RESUMEN
Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.
Asunto(s)
Leishmania major , Proteoma , Transcriptoma , Leishmania major/metabolismo , Leishmania major/genética , Proteoma/metabolismo , Humanos , ARN Polimerasa III/metabolismo , ARN Polimerasa III/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación de la Expresión GénicaRESUMEN
OBJECTIVE: To describe the frequency of anti-RNA polymerase III antibody in patients with Systemic Sclerosis (SSc) of a group of healthcare centres from Argentina and to explore differences among patients with positive and negative anti-RNA polymerase III antibody. PATIENTS AND METHODS: Data from clinical records, anamnesis and physical examination were collected from 135 patients with SSc (ACR/EULAR 2013). A serum sample from each patient was obtained for the detection of anti-RNA polymerase III IgG antibodies by ELISA. RESULTS: In all, 97.8% were women and the median age at diagnosis was 53 years (range 12-87), 77.7% had limited cutaneous SSc (lcSSC), 19,3% patients had diffuse cutaneous SSc (dcSSC) and 2.9% had scleroderma sine scleroderma. The 67.5% of the patients were from a Mestizos or Amerindian ethnic group. Anti-RNA polymerase III was positive in 5.9% of the patients. In 36 patients, the anticentromere (ACA) and anti-Scl70 antibodies were negative; anti-RNA polymerase III was positive in 16.7% of these 36 patients. Pitting scars and pulmonary artery hypertension were more frequent in anti-RNA polymerase III positive patients who were also older at diagnosis. No association with gastric antral vascular ectasia was found. The only patient with scleroderma renal crisis was anti-RNA polymerase III positive. CONCLUSIONS: Anti-RNA polymerase III frequency found in this study was one of the lowest reported, which could be related to the predominance of the Amerindian and Mestizo ethnic group. It is possible that the detection of anti RNA polymerase III allows better classification of SSc patients, to know their prognosis and to improve their follow-up, therefore more studies are needed.
Asunto(s)
ARN Polimerasa III , Esclerodermia Sistémica , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antinucleares , Argentina/epidemiología , Autoanticuerpos , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/diagnóstico , Adulto JovenRESUMEN
Recently, mutations in the RNA polymerase III subunit A (POLR3A) have been described as the cause of the neonatal progeria or Wiedemann-Rautenstrauch syndrome (WRS). POLR3A has important roles in transcription regulation of small RNAs, including tRNA, 5S rRNA, and 7SK rRNA. We aim to describe the cellular and molecular features of WRS fibroblasts. Cultures of primary fibroblasts from one WRS patient [monoallelic POLR3A variant c.3772_3773delCT (p.Leu1258Glyfs*12)] and one control patient were cultured in vitro. The mutation caused a decrease in the expression of wildtype POLR3A mRNA and POLR3A protein and a sharp increase in mutant protein expression. In addition, there was an increase in the nuclear localization of the mutant protein. These changes were associated with an increase in the number and area of nucleoli and to a high increase in the expression of pP53 and pH2AX. All these changes were associated with premature senescence. The present observations add to our understanding of the differences between Hutchinson-Gilford progeria syndrome and WRS and opens new alternatives to study cell senesce and human aging.
Asunto(s)
Retardo del Crecimiento Fetal , Fibroblastos , Progeria , ARN Polimerasa III , Proteína p53 Supresora de Tumor/metabolismo , Nucléolo Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Daño del ADN , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/patología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Expresión Génica , Humanos , Mutación , Progeria/genética , Progeria/patología , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN Ribosómico 5S/metabolismoRESUMEN
MAF1 is a phosphoprotein that plays a critical role in cell growth control as the central regulator of RNA polymerase (Pol) III activity. Citrus MAF1 (CsMAF1) was identified as a direct target of PthA4, a bacterial effector protein required to induce tumors in citrus. CsMAF1 binds to Pol III to restrict transcription; however, exactly how CsMAF1 interacts with the polymerase and how phosphorylation modulates this interaction is unknown. Moreover, how CsMAF1 binds PthA4 is also obscure. Here we show that CsMAF1 binds predominantly to the WH1 domain of the citrus Pol III subunit C34 (CsC34) and that its phosphoregulatory region, comprising loop-3 and α-helix-2, contributes to this interaction. We also show that phosphorylation of this region decreases CsMAF1 affinity to CsC34, leading to Pol III derepression, and that Ser 45, found only in plant MAF1 proteins, is critical for CsC34 interaction and is phosphorylated by a new citrus AGC1 kinase. Additionally, we show that the C-terminal region of the citrus TFIIIB component BRF1 competes with CsMAF1 for CsC34 interaction, whereas the C-terminal region of CsMAF1 is essential for PthA4 binding. Based on CsMAF1 structural data, we propose a mechanism for how CsMAF1 represses Pol III transcription and how phosphorylation controls this process.
