RESUMEN

ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.

RESUMEN

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.

Asunto(s)

ADN Helicasas , Proteínas Nucleares , ARN , Factores de Transcripción , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Empalme Alternativo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN/genética , ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/metabolismo , Hibridación de Ácido Nucleico , Edición de ARN , ARN/metabolismo , Adenosina Desaminasa/genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , ADN Helicasas/metabolismo , Eliminación de Gen , Genes Reporteros , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/metabolismo , Recombinación Homóloga/genética , Humanos , Enzimas Multifuncionales/metabolismo , Estabilidad Proteica , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/genética , Proteína de Replicación A/metabolismo

RESUMEN

This report summarizes an international conference on molecular machines convened at New York University, Abu Dhabi by Piergiorgio Percipalle, George Shubeita and Serdal Kirmizialtin. The meeting was conceived around the epistemological question of what do we understand, or not understand (if we have open minds), about the degree to which cells operate by the individual actions of single enzymes or non-catalytic protein effectors, versus combinations of these in which their heterotypic association creates an entity that is more finely tuned and efficient - a machine. This theme was explored through a vivid series of talks, summarizing the latest findings on macromolecular complexes that operate in the nucleus or cytoplasm.

Asunto(s)

Núcleo Celular , Citoplasma , Citosol , Emiratos Árabes Unidos

RESUMEN

RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.

Asunto(s)

Roturas del ADN de Doble Cadena , ARN , ADN/genética , Reparación del ADN , Recombinación Homóloga , ARN/genética

RESUMEN

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.

Asunto(s)

Roturas del ADN de Doble Cadena , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Recombinación Homóloga , Proteína de Replicación A/metabolismo , ADN/genética , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Exosomas/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Ribonucleasa H/metabolismo

RESUMEN

Recent studies suggest that transcription takes place at DNA double-strand breaks (DSBs), that transcripts at DSBs are processed by Drosha and Dicer into damage-induced small RNAs (diRNAs), and that diRNAs are required for DNA repair. However, diRNAs have been mostly detected in reporter constructs or repetitive sequences, and their existence at endogenous loci has been questioned by recent reports. Using the homing endonuclease I-PpoI, we have investigated diRNA production in genetically unperturbed human and mouse cells. I-PpoI is an ideal tool to clarify the requirements for diRNA production because it induces DSBs in different types of loci: the repetitive 28S locus, unique genes and intergenic loci. We show by extensive sequencing that the rDNA locus produces substantial levels of diRNAs, whereas unique genic and intergenic loci do not. Further characterization of diRNAs emerging from the 28S locus reveals the existence of two diRNA subtypes. Surprisingly, Drosha and its partner DGCR8 are dispensable for diRNA production and only one diRNAs subtype depends on Dicer processing. Furthermore, we provide evidence that diRNAs are incorporated into Argonaute. Our findings provide direct evidence for diRNA production at endogenous loci in mammalian cells and give insights into RNA processing at DSBs.

Asunto(s)

ARN Helicasas DEAD-box/genética , Reparación del ADN , ADN Intergénico/genética , ADN/genética , Endodesoxirribonucleasas/genética , ARN/genética , Ribonucleasa III/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Línea Celular , ARN Helicasas DEAD-box/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Intergénico/metabolismo , Endodesoxirribonucleasas/metabolismo , Sitios Genéticos , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo

RESUMEN

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Asunto(s)

Regiones no Traducidas 3'/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Procesamiento de Término de ARN 3' , Ribonucleoproteína Nuclear Pequeña U1/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

BACKGROUND: Brahma (BRM) is the only catalytic subunit of the SWI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes. RESULTS: Here we have used RNA-seq and ChIP-seq to identify the BRM target genes in S2 cells, and we have used a catalytically inactive BRM mutant (K804R) that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. We show that 49% of the BRM target genes in S2 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. We also show that the catalytic and non-catalytic mechanisms of SWI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes. CONCLUSIONS: This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SWI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes.

