The Regulatory Mechanism Of EIF4G1 On Nonsense Mediated MRNA Decay And Autophagy

Eukaryotic translation initiation factor 4 gamma 1(eIF4G1)as a major hub in translation initiation mediates recruitment of additional initiation factors,providing a scaffold for ribosome/m RNA-bridging.eIF4G1 is involved in the regulation of heat stress,endoplasmic reticulum stress and hypoxia stress,and is closely related to the proliferation and biological energy metabolism and mitochondrial activity,also plays an important role in the regulation of mRNA translation which correlation of these processes.In this study,Earle's balanced salt solution(EBSS)was used to induce the starvation stress and explore the effects of e IF4G1 on autophagy and nonsense-mediated mRNA decay(NMD).This study found that eIF4G1 mRNA and protein levels were significantly reduced with prolonged starvation during starvation stress,further studies show that the downregulation of eIF4G1 can inhibit the NMD,thereby increasing NMD endogenous substrate gene levels.But also contribute to the activation of autophagy during starvation stress,and the activation of autophagy can further degrade the e IF4G1 protein,which may be the stress-enhancing mechanisms during cell stress.This finding is helpful to further understand the mechanism of cellular adaptation to environmental stress,and to analyze the interaction between the autophagy and NMD.The main findings are as follows:1.EBSS-induced starvation stress can activate autophagy and inhibit NMD.The detection of autophagy levels and NMD efficiency in Hela cells treated with EBSS showed that autophagy was activated and NMD was inhibited,There is a negative correlation between the two mechanisms.2.Rapamycin can inhibit NMD activity.The levels of TBL2 and GADD45 B mRNA were significantly up-regulated after treatment with 2.5?M rapamycin for 2h(P?0.05),indicating that autophagic specific activator rapamycin inhibits NMD activity.3.eIF4G1 mRNA and protein levels decreased under starvation stress.With the prolongation of starvation time,the level of eIF4G1 was significantly down-regulated(2h and 4h,P?0.05;6h,P?0.01).Western Blot showed that the protein level was also down-regulated with the prolongation of treatment time.4.Interferenced eIF4G1 inhibits NMD activity.After knockdown eIF4G1,the GADD45 B level was significantly up-regulated(P?0.05)and the level of TPI in the NMD reporter vector was also significantly up-regulated(P?0.01),indicating that NMD was inhibited after eIF4G1 knockdown.5.Interferenced eIF4G1 activates autophagy.The level of SQSTM1 decreased significantly after eIF4G1 knockdown,and the ratio of LC3B?/LC3B? in the RNAi group was significantly increased compared to the NC group.Furthermore the EGFP fluorescence spots of LC3-EGFP-Hela cells increased significantly,indicating that the level of autophagy increased.6.Starvation-induced autophagic degradation of e IF4G1 protein.Blocking autophagy by interfering with autophagy-related gene ATG7,eIF4G1 is no longer degraded by autophagy induced by EBSS.Immunofluorescence co-localization showed that the eIF4G1 protein was co-located with the autophagic membrane protein LC3 in the late stage of EBSS treatment,indicating that the eIF4G1 protein was localized in the autophagosome;suggesting that EBSS-induced starvation stress activates autophagy and degrades eIF4G1 protein.The above results showed that the expression of eIF4G1 was down-regulated in the process of starvation stress.On the one hand,the activation of cell autophagy could increase cell material energy and enhance the ability of cells to cope with environmental stress.On the other hand,inhibition of NMD can up-regulate the level of anti-stress factors to cope with cell stress,while activated autophagy can further degrade e IF4G1 protein to enhance this adaptive mechanism.