Study On The Roles And Mechanisms Of UBAP2L In The Assembly And Disassembly Of Stress Granules

Background and aimsWhen eukaryotic cells are exposed to various types of environmental stress,the assembly of 48S complex is blocked and initiation of protein translation pauses.The stalled translated mRNA,translation initiation factors and a lot of RNA binding proteins concentrate in the cytoplasm to from a non-enveloped high-density structure,called stress granules(SGs).SGs can promote cells better adapt to the environmental pressure.The stress granules are in dynamic,after the environmental stress are eliminated,the SGs will be disassembled,and the translation of proteins will be resumed.The assemble mechanisms of SGs is classified into classical(dependent of eIF2? phosphorylation)and non-classical(independent of eIF2a phosphorylation)pathway.No matter by a classical or non-classical pathway,formation of SGs goes through a series of processes.First step,translation stops upon exposure to environmental stress,and the assembly of the translation initiation complex was suspended due to phosphorylation of eIF2? or inactivation of eIF4A or eIF4E,leading to blockage of translation.In the second step,SGs's core protein-RNA binding proteins aggregate and form the core of SGs,including PABP,TIAl,TIAR,G3BP1,G3BP2,etc.In the third step,mRNP is transported to the microtubule protein,and the SGs gathere further through the interaction between proteins.Finally,some proteins that do not have RNA binding domains enter SGs through the interaction of proteins.There are many kinds of environmental stress can induce stress granules,such as oxidative stress,lack of nutrition,heat shock,cold shock,osmotic shock and infection,etc.The composition of SGs will be slightly different due to different environmental stress.The formation of SGs is influenced by many factors and is very complicated.UBAP2L/NICE4(Ubiquitin Associated Protein 2-Like)is an ubiquitin related protein containing UBA domain and multiple RGG/RG repeats.At present,there are few studies on UBAP2L,which mainly focused on its role in tumorigenesis.UBAP2L was associated with BMI-1,a member of the multi-comb family protein,to maintain the activity of hematopoietic stem cells.PRMT1 can mediate the arginine methylation of the RGG/RG motif of UBAP2L,which is crucial for the precise distribution of chromosomes.Howerve the details of the physiological function of UBAP2L in cells are still not very clear.This paper studies the regulation and mechanism of UBAP2L on stress granules,will be further reveals the molecular mechanism and function of stress granules.Methods1.Hela and HEK293 cells were transfected by siRNA or plasmid by using lipofactemin 3000 or lentivirus transfection system,to knockdown or overexpress UBAP2L.2.Use immunofluorescence to detect the assembly or disassembly of the stress granules,and western blotting to detect the SG-related protein levels,including G3BP2?FXR1?FXR2 and PABPC1.3.Used immunoprecipitation to assess the interaction of UBAP2L with G3BP2?FXR1?FXR2?PABPC1 and rpS6 under various conditions.Results1.UBAP2L interacted with some SGs-related proteins,including G3BP2?FXR1 and FXR2,and co-localized with them under SGs-inducing conditions.2.Both UBAP2L knockdown and overexpression,hardly changed the formation of SGs.3.Knockdown UBAP2L significantly decreased the formation of SGs induce by AS,H2O2 and Sorb,while overexpression UBAP2L significantly increased,and both delayed the degradation of SGs after the stimulus was removed.4.Neither UBAP2L knockdown nor overexpression significantly changed the protein levels of SGs-related proteins,G3BP2?FXR1?FXR2?PABPC1 and rpS6.5.UBAP2L modulated SGs dynamics by interacting with 40S ribosomal small subunit protein rpS6.6.The interaction of UBAP2L with G3BP2?FXR1?FXR2 and PABPC1 disappeared,and interaction with rpS6 enhanced after RNase treatment.Conclusion1.UBAP2L positively regulates the assembly and disassembly of SGs,but do not have independent nucleating capability.2.UBAP2L monitors SGs dynamics by interacting with 40S ribosomal small subunit protein rpS6.3.The association between UBAP2L and SG-related proteins depends on exitence of RNA...