Asunto(s)
Citrus/genética , Proteínas de Plantas/metabolismo , ARN Polimerasa III/metabolismo , Citrus/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , ARN Polimerasa III/genética , Serina/metabolismo , Transcripción Genética , Levaduras/genéticaRESUMEN
Systemic sclerosis (SSc) is a rare autoimmune disease with high mortality, characterized by chronic inflammation and fibrosis, which are processes associated with higher serum tumor necrosis factor-α (sTNF-α) levels. TNFA -308G>A and -238G>A polymorphisms have been associated with higher sTNF-α levels. In this study, we genotyped the TNFA -308G>A and -238G>A polymorphisms in 53 SSc patients and 115 unrelated control subjects (CS) from southern Mexico. The TNFA mRNA expression and sTNF-α levels were also quantified by qPCR and enzyme-linked immunosorbent assays, respectively. TNFA -308GA genotype was associated with disease susceptibility according to a codominant genetic model (OR = 3.2, 95% CI 1.05-9.75, p = 0.03), and with higher anti-fibrillarin antibodies (p = 0.01), and higher skin thickening (p = 0.006). TNFA -238GA was not associated with SSc risk. TNFA mRNA expression and sTNF-α levels were similar between SSc patients and CS and were not statistically associated with the TNFA polymorphisms; however, a correlation (rho = 0.362, p = 0.009) between sTNF-α levels with anti-RNA polymerase III antibodies was observed in the SSc patients. In conclusion, the -308G>A polymorphism is a genetic marker of SSc susceptibility in population from southern Mexico, and it is associated with skin thickening and anti-fibrillarin antibodies. In addition, sTNF-α levels correlate positively with the anti-RNA pol III antibodies levels.
Asunto(s)
Autoanticuerpos/metabolismo , Esclerodermia Sistémica/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Adulto , Estudios de Casos y Controles , Proteínas Cromosómicas no Histona/inmunología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , México , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , ARN Polimerasa III/inmunología , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/inmunologíaRESUMEN
Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.
Asunto(s)
Leishmania major/genética , ARN Polimerasa III/genética , Factor de Transcripción TFIIIB/genética , Transcripción Genética , Simulación por Computador , Secuencia Conservada/genética , Regulación de la Expresión Génica/genética , Recombinación Homóloga/genética , Proteínas Mutantes/genética , Regiones Promotoras Genéticas , Dominios Proteicos/genética , Subunidades de Proteína/genética , ARN Ribosómico 5S/biosíntesis , ARN Nuclear Pequeño/biosíntesis , ARN de Transferencia/biosíntesisRESUMEN
CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. OBJECTIVE: To investigate the genetic etiology of idiopathic POI. PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.
Asunto(s)
Insuficiencia Ovárica Primaria/genética , ARN Polimerasa III/genética , Adolescente , Animales , Sistemas CRISPR-Cas , Niño , Femenino , Proteína Forkhead Box O3/metabolismo , Técnicas de Inactivación de Genes , Heterocigoto , Homocigoto , Humanos , Infertilidad/genética , Tamaño de la Camada , Mutación con Pérdida de Función , Masculino , Ratones , Mutación Missense , Ovario/metabolismo , Maduración Sexual/genética , Tiempo para Quedar Embarazada , Secuenciación del ExomaRESUMEN
The field of tRNA biology, encompassing the functional and structural complexity of tRNAs, has fascinated scientists over the years and is continuously growing. Besides their fundamental role in protein translation, new evidence indicates that tRNA-derived molecules also regulate gene expression and protein synthesis in all domains of life. This review highlights some of the recent findings linking tRNA transcription and modification with plant cell growth and response to pathogens. In fact, mutations in proteins directly involved in tRNA synthesis and modification most often lead to pleiotropic effects on plant growth and immunity. As plants need to optimize and balance their energy and nutrient resources towards growth and defense, regulatory pathways that play a central role in integrating tRNA transcription and protein translation with cell growth control and organ development, such as the auxin-TOR signaling pathway, also influence the plant immune response against pathogens. As a consequence, distinct pathogens employ an array of effector molecules including tRNA fragments to target such regulatory pathways to exploit the plant's translational capacity, gain access to nutrients and evade defenses. An example includes the RNA polymerase III repressor MAF1, a conserved component of the TOR signaling pathway that controls ribosome biogenesis and tRNA synthesis required for plant growth and which is targeted by a pathogen effector molecule to promote disease. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Plantas/genética , ARN de Planta/biosíntesis , ARN de Transferencia/biosíntesis , Transcripción Genética , Secuencia de Aminoácidos , Interacciones Huésped-Patógeno , Ácidos Indolacéticos , Modelos Moleculares , Mutación , Desarrollo de la Planta/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/inmunología , Conformación Proteica , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Planta/genética , ARN de Transferencia/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5' end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5' rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5' end and a poly(A) tail at the 3' end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes.
