RESUMEN

ZNF143 is a ubiquitously expressed transcription factor conserved in all vertebrates, regulating genes involved in primary metabolism and cell growth. It is therefore crucial to tightly maintain the adequate level of this factor in the cell. Although ZNF143 expression is auto-regulated at the transcriptional level, nothing is known about the post-transcriptional events influencing its expression. In this work, performed in mammalian cells, we show that ZNF143 expresses different 3'-untranslated regions (3'-UTR) as a result of alternative polyadenylation. These 3'UTR isoforms have a diverse impact on the ZNF143 transcript fate. Indeed, we show that the longest isoform, unlike the short one, contains a destabilizing AU-Rich element and is targeted by the miRNA 590-3p. Additionally we observed a correlation between ZNF143 downregulation and miR-590-3p up-regulation in retinoic acid treated teratocarcinoma cells. This strongly suggests that ZNF143 post-transcriptional regulation depends on the long 3'UTR isoform during teratocarcinoma cells differentiation. Finally we evidenced that the alternative polyadenylation site usage is independent of the previously identified ZNF143 transcriptional auto-regulation.

Asunto(s)

Regiones no Traducidas 3'/genética , Regulación de la Expresión Génica/genética , Isoformas de ARN/genética , Transactivadores/genética , Elementos Ricos en Adenilato y Uridilato , Secuencia de Bases , Diferenciación Celular/genética , Línea Celular , Regulación hacia Abajo/genética , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Poliadenilación , Transcripción Genética , Regulación hacia Arriba/genética

RESUMEN

A transcriptional feedback loop is the simplest and most direct means for a transcription factor to provide an increased stability of gene expression. In this work performed in human cells, we reveal a new negative auto-regulatory mechanism involving an alternative transcription start site (TSS) usage. Using the activating transcription factor ZNF143 as a model, we show that the ZNF143 low-affinity binding sites, located downstream of its canonical TSS, play the role of protein sensors to induce the up- or down-regulation of ZNF143 gene expression. We uncovered that the TSS switch that mediates this regulation implies the differential expression of two transcripts with an opposite protein production ability due to their different 5' untranslated regions. Moreover, our analysis of the ENCODE data suggests that this mechanism could be used by other transcription factors to rapidly respond to their own aberrant expression level.

Asunto(s)

Regulación de la Expresión Génica , Transactivadores/genética , Sitio de Iniciación de la Transcripción , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Línea Celular , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Homeostasis , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transactivadores/metabolismo

RESUMEN

ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation.

Asunto(s)

Regulación de la Expresión Génica , Receptor Notch1/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células HEK293 , Histonas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Datos de Secuencia Molecular , Motivos de Nucleótidos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo

RESUMEN

In the human genome, ∼ 10% of the genes are arranged head to head so that their transcription start sites reside within <1 kbp on opposite strands. In this configuration, a bidirectional promoter generally drives expression of the two genes. How bidirectional expression is performed from these particular promoters constitutes a puzzling question. Here, by a combination of in silico and biochemical approaches, we demonstrate that hStaf/ZNF143 is involved in controlling expression from a subset of divergent gene pairs. The binding sites for hStaf/ZNF143 (SBS) are overrepresented in bidirectional versus unidirectional promoters. Chromatin immunoprecipitation assays with a significant set of bidirectional promoters containing putative SBS revealed that 93% of them are associated with hStaf/ZNF143. Expression of dual reporter genes directed by bidirectional promoters are dependent on the SBS integrity and requires hStaf/ZNF143. Furthermore, in some cases, functional SBS are located in bidirectional promoters of gene pairs encoding a noncoding RNA and a protein gene. Remarkably, hStaf/ZNF143 per se exhibits an inherently bidirectional transcription activity, and together our data provide the demonstration that hStaf/ZNF143 is indeed a transcription factor controlling the expression of divergent protein-protein and protein-non-coding RNA gene pairs.

