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1.
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
2.
Proc Natl Acad Sci U S A ; 121(11): e2309841121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38442151

RESUMO

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Degrons , Herpesviridae , Apresentação de Antígeno , Citomegalovirus , Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana Transportadoras , Peptídeos , Ubiquitina-Proteína Ligases/genética , Herpesviridae/fisiologia
3.
bioRxiv ; 2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37808699

RESUMO

The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation.

4.
J Vis Exp ; (195)2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37212587

RESUMO

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.


Assuntos
DNA Mitocondrial , Genoma Mitocondrial , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Bromodesoxiuridina/metabolismo , Trifosfato de Adenosina/metabolismo , Replicação do DNA
5.
Nucleic Acids Res ; 50(14): 7991-8007, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35819194

RESUMO

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5'-3' exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.


Assuntos
Endonucleases Flap , RNA , Replicação do DNA , DNA Mitocondrial/genética , Endonucleases/metabolismo , Endonucleases Flap/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA/genética , RNA/metabolismo
6.
Wiley Interdiscip Rev RNA ; 13(3): e1690, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34498404

RESUMO

Mitochondria play a pivotal role in numerous cellular processes. One of them is regulation of the innate immune pathway. In this instance, mitochondria function in two different aspects of regulatory mechanisms. First, mitochondria are part of the antiviral signaling cascade that is triggered in the cytoplasm and transmitted to effector proteins through mitochondria-localized proteins. Second, mitochondria can become an endogenous source of innate immune stimuli. Under some pathophysiological conditions, mitochondria release to the cytoplasm immunogenic factors, such as mitochondrial nucleic acids. Here, we focus on immunogenic mitochondrial double-stranded RNA (mt-dsRNA) and its origin and metabolism. We discuss factors that are responsible for regulating mt-dsRNA and its escape from mitochondria, emphasizing the contribution of polynucleotide phosphorylase (PNPase, PNPT1). Finally, we review current knowledge of the role of PNPase in human health and disease. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Assuntos
Polirribonucleotídeo Nucleotidiltransferase , RNA de Cadeia Dupla , Exorribonucleases/metabolismo , Humanos , Sistema Imunitário/metabolismo , Imunidade Inata , Mitocôndrias/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Mitocondrial/metabolismo
7.
iScience ; 24(9): 103036, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34541468

RESUMO

Processive exoribonucleases are executors of RNA decay. In humans, their physical but not functional interactions were thoughtfully investigated. Here we have screened cells deficient in DIS3, XRN2, EXOSC10, DIS3L, and DIS3L2 with a custom siRNA library and determined their genetic interactions (GIs) with diverse pathways of RNA metabolism. We uncovered a complex network of positive interactions that buffer alterations in RNA degradation and reveal reciprocal cooperation with genes involved in transcription, RNA export, and splicing. Further, we evaluated the functional distinctness of nuclear DIS3 and cytoplasmic DIS3L using a library of all known genes associated with RNA metabolism. Our analysis revealed that DIS3 mutation suppresses RNA splicing deficiency, while DIS3L GIs disclose the interplay of cytoplasmic RNA degradation with nuclear RNA processing. Finally, genome-wide DIS3 GI map uncovered relations with genes not directly involved in RNA metabolism, like microtubule organization or regulation of telomerase activity.

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.
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
10.
Methods Mol Biol ; 2192: 133-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230771

RESUMO

RNA turnover is an essential part of the gene expression pathway, and there are several experimental approaches for its determination. High-throughput measurement of global RNA turnover rates can provide valuable information about conditions or proteins that impact gene expression. Here, we present a protocol for mitochondrial RNA turnover analysis which involves metabolic labeling of RNA coupled with quantitative high-throughput fluorescent microscopy. This approach gives an excellent opportunity to discover new factors involved in mitochondrial gene regulation when combined with loss-of-function screening strategy.