Asunto(s)
Leishmania major/genética , Proteínas Protozoarias/metabolismo , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , Aminoacil-ARN de Transferencia/genética , Leishmania major/enzimología , Leishmania major/metabolismo , Poliadenilación , Empalme del ARNRESUMEN
OBJECTIVES: to identify the number of electro-medical pieces of equipment in a coronary care unit, characterize their types, and analyze implications for the safety of patients from the perspective of alarm fatigue. METHOD: this quantitative, observational, descriptive, non-participatory study was conducted in a coronary care unit of a cardiology hospital with 170 beds. RESULTS: a total of 426 alarms were recorded in 40 hours of observation: 227 were triggered by multi-parametric monitors and 199 were triggered by other equipment (infusion pumps, dialysis pumps, mechanical ventilators, and intra-aortic balloons); that is an average of 10.6 alarms per hour. CONCLUSION: the results reinforce the importance of properly configuring physiological variables, the volume and parameters of alarms of multi-parametric monitors within the routine of intensive care units. The alarms of equipment intended to protect patients have increased noise within the unit, the level of distraction and interruptions in the workflow, leading to a false sense of security. .
OBJETIVOS: identificar o número de alarmes dos equipamentos eletromédicos numa unidade coronariana, caracterizar o tipo e analisar as implicações para a segurança do paciente na perspectiva da fadiga de alarmes. MÉTODO: trata-se de estudo quantitativo observacional descritivo, não participante, desenvolvido numa unidade coronariana de um hospital de cardiologia, com capacidade para 170 leitos. RESULTADOS: registrou-se o total de 426 sinais de alarmes, sendo 227 disparados por monitores multiparamétricos e 199 alarmes disparados por outros equipamentos (bombas infusoras, hemodiálise, ventiladores mecânicos e balão intra-aórtico), nas 40h, numa média total de 10,6 alarmes/hora. CONCLUSÃO: os resultados encontrados reforçam a importância da configuração de variáveis fisiológicas, do volume e dos parâmetros de alarmes dos monitores multiparamétricos à rotina das unidades de terapia intensiva. Os alarmes dos equipamentos destinados a proteger os pacientes têm conduzido ao aumento do ruído na unidade, à fadiga de alarmes, a distrações e interrupções no fluxo de trabalho e à falsa sensação de segurança. .
OBJETIVOS: identificar el número de alarmas de los equipamientos electromédicos en una unidad coronariana, caracterizar el tipo y analizar las implicaciones para la seguridad del paciente en la perspectiva de fatiga de alarmas. MÉTODO: se trata de un estudio cuantitativo, observacional, descriptivo, no participante, desarrollado en una unidad coronariana de un hospital de cardiología, con capacidad de 170 camas. RESULTADOS: se registró un total de 426 señales de alarmas, siendo 227 disparadas por monitores multiparamétricos y 199 disparadas por otros equipamientos (bombas de infusión, hemodiálisis, ventiladores mecánicos y balón intraaórtico), durante 40h, con un promedio total de 10,6 alarmas/hora. CONCLUSIÓN: los resultados encontrados refuerzan la importancia de la configuración de las variables fisiológicas, del volumen y de los parámetros de alarma de los monitores multiparamétricos, a la rutina de las unidades de terapia intensiva. Las alarmas de los equipamientos destinados a proteger a los pacientes, han llevado al aumento del ruido en la unidad, a la fatiga de alarmas, a las distracciones e interrupciones en el flujo de trabajo y a una falsa sensación de seguridad. .
