persisted while others were lost after a specific hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced biased inheritance of a subset of hypersuppressive genomes. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance over a specific hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.


Assuntos
DNA Fúngico/genética , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/genética , Mitocôndrias/genética , RNA Mitocondrial/genética , Replicação do DNA/genética , Genes Fúngicos/genética , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética/genética
5.
Methods Enzymol ; 658: 83-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517961

RESUMO

Unicellular parasite Trypanosoma brucei maintains an elaborate mitochondrial mRNA processing pathway including 3'-5' exonucleolytic trimming of primary precursors, 5' and 3' modifications, and, in most cases, massive U-insertion/deletion editing. Whereas the role of editing in restoring protein coding sequence is apparent, recent developments suggest that terminal modifications are equally critical for generating a stable translationally competent messenger. The enzymatic activities responsible for 5' pyrophosphate hydrolysis, 3' adenylation and uridylation, and 3'-5' decay are positively and negatively regulated by pentatricopeptide repeat-containing (PPR) proteins. These sequence-specific RNA binding factors typically contain arrays of 35-amino acid repeats each of which recognizes a single nucleotide. Here, we introduce a combinatorial CTS affinity tag, which underlies a suite of methods for PPR proteins purification, in vivo RNA binding sites mapping and sub-cellular localization studies. These approaches should be applicable to most trypanosomal RNA binding proteins.


Assuntos
Trypanosoma brucei brucei , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Edição de RNA , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
6.
Int J Mol Sci ; 22(17)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34502411

RESUMO

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Humanos
7.
Nat Commun ; 12(1): 4980, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404792

RESUMO

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.


Assuntos
RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
8.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360765

RESUMO

Mitochondria, often referred to as the powerhouses of cells, are vital organelles that are present in almost all eukaryotic organisms, including humans. They are the key energy suppliers as the site of adenosine triphosphate production, and are involved in apoptosis, calcium homeostasis, and regulation of the innate immune response. Abnormalities occurring in mitochondria, such as mitochondrial DNA (mtDNA) mutations and disturbances at any stage of mitochondrial RNA (mtRNA) processing and translation, usually lead to severe mitochondrial diseases. A fundamental line of investigation is to understand the processes that occur in these organelles and their physiological consequences. Despite substantial progress that has been made in the field of mtRNA processing and its regulation, many unknowns and controversies remain. The present review discusses the current state of knowledge of RNA processing in human mitochondria and sheds some light on the unresolved issues.


Assuntos
Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , RNA Mitocondrial/genética
9.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445229

RESUMO

Symbiosis between the mitochondrion and the ancestor of the eukaryotic cell allowed cellular complexity and supported life. Mitochondria have specialized in many key functions ensuring cell homeostasis and survival. Thus, proper communication between mitochondria and cell nucleus is paramount for cellular health. However, due to their archaebacterial origin, mitochondria possess a high immunogenic potential. Indeed, mitochondria have been identified as an intracellular source of molecules that can elicit cellular responses to pathogens. Compromised mitochondrial integrity leads to release of mitochondrial content into the cytosol, which triggers an unwanted cellular immune response. Mitochondrial nucleic acids (mtDNA and mtRNA) can interact with the same cytoplasmic sensors that are specialized in recognizing genetic material from pathogens. High-energy demanding cells, such as neurons, are highly affected by deficits in mitochondrial function. Notably, mitochondrial dysfunction, neurodegeneration, and chronic inflammation are concurrent events in many severe debilitating disorders. Interestingly in this context of pathology, increasing number of studies have detected immune-activating mtDNA and mtRNA that induce an aberrant production of pro-inflammatory cytokines and interferon effectors. Thus, this review provides new insights on mitochondria-driven inflammation as a potential therapeutic target for neurodegenerative and primary mitochondrial diseases.


Assuntos
Mitocôndrias/imunologia , Doenças Neurodegenerativas/imunologia , Animais , Citocinas/imunologia , DNA Mitocondrial/imunologia , Humanos , Mitocôndrias/patologia , Doenças Neurodegenerativas/patologia , RNA Mitocondrial/imunologia
10.
Cell Mol Life Sci ; 78(19-20): 6585-6592, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34427691

RESUMO

Single-cell RNA-sequencing (scRNA-seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to "low quality" potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes, transcripts per cell, and the fraction of transcripts from mitochondrial genes. While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mitochondrial transcripts is standard and often set as default in scRNA-seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public data set of cells isolated from embryonal murine sinoatrial node tissue (Goodyer William et al. in Circ Res 125:379-397, 2019). Taken together, we recommend omitting this filtering parameter for scRNA-seq in cardiovascular applications whenever possible.


Assuntos
RNA Mitocondrial/genética , RNA Citoplasmático Pequeno/genética , Análise de Célula Única/métodos , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Miócitos Cardíacos/fisiologia , Marca-Passo Artificial , Controle de Qualidade , RNA Mensageiro/genética , Análise de Sequência de RNA , Software , Sequenciamento Completo do Exoma/métodos
11.
J Mol Biol ; 433(18): 167125, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34224750

RESUMO

APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.


Assuntos
Núcleo Celular/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Núcleo Celular/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células HeLa , Humanos , Mitocôndrias/genética , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mitocondrial/genética
12.
Nat Cell Biol ; 23(7): 684-691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34253897

RESUMO

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.


Assuntos
Enzimas AlkB/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Enzimas AlkB/genética , Citidina/análogos & derivados , Citidina/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA Mitocondrial/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo
13.
Methods Mol Biol ; 2275: 227-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118041

RESUMO

Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , RNA Antissenso/farmacologia , RNA Mitocondrial/genética , DNA Mitocondrial/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inativação Gênica , Células HeLa , Humanos , Lipossomos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Mutação , RNA Antissenso/química , RNA Mitocondrial/efeitos dos fármacos
14.
Methods Mol Biol ; 2275: 247-263, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118042

RESUMO

Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.


