Sub-cellular Localization And Phosphorylation Analysis On The Tudor-SN Protein Under Stress Condit1ion

Objectives:Tudor-SN(Tudor Staphylococcal Nuclease)protein,also known as p100 protein or SND1(staphylococcal nuclease domain containing 1),is evolutionarily conserved in human,animals and plants.Tudor-SN participates in several biological responses,including gene transcription,cellular stress,pre-mRNA splicing.Our previous data suggested that Tudor-SN takes part in the formation of SGs(Stress Granules),when the cell was treated with 0.5mM arsenite sodium or heat shock at 45?.SGs is a kind of cytoplasmic RNA foci that aggregates in response to environmental stress stimuli,such as oxidative stress,heat shock,or viral infection,which plays diverse roles in the regulation of mRNA metabolism.Here,we aimed at studying the biological behavior of Tudor-SN granules,including the Sub-cellular localization and phosphorylation analysis,in eukaryotic cell.Methods:The experiment was divided into three parts:1)The immunofluorescence(IF)assays were performed to detect the formation of Tudor-SN formation under different stimuli or in the different cell cycle phase,and the co-localization between Tudor-SN granules and different organelles or autophagy granules.2)LI-COR odyssey infrared imaging system and traditional western Blotting assay were performed to detect the level of expression and total phosphorylation of Tudor-SN at ser,thr,tyr sites under the treatment of 0.5mM arsenite sodium or heat shock at 45?.3)LC-MS/MS(Liquid Chromatography-Mass Spectrometry/Mass Spectrometry,LC-MS/MS)was performed to confirm the stress-induced phosphorylation site of Tudor-SN.The specific phosphorylation antibodies were obtained from the New Zealand rabbits immunized by the corresponding synthesized phosphorylated peptides.Results:1)Tudor-SN granules aggregate under the treatment of 10 mM DTT stress(Dithiothreitol),but not at 4? 1h,32? 20h,20mM H2O2 1h,and l?M thapsigargin 2h.2)Tudor-SN granules fail to co-localize with autophagy granules and organelles,including endoplasmic reticulum,mitochondria,and Golgi apparatus.3)Tudor-SN granules disassemble gradually during the prophase,prometaphase,metaphase and anaphase of the cell cycle and reassemble in the telophase.4)There is no alteration on the expression level of Tudor-SN,but an increased total phosphorylation level of Tudor-SN at Ser,Thr,Tyr sites,after the treatment of 0.5mM arsenite sodium or 45? for 1h.5)Tudor-SN protein is phosphorylated at T73,T103 sites under the oxidative stress.And rabbit polyclonal the anti-pT73,anti-pT103 antibodies are prepared.6)The anti-pT103 antibody is able to recognize the Tudor-SN granules.7)The phosphorylation level of Tudor-SN at T103 site fluctuates during the sodium arsenite-induced oxidative stress and after the removal of stress.Conclusions:1)The formation of Tudor-SN granules differs under different stimuli and in different cell cycle phase.2)Tudor-SN protein containing stress granules fail to co-localize with Golgi apparatus,mitochondria,endoplasmic reticulum and autophagy granules.3)The rabbit polyclonal anti-pT73,anti-pT103 antibodies are prepared successfully.4)The phosphorylation level of Tudor-SN at T103 site increases nonlinearly,and is involved in the stress granules formation during stress.