Assuntos
Regulação da Expressão Gênica , Imuno-Histoquímica/métodos , Mitocôndrias/genética , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Expressão Gênica , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Estabilidade de RNA , RNA Mitocondrial/química , RNA Interferente Pequeno/genética , Coloração e Rotulagem/métodos , Transcrição Gênica , Transfecção , Uridina/análogos & derivados , Uridina/química
11.
FEBS J ; 288(2): 434-436, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32588551

RESUMO

Mitochondria are peculiar organelles because their function depends on genetic information that is present in two genomes: nuclear and mitochondrial. The expression of mitochondrially encoded information requires dedicated machinery. Many efforts have been made to identify this machinery and describe its relevant mechanisms. Recently, Bruni et al. reported a cellular model that they established to investigate the pathway for loading messenger RNAs onto ribosomes in human mitochondria. Their study revealed a role for monosome formation in the stability of mitochondrial mRNAs. Comment on: https://doi.org/10.1111/febs.15342.


Assuntos
Toxinas Bacterianas , Ribossomos Mitocondriais , Toxinas Bacterianas/metabolismo , Humanos , Mitocôndrias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
12.
Nucleic Acids Res ; 48(10): 5572-5590, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365187

RESUMO

RNA decay is a key element of mitochondrial RNA metabolism. To date, the only well-documented machinery that plays a role in mtRNA decay in humans is the complex of polynucleotide phosphorylase (PNPase) and SUV3 helicase, forming the degradosome. REXO2, a homolog of prokaryotic oligoribonucleases present in humans both in mitochondria and the cytoplasm, was earlier shown to be crucial for maintaining mitochondrial homeostasis, but its function in mitochondria has not been fully elucidated. In the present study, we created a cellular model that enables the clear dissection of mitochondrial and non-mitochondrial functions of human REXO2. We identified a novel mitochondrial short RNA, referred to as ncH2, that massively accumulated upon REXO2 silencing. ncH2 degradation occurred independently of the mitochondrial degradosome, strongly supporting the hypothesis that ncH2 is a primary substrate of REXO2. We also investigated the global impact of REXO2 depletion on mtRNA, revealing the importance of the protein for maintaining low steady-state levels of mitochondrial antisense transcripts and double-stranded RNA. Our detailed biochemical and structural studies provide evidence of sequence specificity of the REXO2 oligoribonuclease. We postulate that REXO2 plays dual roles in human mitochondria, 'scavenging' nanoRNAs that are produced by the degradosome and clearing short RNAs that are generated by RNA processing.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mitocondrial/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/fisiologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/fisiologia , Exorribonucleases/química , Exorribonucleases/fisiologia , Células HeLa , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Multimerização Proteica , Especificidade por Substrato
13.
Cells ; 9(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861673

RESUMO

Mitochondria are peculiar organelles whose proper function depends on the crosstalk between two genomes, mitochondrial and nuclear. The human mitochondrial genome (mtDNA) encodes only 13 proteins; nevertheless, its proper expression is essential for cellular homeostasis, as mtDNA-encoded proteins are constituents of mitochondrial respiratory complexes. In addition, mtDNA expression results in the production of RNA molecules, which influence cell physiology once released from the mitochondria into the cytoplasm. As a result, dysfunctions of mtDNA expression may lead to pathologies in humans. Here, we review the mechanisms of mitochondrial gene expression with a focus on recent findings in the field. We summarize the complex turnover of mitochondrial transcripts and present an increasing body of evidence indicating new functions of mitochondrial transcripts. We discuss mitochondrial gene regulation in different cellular contexts, focusing on stress conditions. Finally, we highlight the importance of emerging aspects of mitochondrial gene regulation in human health and disease.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Transcrição Gênica
14.
Nucleic Acids Res ; 47(14): 7502-7517, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31226201

