RESUMEN
The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.
Asunto(s)
Cromatina , Histonas , ARN Largo no Codificante , Cromatina/metabolismo , Cromatina/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Histonas/metabolismo , Histonas/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células HeLa , Transcripción Genética , ARN/metabolismo , ARN/genética , Animales , Regulación de la Expresión GénicaRESUMEN
Cactins constitute a family of eukaryotic proteins broadly conserved from yeast to human and required for fundamental processes such as cell proliferation, genome stability maintenance, organismal development and immune response. Cactin proteins have been found to associate with the spliceosome in several model organisms, nevertheless their molecular functions await elucidation. Here we show that depletion of human cactin leads to premature sister chromatid separation, genome instability and cell proliferation arrest. Moreover, cactin is essential for efficient splicing of thousands of pre-mRNAs, and incomplete splicing of the pre-mRNA of sororin (also known as CDCA5), a cohesin-associated factor, is largely responsible for the aberrant chromatid separation in cactin-depleted cells. Lastly, cactin physically and functionally interacts with the spliceosome-associated factors DHX8 and SRRM2. We propose that cellular complexes comprising cactin, DHX8 and SRRM2 sustain precise chromosome segregation, genome stability and cell proliferation by allowing faithful splicing of specific pre-mRNAs. Our data point to novel pathways of gene expression regulation dependent on cactin, and provide an explanation for the pleiotropic dysfunctions deriving from cactin inactivation in distant eukaryotes.
Asunto(s)
Proteínas Portadoras/metabolismo , Cromátides/metabolismo , ARN Helicasas DEAD-box/metabolismo , Precursores del ARN/genética , Factores de Empalme de ARN/metabolismo , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Forma del Núcleo Celular , Proliferación Celular , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Intrones/genética , Unión Proteica , Precursores del ARN/metabolismoRESUMEN
Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.
Asunto(s)
Rotura Cromosómica , Replicación del ADN/genética , Origen de Réplica/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , ADN/biosíntesis , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Moldes GenéticosRESUMEN
Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a crucial anticancer barrier. DDR inactivation in these conditions promotes genome instability and tumor progression, but the underlying molecular mechanisms are elusive. We found that overexpression of both Cyclin E and Cdc25A rapidly slowed down replication forks and induced fork reversal, suggestive of increased topological stress. Surprisingly, these phenotypes, per se, are neither associated with chromosomal breakage nor with significant DDR activation. Oncogene-induced DNA breakage and DDR activation instead occurred upon persistent G2/M arrest or, in a checkpoint-defective context, upon premature CDK1 activation. Depletion of MUS81, a cell cycle-regulated nuclease, markedly limited chromosomal breakage and led to further accumulation of reversed forks. We propose that nucleolytic processing of unusual replication intermediates mediates oncogene-induced genotoxicity and that limiting such processing to mitosis is a central anti-tumorigenic function of the DNA damage checkpoints.