Asunto(s)
Humanos , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Oncogénicas Virales/genética , ARN Polimerasa III/metabolismo , Sarcosina/análogos & derivados , Factores de Transcripción TFIII , Transcripción Genética , Factores de Transcripción/metabolismo , Proteínas Precoces de Adenovirus , Detergentes , Proteínas de Unión al ADN/genética , Células HeLa , Cinética , Sarcosina/farmacología , Factor de Transcripción TFIIIB , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNA(His) transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA(His) synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.
Asunto(s)
Citrus/fisiología , Enfermedades de las Plantas/genética , Proteínas de Plantas/fisiología , ARN Polimerasa III/antagonistas & inhibidores , Xanthomonas , Secuencia de Aminoácidos , Citrus/genética , Citrus/microbiología , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Represoras/química , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-ß in macrophages and splenocytes. Further, IFN-ß induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-ß expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-ß and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αßR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αßR KO spleen cells and reduced apoptosis.
Asunto(s)
Brucella abortus/aislamiento & purificación , Brucelosis/metabolismo , Factor 3 Regulador del Interferón/fisiología , Interferón beta/biosíntesis , Proteínas de la Membrana/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Animales , Brucella abortus/genética , Brucelosis/microbiología , Línea Celular , ADN Bacteriano/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Polimerasa III/metabolismo , Factor de Transcripción STAT1/metabolismo , Bazo/citología , Bazo/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
OBJECTIVE: To determine serological and clinical variables associated with anti-RNA polymerase III (RNAP-III) antibodies in patients with systemic sclerosis (SSc) using a new ELISA method. METHODS: Sera from 242 patients with SSc were collected from 14 Canadian clinics. Control sera were from 287 blood donors, and 42 patients with infectious disease, 30 with rheumatoid arthritis (RA), and 30 with systemic lupus erythematosus (SLE). Antibodies to RNAP-III were detected by an ELISA kit and antibodies to other cellular antigens were identified by indirect immunofluorescence (IIF) on HEp-2 cell substrate, line immunoassay, immunoprecipitation of recombinant protein, and addressable laser bead immunoassay (ALBIA). RESULTS: Anti-RNAP-III antibodies were detected in 47/242 (19.4%) SSc sera, 0% RA and SLE sera, 1/287 blood donor sera, and 2/42 infectious disease sera. Diffuse disease (59.5%) was more common than limited disease (36.1%) in the anti-RNAP-III-positive patients (p = 0.006) and there was an association between the presence of anti-RNAP-III and kidney and joint/tendon involvement, but there was no association with a nucleolar IIF pattern, lung involvement, or other clinical indicators. There was a negative association between the presence of anti-RNAP-III antibodies and anticentromere by IIF (p = 0.00004) and anti-Scl-70 by ALBIA (p = 0.0005) and line immunoassay (p = 0.003), suggesting a virtually exclusive presence of these antibodies in SSc. CONCLUSION: Anti-RNAP-III autoantibodies were found in nearly 20% of SSc patients but in less than 1% of controls, thus detection of this antibody is a useful marker to help diagnose SSc. As well, this antibody has prognostic utility, since it is associated with scleroderma renal crisis and the diffuse cutaneous form of SSc.
Asunto(s)
Anticuerpos Antinucleares/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , ARN Polimerasa III/inmunología , Esclerodermia Sistémica/sangre , Femenino , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/fisiopatología , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.
Asunto(s)
Leishmania major/genética , Mutagénesis/genética , Mutación/genética , ARN Polimerasa III/genética , Telómero/genética , Animales , Línea Celular , Regulación de la Expresión Génica , Genes Esenciales/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Polimerasa III/metabolismoRESUMEN
In this article we review and summarize salient features of the mechanism of transcription by eukaryotic RNA polymerase III, focusing a considerable part of the account on the work of our laboratory, dealing with Saccharomyces cerevisiae, but emphasizing properties that are common to all eukaryotic RNA polymerases. The following topics are briefly discussed: promoter structure; transcription factors; internal structure of transcription complexes and DNA-protein interactions; U6 genes; promoter opening and RNA chain elongation; hydrolytic RNA retraction.
Asunto(s)
Regiones Promotoras Genéticas/genética , ARN Polimerasa III/genética , ARN de Hongos/biosíntesis , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , AnimalesRESUMEN
A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.