RESUMEN
RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
Asunto(s)
Factores de Transcripción , Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , ADN/metabolismoRESUMEN
Deregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the factors and mechanisms that modulate Pol III-directed transcription remain to be found, although massive advances have been achieved. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3' UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the miRNA promoter, indicating that the miR-106a-5p links STAT3 with TP73 to regulate Pol III-directed transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.
Asunto(s)
MicroARNs , Neoplasias , Humanos , Proliferación Celular , MicroARNs/genética , Neoplasias/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismoRESUMEN
BACKGROUND: TFIIIB, an RNA polymerase III specific transcription factor has been found to be deregulated in human cancers with much of the research focused on the TBP, BRF1, and BRF2 subunits. To date, the TFIIIB specific subunit BDP1 has not been investigated in ovarian cancer but has previously been shown to be deregulated in neuroblastoma, breast cancer, and Non-Hodgkins lymphoma. RESULTS: Using in silico analysis of clinically derived platforms, we report a decreased BDP1 expression as a result of deletion in serous ovarian cancer and a correlation with higher and advanced ovarian stages. Further analysis in the context of TP53 mutations, a major contributor to ovarian tumorigenesis, suggests that high BDP1 expression is unfavorable for overall survival and high BDP1 expression occurs in stages 2, 3 and 4 serous ovarian cancer. Additionally, high BDP1 expression is disadvantageous and unfavorable for progression-free survival. Lastly, BDP1 expression significantly decreased in patients treated with first-line chemotherapy, platin and taxane, at twelve-month relapse-free survival. CONCLUSIONS: Taken together with a ROC analysis, the data suggest BDP1 could be of clinical relevance as a predictive biomarker in serous ovarian cancer. Lastly, this study further demonstrates that both the over- and under expression of BDP1 warrants further investigation and suggests BDP1 may exhibit dual function in the context of tumorigenesis.
Asunto(s)
Neoplasias Ováricas , Proteínas de Saccharomyces cerevisiae , Factores Asociados con la Proteína de Unión a TATA , Humanos , Femenino , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Recurrencia Local de Neoplasia , Biomarcadores , Neoplasias Ováricas/genética , Carcinogénesis , Factores Asociados con la Proteína de Unión a TATA/genéticaRESUMEN
In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.
Asunto(s)
Leishmania major/genética , Leishmaniasis Cutánea/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIIIB/genética , Animales , Regulación de la Expresión Génica/genética , Humanos , Leishmania major/patogenicidad , Leishmaniasis Cutánea/parasitología , Regiones Promotoras Genéticas/genética , ARN Polimerasa III/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 5S/genética , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
BACKGROUND: Deregulation of the RNA polymerase III specific TFIIIB subunit BRF2 occurs in subtypes of human cancers. However, correlations between BRF2 alterations and clinical outcomes in breast cancer are limited. We conducted this review to analyze BRF2 alterations in genomic data sets housed in Oncomine and cBioPortal to identify potential correlations between BRF2 alterations and clinical outcomes. METHODS: The authors queried both Oncomine and cBioPortal for alterations in BRF2 in human cancers and performed meta-analyses identifying significant correlations between BRF2 and clinical outcomes in invasive breast cancer (IBC). RESULTS: A meta cancer outlier profile analysis (COPA) of 715 data sets (86,733 samples) in Oncomine identified BRF2 as overexpressed in 60% of breast cancer data sets. COPA scores in IBC data sets (3594 patients) are comparable for HER2 (24.211, median gene rank 60) and BRF2 (29.656, median gene rank 36.5). Overall survival in IBC patients with BRF2 alterations (21%) is significantly decreased (p = 9.332e-3). IBC patients with BRF2 alterations aged 46 to 50 have a significantly poor survival outcome (p = 7.093e-3). Strikingly, in metastatic breast cancer, BRF2 is altered in 33% of women aged 45-50. BRF2 deletions are predominant in this age group. CONCLUSION: This study suggests BRF2 may be an prognostic biomarker in invasive breast carcinoma.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Eliminación de Gen , Factor de Transcripción TFIIIB/genética , Neoplasias de la Mama/genética , Femenino , Humanos , Invasividad Neoplásica , Pronóstico , Tasa de SupervivenciaRESUMEN
Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN Polimerasa III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/genética , Factores de Transcripción TFIII/ultraestructura , Animales , Línea Celular , Microscopía por Crioelectrón , ADN de Hongos/genética , ADN de Hongos/metabolismo , Genes Fúngicos/genética , Insectos , Dominios Proteicos , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/aislamiento & purificación , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción TFIII/genética , Factores de Transcripción TFIII/aislamiento & purificación , Factores de Transcripción TFIII/metabolismo , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción GenéticaRESUMEN