Asunto(s)

Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Nucleosomas/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transactivadores/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Genómica , Regiones Promotoras Genéticas/genética

RESUMEN

Protein-DNA interactions in vivo can be detected and quantified by chromatin immunoprecipitation (ChIP). ChIP has been instrumental for the advancement of epigenetics and has set the groundwork for the development of a number of ChIP-related techniques that have provided valuable information about the organization and function of genomes. Here, we provide an introduction to ChIP and discuss the applications of ChIP in different research areas. We also review some of the strategies that have been devised to improve ChIP performance.

Asunto(s)

Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN , Epigénesis Genética , Epigenómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/metabolismo , Humanos , Unión Proteica , Reproducibilidad de los Resultados

RESUMEN

Arguably one of the most valuable techniques to study chromatin organization, ChIP is the method of choice to map the contacts established between proteins and genomic DNA. Ever since its inception, more than 30 years ago, ChIP has been constantly evolving, improving, and expanding its capabilities and reach. Despite its widespread use by many laboratories across a wide variety of disciplines, ChIP assays can be sometimes challenging to design, and are often sensitive to variations in practical implementation.In this chapter, we provide a general overview of the ChIP method and its most common variations, with a special focus on ChIP-seq. We try to address some of the most important aspects that need to be taken into account in order to design and perform experiments that generate the most reproducible, high-quality data. Some of the main topics covered include the use of properly characterized antibodies, alternatives to chromatin preparation, the need for proper controls, and some recommendations about ChIP-seq data analysis.

Asunto(s)

Inmunoprecipitación de Cromatina , Interpretación Estadística de Datos , Secuenciación de Nucleótidos de Alto Rendimiento , Proyectos de Investigación , Animales , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , Inmunoprecipitación de Cromatina/normas , ADN , Proteínas de Unión al ADN , Guías como Asunto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Control de Calidad , Reproducibilidad de los Resultados , Flujo de Trabajo

RESUMEN

Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that ß-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of ß-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In ß-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type ß-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of ß-actin in Pol I transcription. The rRNA synthesis defects in the ß-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (monomethylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated ß-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.-Almuzzaini, B., Sarshad, A. A. , Rahmanto, A. S., Hansson, M. L., Von Euler, A., Sangfelt, O., Visa, N., Farrants, A.-K. Ö., Percipalle, P. In ß-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.

Asunto(s)

Actinas/metabolismo , Reprogramación Celular/fisiología , ADN Ribosómico/metabolismo , Epigénesis Genética/fisiología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Actinas/genética , Animales , Células Cultivadas , Cromatina , ADN Ribosómico/genética , Ratones , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/fisiología , Transcripción Genética/fisiología

RESUMEN

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Heterocromatina/metabolismo , Proteínas Represoras/metabolismo , Animales , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Silenciador del Gen , Genoma , Heterocromatina/genética , Unión Proteica , ARN Mensajero/genética

RESUMEN

Phenomenological screening of small molecule libraries for anticancer activity yields potentially interesting candidate molecules, with a bottleneck in the determination of drug targets and the mechanism of anticancer action. We have found that, for the protein target of a small-molecule drug, the abundance change in late apoptosis is exceptional compared to the expectations based on the abundances of co-regulated proteins. Based on this finding, a novel method to drug target deconvolution is proposed. In a proof of principle experiment, the method yielded known targets of several common anticancer agents among a few (often, just one) likely candidates identified in an unbiased way from cellular proteome comprising more than 4,000 proteins. A validation experiment with a different set of cells and drugs confirmed the findings. As an additional benefit, mapping most specifically regulated proteins on known protein networks highlighted the mechanism of drug action. The new method, if proven to be general, can significantly shorten drug target identification, and thus facilitate the emergence of novel anticancer treatments.