Assuntos
DNA Mitocondrial/química , Mitocôndrias/genética , Doenças Mitocondriais/genética , Análise de Célula Única/métodos , Animais , Humanos , Hibridização in Situ Fluorescente , Camundongos , RNA Mitocondrial/química , Transcrição Genética
15.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071057

RESUMO

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.


Assuntos
Transformação Celular Neoplásica , Neoplasias/metabolismo , Biogênese de Organelas , Ribossomos , Animais , Apoptose , Autofagia , Ciclo Celular , Movimento Celular , Nucléolo Celular/metabolismo , Citosol/metabolismo , Reparo do DNA , Estresse do Retículo Endoplasmático , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia
16.
Methods Mol Biol ; 2277: 203-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080154

RESUMO

Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result of mutations in genes encoding OXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis. Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis (HIA ) that can be used to make predictions concerning the disruptive potential of a large subset of mt-rRNA variants. We have used HIA to explore the mutational landscape of 12S and 16S mt-rRNA genes. Our HIA studies include a thorough classification of all rare variants reported in the literature as well as others obtained from studies performed in collaboration with physicians. HIA has also been used with non-mammalian mt-rRNA genes to elucidate how mitotypes influence the interaction of the individual and the environment. Regarding mt-tRNA variations, rapidly growing evidence shows that the spectrum of mutations causing mitochondrial disease might differ between the different mitochondrial haplogroups seen in human populations.


Assuntos
Biologia Computacional/métodos , DNA Mitocondrial/genética , Genômica/métodos , Doenças Mitocondriais/genética , RNA Mitocondrial/genética , Humanos , Mutação , Biossíntese de Proteínas , RNA Ribossômico , RNA Ribossômico 16S , RNA de Transferência/genética
17.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
18.
J Biol Chem ; 297(1): 100816, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023389

RESUMO

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.


Assuntos
Mitocôndrias/metabolismo , RNA de Transferência de Alanina/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Sequência de Bases , Citocromos c/metabolismo , Transporte de Elétrons , Humanos , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Mutação/genética , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mitocondrial/genética , RNA de Transferência de Alanina/química , Espécies Reativas de Oxigênio/metabolismo , Aminoacilação de RNA de Transferência
19.
Plant Mol Biol ; 106(4-5): 335-348, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33909186

RESUMO

KEY MESSAGE: RNA PROCESSING FACTORs 1 AND 8 (RPF1 and RPF8), both restorer of fertility like pentatricopeptide repeat proteins, are required for processing of dicistronic nad4L-atp4 and nad3-rps12 transcripts in Arabidopsis mitochondria. In mitochondria of Arabidopsis thaliana (Arabidopsis), the 5' termini of many RNAs are generated on the post-transcriptional level. This process is still poorly understood in terms of both the underlying mechanism as well as proteins required. Our studies now link the generation of polymorphic 5' extremities of the dicistronic nad3-rps12 and nad4L-atp4 transcripts to the function of the P-type pentatricopeptide repeat proteins RNA PROCESSING FACTORs 8 (RPF8) and 1 (RPF1). RPF8 is required to generate the nad3-rps12 -141 5' end in ecotype Van-0 whereas the RPF8 allele in Col has no function in the generation of any 5' terminus of this transcript. This observation strongly suggests the involvement of an additional factor in the generation of the -229 5' end of nad3-rps12 transcripts in Col. RPF1, previously found to be necessary for the generation of the -228 5' end of the major 1538 nucleotide-long nad4 mRNAs, is also important for the formation of nad4L-atp4 transcripts with a 5' end at position -318 in Col. Many Arabidopsis ecotypes contain inactive RPF1 alleles resulting in the accumulation of various low abundant nad4L-atp4 RNAs which might represent precursor and/or degradation products. Some of these ecotypes accumulate major, but slightly smaller RNA species. The introduction of RPF1 into these lines not only establishes the formation of the major nad4L-atp4 dicistronic mRNA with the -318 5' terminus, the presence of this gene also suppresses the accumulation of most alternative nad4L-atp4 RNAs. Beside RPF1, several other factors contribute to nad4L-atp4 transcript formation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Mitocôndrias/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/metabolismo , Técnicas de Inativação de Genes , Mitocôndrias/genética , Polimorfismo Genético , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Plantas/genética , Transcrição Genética
20.
Oxid Med Cell Longev ; 2021: 5517777, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33824695

RESUMO

Triple-negative breast cancer (TNBC) is characterized by poor outcome and the most challenging breast cancer type to treat worldwide. TNBC manifests distinct profile of mitochondrial functions, which dictates reprogrammed metabolism, fosters tumor progression, and notably serves as therapeutic targets. Mitochondrial microRNAs (mitomiRs) are a group of microRNAs that critically modulate mitochondrial homeostasis. By a pathway-centric manner, mitomiRs tightly orchestrate metabolic reprogramming, redox status, cell apoptosis, mitochondrial dynamics, mitophagy, mitochondrial DNA (mtDNA) maintenance, and calcium balance, leading to an emerging field of study in various cancer types, including TNBC. We herein review the recent insights into the roles and mechanism of mitomiRs in TNBC and highlight its clinical value in diagnosis and prognosis as well as vital advances on therapeutics of preclinical and clinical studies.


Assuntos
MicroRNAs/genética , RNA Mitocondrial/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , MicroRNAs/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo , RNA Mitocondrial/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
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