Asunto(s)

Regiones Promotoras Genéticas , Transactivadores/fisiología , Transcripción Genética , Sitios de Unión , ADN/química , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Genoma Humano , Células HeLa , Humanos , Proteínas/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Análisis de Secuencia de ADN , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

The C/D box scaRNA2 is predicted to guide specific 2'-O-methylation of U2 snRNA. In contrast to other SCARNA genes, SCARNA2 appears to be independently transcribed. By transient expression of SCARNA2-reporter gene constructs, we have demonstrated that this gene is transcribed by RNA polymerase II and that the promoter elements responsible for its transcription are contained within a 161 bp region upstream of the transcription start site. In mammals, we have identified four cross species conserved promoter elements, a TATA motif, an hStaf/ZNF143 binding site and two novel elements that are required for full promoter activity. Binding of the human hStaf/ZNF143 transcription factor to its target sequence is required for promoter activity, suggesting that hStaf/ZNF143 is a fundamental regulator of the SCARNA2 gene. We also showed that RNA polymerase II continues transcription past the 3'-end of the mature RNA, irrespective of the identity of the Pol II promoter. The 3'-end processing and accumulation are governed by the sole information contained in the scaRNA2 encoding region, the maturation occurring via a particular pathway incompatible with that of mRNA or snRNA production.

Asunto(s)

ARN/genética , Transcripción Genética , Sitios de Unión , Células HeLa , Humanos , Regiones Promotoras Genéticas , ARN/biosíntesis , ARN/metabolismo , Procesamiento de Término de ARN 3' , ARN Polimerasa II/metabolismo , Transactivadores/metabolismo , ARN Pequeño no Traducido

RESUMEN

The mitochondrial transcription factor A (Tfam) is essential for transcription initiation and replication of mitochondrial DNA. It was previously reported that transcription factors Sp1, NRF-1, NRF-2 were critical for maintaining the normal transcription levels of the mammalian TFAM gene. In this work, investigation of the transcriptional regulation of the human TFAM gene revealed the presence of two cross-species conserved binding sites for the transcription factor hStaf/ZNF143. By using promoter binding assays, transient expression of mutant TFAM reporter gene constructs and chromatin immunoprecipitation experiments, we provided insight into the involvement of hStaf/ZNF143 in promoter activity. Furthermore, we reported the identification of two other functionally important elements. Altogether, our data led to the conclusion that the promoter of the human TFAM gene harbors a complex organization with at least six transcriptional regulatory elements.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Mitocondriales/metabolismo , Transactivadores/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Reporteros , Humanos , Luciferasas/metabolismo , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , beta-Galactosidasa/metabolismo

RESUMEN

BubR1 is a key protein mediating spindle checkpoint activation. Loss of this checkpoint control results in chromosomal instability and aneuploidy. The transcriptional regulation of the cell cycle regulated human BUB1B gene, which encodes BubR1, was investigated in this report. A minimal BUB1B gene promoter containing 464 bp upstream from the translation initiation codon was sufficient for cell cycle regulated promoter activity. A pivotal role for transcription factor hStaf/ZNF143 in the expression of the BUB1B gene was demonstrated through gel retardation assays, transient expression of mutant BUB1B promoter-reporter gene constructs and chromatin immunoprecipitation assay. Two phylogenetically conserved hStaf/ZNF143-binding sites (SBS) were identified which are indispensable for BUB1B promoter activity. In addition, we found that the domain covering the transcription start sites contains conserved boxes homologous to initiator (Inr), cell cycle dependent (CDE) and cell cycle genes homology regions (CHR) elements. Mutations within the CDE and CHR elements led to diminished cell cycle regulation of BUB1B transcription. These results demonstrate that BUB1B gene transcription is positively regulated by hStaf/ZNF143, a ubiquitously expressed factor, and that the CDE-CHR tandem element was essential for G2/M-specific transcription of the BUB1B gene.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Transactivadores/fisiología , Activación Transcripcional , Región de Flanqueo 5' , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Ciclo Celular/genética , Línea Celular , Chlorocebus aethiops , Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Drosophila/citología , Drosophila/genética , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

