RESUMO
Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.
Assuntos
Apoptose/efeitos da radiação , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , RNA Ribossômico/biossíntese , Ribossomos/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Células Germinativas/efeitos da radiação , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação Puntual , RNA Polimerase I/genética , RNA Ribossômico/efeitos da radiação , Radiação Ionizante , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: The inability to efficiently repair DNA damage or remove cells with severely damaged genomes has been linked to several human cancers. Studies in yeasts and mammals have identified several genes that are required for proper activation of cell cycle checkpoints following various types of DNA damage. However, in metazoans, DNA damage can induce apoptosis as well. How DNA damage activates the apoptotic machinery is not fully understood. RESULTS: We demonstrate here that the Caenorhabditis elegans gene hus-1 is required for DNA damage-induced cell cycle arrest and apoptosis. Following DNA damage, HUS-1 relocalizes and forms distinct foci that overlap with chromatin. Relocalization does not require the novel checkpoint protein RAD-5; rather, relocalization appears more frequently in rad-5 mutants, suggesting that RAD-5 plays a role in repair. HUS-1 is required for genome stability, as demonstrated by increased frequency of spontaneous mutations, chromosome nondisjunction, and telomere shortening. Finally, we show that DNA damage increases expression of the proapoptotic gene egl-1, a response that requires hus-1 and the p53 homolog cep-1. CONCLUSIONS: Our findings suggest that the RAD-5 checkpoint protein is not required for HUS-1 to relocalize following DNA damage. Furthermore, our studies reveal a new function of HUS-1 in the prevention of telomere shortening and mortalization of germ cells. DNA damage-induced germ cell death is abrogated in hus-1 mutants, in part, due to the inability of these mutants to activate egl-1 transcription in a cep-1/p53-dependent manner. Thus, HUS-1 is required for p53-dependent activation of a BH3 domain protein in C. elegans.
Assuntos
Apoptose/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Mutação , Proteínas Repressoras/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Primers do DNA , Genoma , Genótipo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Schizosaccharomyces pombe , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.