Asunto(s)

Antineoplásicos/farmacología , Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Proteoma/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Línea Celular Tumoral , Doxorrubicina/farmacología , Fluorouracilo/farmacología , Células HCT116 , Humanos , Metotrexato/farmacología , Paclitaxel/farmacología , Proteómica , Quinazolinas/farmacología , Tiofenos/farmacología

RESUMEN

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Recombinación Homóloga/genética , Animales , Western Blotting , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/antagonistas & inhibidores , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Células HeLa , Histonas/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/genética , Recombinasa Rad51/metabolismo

RESUMEN

BACKGROUND: The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes. RESULTS: The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150-250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations. CONCLUSIONS: We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.

Asunto(s)

Chironomidae/genética , Puffs Cromosómicos , Genoma , Animales , Mapeo Contig , Drosophila melanogaster/genética , Sitios Genéticos , Microscopía Electrónica de Transmisión , Anotación de Secuencia Molecular , Análisis de Secuencia de ADN , Transcriptoma

RESUMEN

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3ß phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3ß selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3ß-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3ß directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3ß-mediated phosphorylation of NM1 is required for pol I transcription activation.

Asunto(s)

Fase G1/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Miosina Tipo I/metabolismo , Activación Transcripcional/genética , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Animales , Línea Celular , Cromatina/genética , ADN Ribosómico/genética , Proteínas F-Box/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Ratones , Ratones Noqueados , Fosforilación , Proteolisis , Interferencia de ARN , ARN Polimerasa I/genética , ARN Interferente Pequeño , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Factores de Transcripción p300-CBP/metabolismo

RESUMEN

Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or eliminate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the quality controls can sense defects in the assembly of mRNA-protein complexes, in the processing of the precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by different specialized mechanisms. Some of them involve dedicated factors whose function is to identify faulty molecules and target them for degradation. Others are the result of a more subtle balance in the kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear retention and transcriptional silencing. Three major degradation systems are responsible for the destruction of the defective transcripts: the exosome, the 5'-3' exoribonucleases, and the nonsense-mediated mRNA decay (NMD) machinery. This review summarizes recent findings on the cotranscriptional quality control of mRNA biogenesis, and speculates that a protein-protein interaction network integrates multiple mRNA degradation systems with the transcription machinery.

Asunto(s)

Regulación de la Expresión Génica , Redes Reguladoras de Genes , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Humanos , Modelos Genéticos , Unión Proteica , ARN Mensajero/genética , Ribonucleoproteínas/genética , Transcripción Genética

RESUMEN

The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interferencia de ARN , ARN/metabolismo , Transactivadores/metabolismo , Trans-Empalme , Factores de Transcripción/genética , Empalme Alternativo , Animales , Línea Celular , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación de la Expresión Génica , División del ARN , Isoformas de ARN/metabolismo , ARN Polimerasa II/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

The pyrimidine analogue 5-fluorouracil (5FU) is used as a treatment for solid tumors, but its mechanism of action is not fully understood. We have used mass spectrometry to study the mechanism of action of 5FU, and we have measured the effects of this drug on the composition and on the turnover of the proteome of RKO cancer cells. We have identified novel potential targets of 5FU that are affected after very short exposure times. We have also shown that 5FU has a massive effect on the proteins involved in RNA metabolism. After only 1 h of treatment, 5FU causes a post-transcriptional reduction in the abundance of components of the translation machinery (mostly ribosomal proteins), and this reduction is accompanied by a down-regulation of the translational capacity of the cells. Neither rapamycin nor raltitrexed, two drugs that also block cell proliferation, reduce the abundances of ribosomal proteins as 5FU does, which suggests that the down-regulation of ribosomal proteins is coupled to the mechanism of action of 5FU. Some of our observations conflict with previous reports based on RNA quantification. This shows how important it is to complement RNA profiling studies with analyses of drug toxicity at the protein level.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Fluorouracilo/farmacología , Proteoma/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Espectrometría de Masas en Tándem