Staf was originally identified as the transcriptional activator of Xenopus tRNA(Sec) and small nuclear (sn) RNA-type genes. Recently, transcription of seven human (h) protein coding genes was reported to be activated by the human ortholog hStaf/ZNF143. Here we have used a combined in silico and biochemical approach to identify 1175 conserved hStaf/ZNF143-binding sites (SBS) distributed in 938 promoters of four mammalian genomes. The SBS shows a significant positional preference and occurs mostly within 200 bp upstream of the transcription start site. Chromatin immunoprecipitation assays with 295 of the promoters established that 90% contain bona fide SBS. By extrapolating the values of this mapping to the full sizes of the mammalian genomes, we can infer the existence of at least 2500 SBS distributed in 2000 promoters. This unexpected large number strongly suggests that SBS constitutes one of the most widespread transcription factor-binding sites in mammalian promoters. Furthermore, we demonstrated that the presence of the SBS alone is sufficient to direct expression of a luciferase reporter gene, suggesting that hStaf/ZNF143 can recruit per se the transcription machinery.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Humano , Regiones Promotoras Genéticas/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Secuencias de Aminoácidos/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Chlorocebus aethiops , Biología Computacional , Secuencia Conservada , Islas de CpG/genética , Proteínas de Unión al ADN/fisiología , Variación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Transactivadores/fisiología

RESUMEN

Vertebrate snRNA and snRNA-type genes occur in independent transcription units with external promoters. The transcription level from the basal promoter is enhanced by the distal sequence element DSE. This element contains almost invariably two activator submotifs, the Staf binding site and the octamer motif, recruiting the Staf and Oct-1 transcriptional activators. In the present work, database search identified 35 snRNA and snRNA-type genes in the genome sequence of the pufferfish Fugu rubripes. Sequence comparisons of promoter elements, determination of template activities by microinjection into Xenopus oocytes and DNA binding assays of the transcriptional activators led to the surprising finding that only two Fugu genes conform to the general scheme with the expected two submotifs in the DSE. Distinctively, all the other DSEs harbor a unique Staf binding site. Also striking was the observation that the tRNA(Sec), and the snRNA genes that are tandemly repeated, are transcribed from promoter-less DSEs. Evolutionary implications of these results are discussed.

Asunto(s)

ARN Nuclear Pequeño/genética , Takifugu/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genoma , Factor C1 de la Célula Huésped , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , Oocitos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Elementos de Respuesta/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Xenopus laevis

RESUMEN

BACKGROUND: Fatigue-related incidents in aviation may be self-reported by pilots in confidential systems. The aim of this study was to clarify what fatigue means to pilots on short- and long-haul flights (SHF and LHF, respectively). METHODS: Questionnaires were distributed to pilots through four airlines. Questions concerned the perceived causes of fatigue, its signs and symptoms in the reporting pilot and observed in others, as well as the strategies used to minimize its impact. RESULTS: Of 3,436 questionnaires distributed, 739 (21.5%) were returned. For LHF, fatigue was seen as mainly due to night flights (59%) and jet lag (45%). For SHF, fatigue was caused by prolonged duty periods (multi-segment flights over a sequence of 4 to 5 d) (53%) and successive early wake-ups (41%). Self-reported manifestations of fatigue in 60% of LHF pilots and 49% of SHF pilots included reduction in alertness and attention, and a lack of concentration. Signs observed in other crewmembers included an increase in response times and small mistakes (calculation, interpretation). When pilots were tired, all the flying tasks seemed to be more difficult than usual. In both LHF and SHF, rest and sleep management were the primary strategies used to cope with fatigue. Analysis showed that duty time is a major predictor of fatigue, but that it cannot be considered independently from the other contributory factors. CONCLUSION: For both LHF and SHF, pilots reported acute fatigue related to sleep deprivation, due mainly to work schedules: night flights, jet-lag, and successive early wake-ups. These causal factors could easily be assessed in investigation of accidents and incidents.

Asunto(s)

Medicina Aeroespacial , Fatiga , Privación de Sueño , Adulto , Aeronaves , Atención , Recolección de Datos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Admisión y Programación de Personal , Factores de Tiempo

RESUMEN

Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3' untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.

Asunto(s)

ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Selenocisteína/metabolismo , Homología de Secuencia de Aminoácido , Empalmosomas/metabolismo , Homología Estructural de Proteína

RESUMEN

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.

Asunto(s)

Proteínas Bacterianas/metabolismo , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas , Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas Bacterianas/genética , Guanosina Trifosfato/metabolismo , Modelos Biológicos , Unión Proteica , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Selenoproteínas

RESUMEN

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.

Asunto(s)

Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Factores de Elongación de Péptidos/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido