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1.
Mol Cell ; 76(5): 784-796.e6, 2019 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-31588022

RESUMEN

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.


Asunto(s)
Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/metabolismo , Exorribonucleasas/metabolismo , Mitocondrias/enzimología , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mitocondrial/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Biomarcadores de Tumor/genética , Exorribonucleasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mitocondrial/genética , Células Sf9 , Spodoptera
2.
Mol Cell ; 59(2): 258-69, 2015 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-26186291

RESUMEN

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.


Asunto(s)
ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Bases , Cristalografía por Rayos X , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína , ARN Polimerasa II/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Electricidad Estática , Transcripción Genética
3.
Nucleic Acids Res ; 47(17): 9386-9399, 2019 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-31396629

RESUMEN

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.


Asunto(s)
Mitocondrias/genética , Proteínas Mitocondriales/biosíntesis , ARN Mitocondrial/genética , Proteínas de Unión al ARN/genética , Animales , Células HEK293 , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Ribosomas Mitocondriales/metabolismo , ARN Mensajero/genética , ARN Ribosómico/genética , Proteínas de Unión al ARN/fisiología
4.
Nucleic Acids Res ; 44(14): 6868-82, 2016 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-27353330

RESUMEN

LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA. The LRPPRC-SLIRP complex comprises a hetero-dimer via interactions by polar amino acids in the single RRM domain of SLIRP and three neighbouring PPR motifs in the second quarter of LRPPRC, which critically contribute to the LRPPRC-SLIRP binding interface to enhance its stability. Unexpectedly, specific amino acids at this interface are located within the PPRs of LRPPRC at positions predicted to interact with RNA and within the RNP1 motif of SLIRP's RRM domain. Our findings thus unexpectedly establish that despite the prediction that these residues in LRPPRC and SLIRP should bind RNA, they are instead used to facilitate protein-protein interactions, enabling the formation of a stable complex between these two proteins.


Asunto(s)
Proteínas de Neoplasias/metabolismo , Motivo de Reconocimiento de ARN , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Aminoácidos/genética , Secuencia Conservada , Reactivos de Enlaces Cruzados/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Mutación/genética , Proteínas de Neoplasias/química , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , ARN/metabolismo , Proteínas de Unión al ARN/química
5.
Proc Natl Acad Sci U S A ; 112(36): 11288-93, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26305956

RESUMEN

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.


Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Mitocondrias/metabolismo , Nucleoproteínas/metabolismo , Animales , Células Cultivadas , Microscopía por Crioelectrón , ADN Mitocondrial/genética , ADN Mitocondrial/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Tomografía con Microscopio Electrónico , Genoma Mitocondrial/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/ultraestructura , Ratones , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/ultraestructura , Mutación , Nucleoproteínas/genética , Nucleoproteínas/ultraestructura , Unión Proteica
6.
PLoS Genet ; 11(8): e1005423, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26247782

RESUMEN

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.


Asunto(s)
Proteínas Mitocondriales/biosíntesis , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al ARN/fisiología , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Poliadenilación , Biosíntesis de Proteínas , Proteolisis , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo
7.
PLoS Genet ; 10(2): e1004110, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24516400

RESUMEN

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.


Asunto(s)
Proteínas Portadoras/genética , Metiltransferasas/genética , Mitocondrias/genética , ARN Ribosómico/genética , Ribosomas/genética , Animales , Proteínas Portadoras/metabolismo , Metilación de ADN/genética , Metiltransferasas/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Unión Proteica , ARN Ribosómico/biosíntesis , Ribosomas/ultraestructura , Factores de Transcripción/metabolismo
8.
Hum Mol Genet ; 23(23): 6345-55, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25008111

RESUMEN

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.


Asunto(s)
Proteínas Mitocondriales/metabolismo , Polinucleotido Adenililtransferasa/metabolismo , ARN Mensajero/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Humanos , Proteínas Mitocondriales/genética , Mutación , Proteínas de Neoplasias/metabolismo , Fosforilación Oxidativa , Polinucleotido Adenililtransferasa/genética , Cultivo Primario de Células , Procesamiento Postranscripcional del ARN , ARN Mitocondrial , Proteínas de Unión al ARN/metabolismo
9.
PLoS Genet ; 9(1): e1003178, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23300484

RESUMEN

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.


Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Mitocondrias , Proteínas Mitocondriales , Ribosomas , Animales , ADN Mitocondrial/genética , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Invertebrados/genética , Invertebrados/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Ribosomas/genética , Ribosomas/metabolismo , Transcripción Genética
10.
Proc Natl Acad Sci U S A ; 109(38): 15253-8, 2012 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-22949673

RESUMEN

Proteins crucial for the respiratory chain are translated by the mitochondrial ribosome. Mitochondrial ribosome biogenesis is therefore critical for oxidative phosphorylation capacity and disturbances are known to cause human disease. This complex process is evolutionary conserved and involves several RNA processing and modification steps required for correct ribosomal RNA maturation. We recently showed that a member of the mitochondrial transcription termination factor (MTERF) family of proteins, MTERF4, recruits NSUN4, a 5-methylcytosine RNA methyltransferase, to the large ribosomal subunit in a process crucial for mitochondrial ribosome biogenesis. Here, we describe the 3D crystal structure of the human MTERF4-NSUN4 complex determined to 2.9 Å resolution. MTERF4 is composed of structurally repeated MTERF-motifs that form a nucleic acid binding domain. NSUN4 lacks an N- or C-terminal extension that is commonly used for RNA recognition by related RNA methyltransferases. Instead, NSUN4 binds to the C-terminus of MTERF4. A positively charged surface forms an RNA binding path from the concave to the convex side of MTERF4 and further along NSUN4 all of the way into the active site. This finding suggests that both subunits of the protein complex likely contribute to RNA recognition. The interface between MTERF4 and NSUN4 contains evolutionarily conserved polar and hydrophobic amino acids, and mutations that change these residues completely disrupt complex formation. This study provides a molecular explanation for MTERF4-dependent recruitment of NSUN4 to ribosomal RNA and suggests a unique mechanism by which other members of the large MTERF-family of proteins can regulate ribosomal biogenesis.


Asunto(s)
Proteínas Portadoras/química , Metiltransferasas/química , Mitocondrias/metabolismo , Ribosomas/química , Factores de Transcripción/química , Dominio Catalítico , Biología Computacional/métodos , Cristalografía por Rayos X/métodos , Análisis Mutacional de ADN , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Unión Proteica , Estructura Terciaria de Proteína , ARN Ribosómico/química , Proteínas Recombinantes/química
11.
J Biol Chem ; 288(22): 15510-9, 2013 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-23599432

RESUMEN

Regulation of mtDNA expression is critical for controlling oxidative phosphorylation capacity and has been reported to occur at several different levels in mammalian mitochondria. LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) has a key role in this regulation and acts at the post-transcriptional level to stabilize mitochondrial mRNAs, to promote mitochondrial mRNA polyadenylation, and to coordinate mitochondrial translation. However, recent studies have suggested that LRPPRC may have an additional intramitochondrial role by directly interacting with the mitochondrial RNA polymerase POLRMT to stimulate mtDNA transcription. In this study, we have further examined the intramitochondrial roles for LRPPRC by creating bacterial artificial chromosome transgenic mice with moderately increased LRPPRC expression and heterozygous Lrpprc knock-out mice with moderately decreased LRPPRC expression. Variation of LRPPRC levels in mice in vivo, occurring within a predicted normal physiological range, strongly affected the levels of an unprocessed mitochondrial precursor transcript (ND5-cytochrome b) but had no effect on steady-state levels of mitochondrial transcripts or de novo transcription of mtDNA. We further assessed the role of LRPPRC in mitochondrial transcription by performing size exclusion chromatography and immunoprecipitation experiments in human cell lines and mice, but we found no interaction between LRPPRC and POLRMT. Furthermore, addition of purified LRPPRC to a recombinant human in vitro transcription system did not activate mtDNA transcription. On the basis of these data, we conclude that LRPPRC does not directly regulate mtDNA transcription but rather acts as a post-transcriptional regulator of mammalian mtDNA expression.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Citocromos b/genética , Citocromos b/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Células HeLa , Humanos , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Hepáticas/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Proteínas de Neoplasias/genética , Transcripción Genética/fisiología
12.
Biochem Biophys Res Commun ; 443(1): 7-12, 2014 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-24211586

RESUMEN

UBTD1 is a previously uncharacterized ubiquitin-like (UbL) domain containing protein with high homology to the mitochondrial Dc-UbP/UBTD2 protein. Here we show that UBTD1 and UBTD2 belong to a family of proteins that is conserved through evolution and found in metazoa, funghi, and plants. To gain further insight into the function of UBTD1, we screened for interacting proteins. In a yeast-2-hybrid (Y2H) screen, we identified several proteins involved in the ubiquitylation pathway, including the UBE2D family of E2 ubiquitin conjugating enzymes. An affinity capture screen for UBTD1 interacting proteins in whole cell extracts also identified members of the UBE2D family. Biochemical characterization of recombinant UBTD1 and UBE2D demonstrated that the two proteins form a stable, stoichiometric complex that can be purified to near homogeneity. We discuss the implications of these findings in light of the ubiquitin proteasome system (UPS).


Asunto(s)
Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinación , Ubiquitinas/clasificación , Ubiquitinas/genética
13.
Proc Natl Acad Sci U S A ; 108(33): 13534-9, 2011 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-21808029

RESUMEN

Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of ∼100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ∼1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging.


Asunto(s)
ADN Mitocondrial/ultraestructura , Microscopía/instrumentación , Animales , Anticuerpos , ADN Mitocondrial/inmunología , Proteínas de Unión al ADN/inmunología , Humanos , Mitocondrias , Proteínas Mitocondriales/inmunología , Factores de Transcripción/inmunología
14.
Protein Expr Purif ; 87(2): 111-9, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23137940

RESUMEN

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.


Asunto(s)
Biotecnología/métodos , Detergentes/química , Proteínas de la Membrana/aislamiento & purificación , Complejos Multiproteicos/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Proteínas de la Membrana/química , Complejos Multiproteicos/química , Proteínas Recombinantes/química , Saccharomyces cerevisiae , Células Sf9 , Solubilidad
15.
Nat Commun ; 14(1): 30, 2023 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-36596788

RESUMEN

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.


Asunto(s)
Codón de Terminación , Mitocondrias , Factores de Terminación de Péptidos , Humanos , Codón de Terminación/metabolismo , Mitocondrias/metabolismo , Factores de Terminación de Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo
16.
Methods Mol Biol ; 2661: 217-232, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37166640

RESUMEN

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.


Asunto(s)
Mitocondrias , Biosíntesis de Proteínas , Animales , Mitocondrias/genética , Mitocondrias/metabolismo , Fosforilación Oxidativa , Procesamiento Proteico-Postraduccional , Citosol/metabolismo , Proteínas Mitocondriales/metabolismo , Ribosomas Mitocondriales/metabolismo , Mamíferos/metabolismo
17.
Proc Natl Acad Sci U S A ; 106(23): 9185-90, 2009 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-19458260

RESUMEN

The second structure of a eukaryotic RNA polymerase II so far determined, that of the enzyme from the fission yeast Schizosaccharomyces pombe, is reported here. Comparison with the previous structure of the enzyme from the budding yeast Saccharomyces cerevisiae reveals differences in regions implicated in start site selection and transcription factor interaction. These aspects of the transcription mechanism differ between S. pombe and S. cerevisiae, but are conserved between S. pombe and humans. Amino acid changes apparently responsible for the structural differences are also conserved between S. pombe and humans, suggesting that the S. pombe structure may be a good surrogate for that of the human enzyme.


Asunto(s)
ARN Polimerasa II/química , Schizosaccharomyces/enzimología , Transcripción Genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , TATA Box
18.
Biochim Biophys Acta Mol Basis Dis ; 1868(10): 166467, 2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-35716868

RESUMEN

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression, and packaging of mitochondrial DNA (mtDNA). Recently, a pathogenic homozygous variant in TFAM (P178L) has been associated with a severe mtDNA depletion syndrome leading to neonatal liver failure and early death. We have performed a biochemical characterization of the TFAM variant P178L in order to understand the molecular basis for the pathogenicity of this mutation. We observe no effects on DNA binding, and compaction of DNA is only mildly affected by the P178L amino acid change. Instead, the mutation severely impairs mtDNA transcription initiation at the mitochondrial heavy and light strand promoters. Molecular modeling suggests that the P178L mutation affects promoter sequence recognition and the interaction between TFAM and the tether helix of POLRMT, thus explaining transcription initiation deficiency.


Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales , Mutación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
Nat Commun ; 13(1): 5750, 2022 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-36180430

RESUMEN

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.


Asunto(s)
Procesamiento Postranscripcional del ARN , ARN , Animales , Carbono/metabolismo , Drosophila , Exorribonucleasas , Mamíferos/genética , Ratones , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , ARN/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismo
20.
Biochem Biophys Res Commun ; 397(3): 386-90, 2010 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-20430012

RESUMEN

In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. MTERF family members share a approximately 270 residues long MTERF-domain required for DNA binding and transcription regulation. However, the structure of this widely conserved domain is unknown. Here, we show that the MTERF-domain of human MTERF3 forms a half-doughnut-shaped right-handed superhelix. The superhelix is built from alpha-helical tandem repeats that display a novel triangular three-helix motif. This repeat motif, which we denote the MTERF-motif, is a conserved structural element present in proteins from metazoans, plants, and protozoans. Furthermore, a narrow, strongly positively charged nucleic acid-binding path is found in the middle of the concave side of the half-doughnut. This arrangement suggests a half clamp nucleic acid-binding mode for MTERF-domains.


Asunto(s)
ADN/química , Proteínas Mitocondriales/química , ARN Bicatenario/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN , Humanos , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Factores de Transcripción/genética
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