The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells.
This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm.
The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
方法:邻近连接测定(PLA)已被广泛用于揭示蛋白质-mRNA和蛋白质-蛋白质相互作用的亚细胞定位。ELISA和免疫共沉淀证实了体外和细胞中的相互作用。
结果:这项研究提供了一种新的机制,使p53mRNA与ATM激酶相互作用,并表明L22L同义突变体,已知改变p53mRNA的二级结构,阻止互动。在DNA损伤感知途径中的相关机制作用,这与下游的DNA损伤反应有关,正在探索。DNA损伤(双链DNA断裂激活ATM)后,激活的MDMX蛋白竞争ATM-p53mRNA相互作用,并阻止p53mRNA与NBS1(MRN复合物)的关联。这些数据还揭示了ATM上的结合结构域和磷酸化事件,其调节复合物到细胞质的相互作用和运输。
结论:提出的模型显示了ATM与p53mRNA的新型相互作用,并描述了DNA损伤传感与下游p53激活途径之间的联系;支持改变二级mRNA结构的同义突变的功能含义上升。