The Molecular Mechanism For The Regulation Of Tudor-SN Protein In Stress Granules Assembly

Objectives: Stress Granules(SGs) and Processing Bodies(PBs) are two types of granules that dynamically accumulate in the cytoplasm under stress stimuli, such as oxidative stress, heat shock, UV irradiation or viral infection and are closely related with RNA metabolism. Our previous data suggested that Tudor-SN takes part in the formation of SGs, when the cells are treated with 0.5m M arsenite sodium or heat shock at 45°C, However, the precise molecular mechanism has not been fully elucidated. Here, we aimed at further studying the biological behavior of Tudor-SN granules, and the role of Tudor-SN in the stress granule aggregation dynamics.Methods: The experiment was divided into three parts, PART1:①The immunofluorescence(IF) assay and plasmids transfection-confocal microscopy analysis were performed to detect the localization of Tudor-SN and SGs/PBs. ② Fluorescence recovery after photobleaching(FRAP) assay was performed to study the kinetics of Tudor-SN granules. ③ Fluorescence loss in photobleaching(FLIP) assay was performed to study the nucleocytoplasmic trafficking nature of Tudor-SN and whether Tudor-SN granule communicates with the nuclear pool during stress. PART2: ① The AKTA purification system, Co-immunoprecipitation(Co-IP), plasmids transfection and GST-pulldown assays were performed to analyze the stress-associated Tudor-SN-containing protein complex. ② Oligo(d T) chromatography, Fluorescence in situ hybridization(FISH) and RNA-binding protein immunoprecipitation(RIP) assays were performed to detect the binding and colocalization of Tudor-SN with poly(A)+ m RNA/AGTR1-3’UTR. PART3: ①GFP-sh RNA transfection and FRAP assay were used to study the effect of Tudor-SN on the aggregation dynamics of SGs/PBs ② Tudor-SN-/- mouse embryonic fibroblasts(MEFs) were used to detect the effect of Tudor-SN on the poly(A)+ m RNA granules formation. ③si RNA transfection and sucrose gradient analysis of polysomes were performed to study the relationship between Tudor-SN and polysome disassembly; ④AGTR1-3’UTR(3’-untranslated region of angiotensin II receptor, type 1 m RNA) was used as the target gene and labeled with GFP-MS2 labeling system in the living cell. Combination of the GFP-MS2 labeling system, si RNA/ds Red-sh RNA, FRAP, IF and live-cell imaging assay was performed to study the role of Tudor-SN in the aggregation kinetics of AGTR1-3’UTR.Results: PART1: ①The endogenous Tudor-SN protein co-localizes with SGs marker proteins(Hu R, PABP1, G3BP), partially with the SGs/PBs sharing protein(AGO1/2),but not the PBs marker proteins(DCP1a, GW182). RFP-tagged Tudor-SN partially co-localizes with GFP-AGO2, but not GFP-DCP1a/b, GFP-DCP2, GW182, DCP2 or GFP control protein under stress conditions. ② The fluorescence intensity of RFP-tagged Tudor-SN granule ROI(region of interest) is reduced to ~23% of the pre-bleach intensity and then rapidly recovered to a maximum level of ~66.3% with a half-time of fluorescence recovery(t1/2) of 9.22±1.72 sec, after the photobleaching with a high-power laser. ③The fluorescence intensity of the cytosolic pulse bleach ROI is eliminated effectively, the fluorescence intensity of the cytosolic/nuclear detection ROI in the same cell is reduced significantly, after the treatment of pulse bleaching. PART2: ①Tudor-SN protein interacts with TIAR, AGO1/2, PABP1, e IF5 A, Hu R, TTP protein, but not DCP1 a protein. And the SN domain mediates the binding of Tudor-SN and PABP1, TIAR, AGO1/2 protein. ②Tudor-SN interacts with PABP1 in a partially RNA-dependent manner. In addition, Tudor-SN protein binds and colocalizes with poly(A)+ m RNA, AGTR1-3’UTR into SGs. PART3: ①When Tudor-SN is knocked down, t1/2 value of the SGs ROI increases(from 4.35±1.26 sec to 7.01±1.55 sec, P<0.05), compared with the control group, but no significant difference is observed in PBs ROI. ②Knockout of Tudor-SN impairs the efficient recruitment of poly(A)+ m RNA into SGs. ③The alteration of polysome disassembly induced by cycloheximide or puromycin can affect the Tudor-SN granules formation, however, transfection of Tudor-SN si RNA has no influence on the polysome collapse process. ④ The recombinant eukaryotic plasmid of p T7-AGTR1-3’UTR-24×MS2 is constructed successfully. AGTR1-3’UTR was labeled with GFP-MS2 labeling system in the living cell, and the SGs recruitment of the AGTR1 transcript is accompanied by the formation of Tudor-SN granules. Knockdown of Tudor-SN hinders the emergence of AGTR1-3’UTR granules and increased the t1/2 value of the AGTR1-3’UTR granule ROI(from 9.96±0.78 sec to 16.77±1.94 sec, P<0.05) in FRAP assay.Conclusions: 1) Tudor-SN, an SG-specific protein, possesses nucleocytoplasmic trafficking nature, which is associated with the fast mobility of Tudor-SN granules. 2) Tudor-SN interacts with SG-related proteins(e.g., PABP1 and TIAR) and poly(A)+ m RNA to form a complicated RNA-protein complex, participating in the cytoplasmic SGs formation. 3) Tudor-SN fails to inhibit stress-mediated polysome disassembly, but modulates the aggregation dynamics of SGs components(such as G3 BP protein, AGTR1-3’UTR) under stress conditions.