The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN/metabolismo , Exonucleasas/química , ARN de Transferencia/genética , Proteínas Ribosómicas/genética , Alineación de Secuencia/métodos , Factores de Transcripción/metabolismo , Algoritmos , Animales , Sitios de Unión , Simulación por Computador , Proteínas de Unión al ADN/química , Bases de Datos Genéticas , Drosophila/química , Drosophila/genética , Drosophila/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/química , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN/métodos , Factor de Transcripción TFIIIB/química , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción TFIII/química , Factores de Transcripción TFIII/genética , Factores de Transcripción TFIII/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Transcription factor II B (TFIIB)related factor 2 (BRF2) is involved in the development of cancer, but its role in lung cancer is underreported. The present study aimed to explore the role of BRF2 in the regulation of lung cancer cells. Immunofluorescence staining and immunohistochemistry were performed to detect BRF2 protein expression in human lung cancer cells and tissues. Following cell transfection with small interfering RNA for silencing BRF2, the cell proliferation was examined by Cell Counting Kit8 and MTT assays. Cell apoptosis, migration and invasion were determined by flow cytometry, woundhealing and Transwell assay. The expression levels of Akt, phosphorylated (p)Akt, Bax, Ecadherin, Bcl2, Ncadherin, Snail and epidermal growth factor receptor (EGFR) in human lung cancer A549 cells were detected by western blotting. The results demonstrated that BRF2 expression was increased in human lung cancer cells and tissues, and that silencing of BRF2 promoted cell apoptosis but inhibited cell proliferation and migration. The protein expression levels of Akt, Ecadherin, pAkt, Bcl2, Ncadherin, Snail and EGFR in A549 cells were inhibited by silencing of BRF2, while expression levels of Bax and Ecadherin were increased by silencing BRF2. In conclusion, BRF2 demonstrates high expression in lung cancer and silencing of BRF2 inhibits the growth and metastasis of lung cancer cells. The current findings provide a novel approach for the treatment of lung cancer.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Regulación hacia Arriba , Células A549 , Adulto , Anciano , Movimiento Celular , Proliferación Celular , Femenino , Silenciador del Gen , Humanos , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-Å-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex.
Asunto(s)
ARN Polimerasa III/química , Proteínas Represoras/química , Proteínas de Saccharomyces cerevisiae/química , Factor de Transcripción TFIIIB/química , Factores de Transcripción/química , Transcripción Genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.
Asunto(s)
Genoma Fúngico , Integrasas/genética , ARN Polimerasa III/genética , Retroelementos , Saccharomyces cerevisiae/genética , Transcripción Genética , Integrasas/metabolismo , Mutagénesis Insercional , Motivos de Nucleótidos , Plásmidos/química , Plásmidos/metabolismo , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
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
PURPOSE: Radical lymph-node dissection surgery in patients with cN0 middle thoracic esophageal squamous cell carcinoma (ESCC) remains controversial. We sought a novel biomarker that could be used for decision-making in relation to radical lymph-node dissection. METHODS: One hundred and nineteen patients with cN0 middle thoracic ESCC undergoing three-field lymph-node dissection (3FLND) or two-field lymph-node dissection (Ivor Lewis) esophagectomy were reviewed. A survival analysis, and Chi-square and parametric tests were performed. RESULTS: A Cox regression analysis revealed that the expression of BRF2 was an independent prognostic factor for overall survival (P = 0.014) and progression-free survival (P = 0.014). The survival of patients who underwent 3FLND was better than that of patients who underwent Ivor Lewis esophagectomy in the BRF2 overexpression group (P = 0.002), but not in the BRF2 nonoverexpression group (P = 0.386). The risk of lymph-node recurrence and the number of recurrent lymph nodes in patients with the overexpression of BRF2 were increased in the Ivor Lewis group in comparison to the 3FLND group (P = 0.01 and P < 0.001). The risk of cervical and superior mediastinal lymph-node recurrence was positively correlated with the overexpression of BRF2 (P = 0.027). Furthermore, in the Ivor Lewis group, a significant correlation was found between the risk of lymph-node recurrence or the number of recurrent lymph nodes and the expression of BRF2 (P = 0.002 and P = 0.004), but not in the 3FLND group (P = 0.193 and P = 0.694). CONCLUSIONS: 3FLND generated better survival outcomes and reduced the rate of lymph-node recurrence in comparison to Ivor Lewis in patients with the overexpression of BRF2. BRF2 can be used as an indicator for radical lymph-node dissection surgery in cN0 ESCC patients.
Asunto(s)
Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/cirugía , Toma de Decisiones Clínicas/métodos , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/cirugía , Esofagectomía/métodos , Expresión Génica , Escisión del Ganglio Linfático/métodos , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Anciano , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/prevención & control , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Resultado del TratamientoRESUMEN
tRNA genes are transcribed by RNA polymerase III (RNAPIII). During recent years it has become clear that RNAPIII activity is strictly regulated by the cell in response to environmental cues and the homeostatic status of the cell. However, the molecular mechanisms that control RNAPIII activity to regulate the amplitude of tDNA transcription in normally cycling cells are not well understood. Here, we show that tRNA levels fluctuate during the cell cycle and reveal an underlying molecular mechanism. The cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to boost tDNA transcription during late S phase. At tDNA genes, Cdk1 promotes the recruitment of TFIIIC, stimulates the interaction between TFIIIB and TFIIIC, and increases the dynamics of RNA polymerase III in vivo. Furthermore, we identified Bdp1 as a putative Cdk1 substrate in this process. Preventing Bdp1 phosphorylation prevented cell cycle-dependent recruitment of TFIIIC and abolished the cell cycle-dependent increase in tDNA transcription. Our findings demonstrate that under optimal growth conditions Cdk1 gates tRNA synthesis in S phase by regulating the RNAPIII machinery, revealing a direct link between the cell cycle and RNAPIII activity.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , ARN Polimerasa III/genética , ARN de Transferencia/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosforilación , Unión Proteica , ARN Polimerasa III/metabolismo , ARN de Transferencia/metabolismo , Fase S/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción TFIII/genética , Factores de Transcripción TFIII/metabolismoRESUMEN
Transcription of transfer RNA genes by RNA polymerase III (Pol III) is controlled by general factors, TFIIIB and TFIIIC, and a negative regulator, Maf1. Here we report the interplay between TFIIIC and Maf1 in controlling Pol III activity upon the physiological switch of yeast from fermentation to respiration. TFIIIC directly competes with Pol III for chromatin occupancy as demonstrated by inversely correlated tDNA binding. The association of TFIIIC with tDNA was stronger under unfavorable respiratory conditions and in the presence of Maf1. Induction of tDNA transcription by glucose-activated protein kinase A (PKA) was correlated with the down-regulation of TFIIIC occupancy on tDNA. The conditions that activate the PKA signaling pathway promoted the binding of TFIIIB subunits, Brf1 and Bdp1, with tDNA, but decreased their interaction with TFIIIC. Association of Brf1 and Bdp1 with TFIIIC was much stronger under repressive conditions, potentially restricting TFIIIB recruitment to tDNA and preventing Pol III recruitment. Altogether, we propose a model in which, depending on growth conditions, TFIIIC promotes activation or repression of tDNA transcription.
Asunto(s)
ARN de Transferencia/genética , Factores de Transcripción TFIII/fisiología , Transcripción Genética , Respiración de la Célula/genética , Fermentación/genética , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Sistema de Señalización de MAP Quinasas/fisiología , ARN de Transferencia/biosíntesis , Proteínas Represoras/fisiología , Elongación de la Transcripción Genética , Proteínas ras/fisiología , Animales , Animales Modificados Genéticamente , Proliferación Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Embrión no Mamífero , Biosíntesis de Proteínas/genética , ARN Polimerasa III/antagonistas & inhibidores , ARN de Transferencia/genética , Proteínas Represoras/genética , Transducción de Señal/fisiología , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/fisiología , Alas de Animales/embriología , Alas de Animales/metabolismoRESUMEN
RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.
Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Animales , Humanos , Modelos Biológicos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Conformación Proteica , ARN Polimerasa III/química , Procesamiento Postranscripcional del ARN , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estrés Fisiológico , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The synthesis of transfer RNA (tRNA) is directed by RNA polymerase III (Pol III) specialized in high-level transcription of short DNA templates. Pol III recruitment to tRNA genes is controlled by two general initiation factors, TFIIIB and TFIIIC. They are multi-protein complexes regulated at the level of expression of individual subunits, as well as through phosphorylation and interaction with partner proteins. Here, we describe particular aspects of TFIIIB and TFIIIC control in yeast and human cells. Under stress conditions, tRNA synthesis is negatively regulated by the MAF1 protein, which interacts directly with Pol III. Sequence and function of MAF1 are conserved among eukaryotic organisms from yeast to humans. MAF1 is a phosphoprotein which mediates diverse regulatory signals to Pol III. Interestingly, there is a subset of housekeeping tRNA genes, both in the yeast and human genome, which are less sensitive to MAF1-dependent repression. The possible mechanisms responsible for this differential regulation of tRNA synthesis by MAF1 are discussed.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción Maf/genética , ARN de Transferencia/biosíntesis , Factor de Transcripción TFIIIB/genética , Factores de Transcripción TFIII/genética , Transcripción Genética , Animales , Regulación Fúngica de la Expresión Génica , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
RNA polymerase III (RNAPIII) transcribes tRNA genes, 5S RNA as well as a number of other non-coding RNAs. Because transcription by RNAPIII is an energy-demanding process, its activity is tightly linked to the stress levels and nutrient status of the cell. Multiple signaling pathways control RNAPIII activity in response to environmental cues, but exactly how these pathways regulate RNAPIII is still poorly understood. One major target of these pathways is the transcriptional repressor Maf1, which inhibits RNAPIII activity under conditions that are detrimental to cell growth. However, recent studies have found that the cell can also directly regulate the RNAPIII machinery through phosphorylation and sumoylation of RNAPIII subunits. In this review we summarize post-translational modifications of RNAPIII subunits that mainly have been identified in large-scale proteomics studies, and we highlight several examples to discuss their relevance for regulation of RNAPIII.
Asunto(s)
Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , ARN Polimerasa III/metabolismo , ARN de Transferencia/biosíntesis , Animales , Caseína Quinasas/genética , Proteína Coatómero/genética , Regulación Fúngica de la Expresión Génica , Fosforilación , Subunidades de Proteína , ARN Polimerasa III/química , ARN Polimerasa III/genética , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN de Transferencia/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Sumoilación , Proteína de Unión a TATA-Box/genética , Factor de Transcripción TFIIIB/genética , Factores de Transcripción/genéticaRESUMEN
PURPOSE: The purpose of this study was to investigate the biological role and mechanism of miR-373 targeting of TFIIB-related factor 2 (BRF2) in the regulation of non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: miRNA microarray chip analysis of four paired NSCLC and adjacent non-tumor tissues was performed. Quantitative real-time polymerase chain reaction (qRT-PCR) andwestern blotting were used to detect the expression levels of miR-373 and BRF2 in NSCLC tissues and cell lines. The dual-luciferase reporter method was performed to determine if BRF2 is a target of miR-373. MTT, wound-healing, Transwell, and flow cytometric assays were conducted to examine the proliferation, migration, invasion, and cell cycle progression of NSCLC A549 cells, respectively; western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins. RESULTS: The miRNA microarray chip analysis demonstrated that miR-373 was down-regulated in NSCLC tissues, and this result was confirmed by qRT-PCR. Additionally, miR-373 was confirmed to target BRF2. Moreover, miR-373 expression was inversely correlated with BRF2 expression in NSCLC tissues and cell lines; both miR-373 down-regulation and BRF2 up-regulation were strongly associated with the clinicopathological features and prognosis of NSCLC patients. In vitro, overexpression of miR-373 markedly inhibited cell proliferation, migration, and invasion; up-regulated the expression of E-cadherin; and down-regulated the expression of N-cadherin and Snail in A549 cell. Knockdown BRF2 by siRNA resulted in effects similar to those caused by overexpression of miR-373. CONCLUSION: MiR-373 is decreased in NSCLC, and overexpression of miR-373 can suppress cell EMT, and inhibit the proliferation, migration, and invasion of NSCLC A549 cells by targeting BRF2.