RESUMO
Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.
Assuntos
Apoptose/genética , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Estresse Oxidativo/genética , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/química , Precursores de RNA/química , Precursores de RNA/genética , Análise de Sequência de Proteína , Serina/genética , Serina/metabolismo , Proteínas Supressoras de Tumor/químicaRESUMO
The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin-proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEV(hPrp19/hPso4) and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.