RESUMO
Cell survival to replication stress depends on the activation of the Mec1ATR-Rad53 checkpoint response that protects the integrity of stalled forks and controls the origin firing program. Here we found that Mad2, a member of the spindle assembly checkpoint (SAC), contributes to efficient origin firing and to cell survival in response to replication stress. We show that Rad53 and Mad2 promote S-phase cyclin expression through different mechanisms: while Rad53 influences Clb5,6 degradation, Mad2 promotes their protein synthesis. We found that Mad2 co-sediments with polysomes and modulates the association of the translation inhibitor Caf204E-BP with the translation machinery and the initiation factor eIF4E. This Mad2-dependent translational regulatory process does not depend on other SAC proteins. Altogether our observations indicate that Mad2 has an additional function outside of mitosis to control DNA synthesis and collaborates with the Mec1-Rad53 regulatory axis to allow cell survival in response to replication stress.
Assuntos
Ciclinas/genética , Replicação do DNA , Proteínas Mad2/metabolismo , Mitose , Biossíntese de Proteínas , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclinas/metabolismo , Proteínas Mad2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Translation initiation factor eIF4E forms eIF4E-eIF4G complex at the 5' cap of mRNA. This interaction can be inhibited by the family of 4E-binding proteins (4E-BP). In yeast Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete with eIF4G for binding to eIF4E via the shared conserved interaction motif. In order to investigate the roles of Caf20 in gene-specific translational regulation and the formation of mRNA granules (P-bodies), we introduced substitution mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding motif for CAF20. Overexpression of the wild-type CAF20 showed an increased protein level of Ste12 transcription factor as well as highly developed P-body formation. However, 4E-binding site mutations of CAF20 led to a reduced number of P-body foci and decreased levels of Ste12 protein. The phenotypes of the caf20 deletion mutation were also analyzed, and we suggest that Caf20 plays a critical role in Ste12 protein expression and in the control of P-body formation.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Sítios de Ligação , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
MAD2L1 (Mitotic Arrest Deficient 2 Like 1), a member of the mitotic checkpoint, maintains the genomic stability by insuring the proper segregation of the sister chromatids. Deregulation of MAD2L1 protein expression is a recurrent feature in cancer cells. In our recent publication, we uncovered a role for its yeast homolog, Mad2p, in protein synthesis during S-phase.