RESUMO

Maintenance of mitochondrial gene expression is crucial for cellular homeostasis. Stress conditions may lead to a temporary reduction of mitochondrial genome copy number, raising the risk of insufficient expression of mitochondrial encoded genes. Little is known how compensatory mechanisms operate to maintain proper mitochondrial transcripts levels upon disturbed transcription and which proteins are involved in them. Here we performed a quantitative proteomic screen to search for proteins that sustain expression of mtDNA under stress conditions. Analysis of stress-induced changes of the human mitochondrial proteome led to the identification of several proteins with poorly defined functions among which we focused on C6orf203, which we named MTRES1 (Mitochondrial Transcription Rescue Factor 1). We found that the level of MTRES1 is elevated in cells under stress and we show that this upregulation of MTRES1 prevents mitochondrial transcript loss under perturbed mitochondrial gene expression. This protective effect depends on the RNA binding activity of MTRES1. Functional analysis revealed that MTRES1 associates with mitochondrial RNA polymerase POLRMT and acts by increasing mitochondrial transcription, without changing the stability of mitochondrial RNAs. We propose that MTRES1 is an example of a protein that protects the cell from mitochondrial RNA loss during stress.


Assuntos
Perfilação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Genes Mitocondriais/genética , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/genética , Proteoma/metabolismo , RNA Mitocondrial/genética , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Estresse Fisiológico
15.
Mol Cell Oncol ; 5(6): e1516452, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30525095

RESUMO

Transcription of the human mitochondrial genome produces a vast amount of non-coding antisense RNAs. These RNA species can form G-quadraplexes (G4), which affect their decay. We found that the mitochondrial degradosome, a complex of RNA helicase SUPV3L1 (best known as SUV3) and the ribonuclease PNPT1 (also known as PNPase), together with G4-melting protein GRSF1, is a key player in restricting antisense mtRNAs.

16.
Nature ; 560(7717): 238-242, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30046113

RESUMO

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.


Assuntos
Herpesvirus Humano 1/imunologia , RNA de Cadeia Dupla/imunologia , RNA Mitocondrial/imunologia , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/deficiência , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica/imunologia , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , Mutação , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Análise de Célula Única , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
17.
Nat Commun ; 9(1): 2558, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29967381

RESUMO

The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in human mitochondria. In vitro reconstitution experiments show that GRSF1 promotes G4 melting that facilitates degradosome-mediated decay. Among degradosome and GRSF1 regulated transcripts we identified one that undergoes post-transcriptional modification. We show that GRSF1 proteins form a distinct qRRM group found only in vertebrates. The appearance of GRSF1 coincided with changes in the mitochondrial genome, which allows the emergence of G4-containing RNAs. We propose that GRSF1 appearance is an evolutionary adaptation enabling control of G4 RNA.


Assuntos
Quadruplex G , Genoma Mitocondrial/genética , Mitocôndrias/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA não Traduzido/metabolismo , Animais , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/genética , Complexos Multienzimáticos/metabolismo , Filogenia , Proteínas de Ligação a Poli(A)/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/genética
18.
PLoS One ; 13(3): e0194887, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29590189

RESUMO

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq.


Assuntos
DNA Nucleotidiltransferases/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Proteínas/metabolismo , Recombinação Genética , Terminação da Transcrição Genética , Clonagem Molecular , DNA Nucleotidiltransferases/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Nucleofosmina , Regiões Promotoras Genéticas , Proteínas/genética
19.
BMC Med Genet ; 18(1): 65, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28595636

RESUMO

BACKGROUND: The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. ß-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the ß-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. METHODS: Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. RESULTS: We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. CONCLUSION: Our results show that a lack of natural stop codon due to the frameshift in exon 3 of ß-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.


Assuntos
Mutação da Fase de Leitura , Estabilidade de RNA/genética , Globinas beta/genética , Talassemia beta/genética , Linhagem Celular , Criança , Análise Mutacional de DNA , Éxons , Humanos , Masculino , Polônia
20.
Hum Mol Genet ; 23(23): 6147-62, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24986917

RESUMO

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Exodesoxirribonucleases/genética , Rearranjo Gênico , Doenças Mitocondriais/genética , Linhagem Celular , DNA Polimerase gama , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Doenças Mitocondriais/enzimologia , Mutação
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