RESUMO
Altered cellular responses to DNA damage can contribute to cancer development, progression, and therapeutic resistance. Mutations in key DNA damage response factors occur across many cancer types, and the DNA damage-responsive gene, TP53, is frequently mutated in a high percentage of cancers. We recently reported that an alternative splicing pathway induced by DNA damage regulates alternative splicing of TP53 RNA and further modulates cellular stress responses. Through damage-induced inhibition of the SMG1 kinase, TP53 pre-mRNA is alternatively spliced to generate TP53b mRNA and p53b protein is required for optimal induction of cellular senescence after ionizing radiation-induced DNA damage. Herein, we confirmed and extended these observations by demonstrating that the ATM protein kinase is required for repression of SMG1 kinase activity after ionizing radiation. We found that the RNA helicase and splicing factor, DDX5, interacts with SMG1, is required for alternative splicing of TP53 pre-mRNA to TP53b and TP53c mRNAs after DNA damage, and contributes to radiation-induced cellular senescence. Interestingly, the role of SMG1 in alternative splicing of p53 appears to be distinguishable from its role in regulating nonsense-mediated RNA decay. Thus, ATM, SMG1, and DDX5 participate in a DNA damage-induced alternative splicing pathway that regulates TP53 splicing and modulates radiation-induced cellular senescence.
Assuntos
Processamento Alternativo , Neoplasias , Humanos , Proteínas Serina-Treonina Quinases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Dano ao DNA , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
IMPLICATIONS: Multiple members of the cohesin complex are involved in the regulation of DNA replication and transcription in the vicinity of DNA double-strand breaks and their role(s) are regulated by the ATM kinase.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Fosforilação , CoesinasRESUMO
The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Segregação de Cromossomos , Mitose , Antígenos de Superfície/genética , Antineoplásicos Fitogênicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Células HCT116 , Células HEK293 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Mitose/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Paclitaxel/farmacologia , Fosforilação , Mapas de Interação de Proteínas , Proteômica/métodos , Transdução de SinaisRESUMO
The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.
Assuntos
Fase G2/genética , Mitose/genética , Fase S/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Ciclina B1/antagonistas & inibidores , Ciclina B1/metabolismo , Dano ao DNA/genética , Replicação do DNA/genética , Proteína Forkhead Box M1/metabolismo , Redes Reguladoras de Genes , Células HCT116 , Humanos , Fosforilação , TelomeraseRESUMO
The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hypersensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR-activating proteins.
