Effect Of Molecular Mutations Of Cold Shock Protein RBM3 On Its Nuclear Localization And Neuroprotective Roles

BackgroundThe RNA-binding motif protein 3(RBM3)is a cold shock protein that is mainly distributed in the nucleus.It has many important biological functions,such as neuroprotection.However,the association between RBM3 molecule and the neuroprotective effect is unclear.It has been shown that some RNA-binding proteins bearing RNA binding motifs(RRM)or arginine-glycine-glycine-rich domains(RGG)are implicated in various biological processes,such as subcellular localization,gene transcription,mRNA translation,and apoptosis.Meanwhile,the posttranslational modification(PTM)of RNA-binding proteins,such as phosphorylation or methylation,plays an important role in fulfilling their biological functions.RBM3 consists of two RRM domains at N-terminus and two RGG domains at C-terminus,along with some potential PTM sites in RGG domains.However,the molecular function of aboved-mentioned structures remains largely unknown.In this study,molecular mutants of RBM3 protein in different domains and potential PTM sites were developed,in order to investigate their effects on RBM3 subcellular localization and neuroprotective function.Objectives1.To explore the effect of various RBM3 mutants on its cellular localization.2.To evaluate the effect of various RBM3 mutants on its neuroprotective role.Methods1.Construction of recombinant plasmids containing mutations in different domains and potential PTM sites of RBM3.Constructionof plasmids of mutations of RBM3 with RRM or RGG domains deleted(pXJ40-myc-RGG,pXJ40-myc-RRM)and mutations of RBM3 with arginines deleted(pXJ40-myc-RGG 1 mut,pXJ40-myc-RGG 2 mut).2.Analysis of the effect of different mutations of RBM3 on its cell localizationSH-SY5Y cells were cultured at 37~oC for 24 h,and transfected with different plasmids for 48 hours.Followed by detection of the cellular localization of different mutants with Western blotting and immunofluorescence.Cells transfected with plasmids pXJ40-myc or pXJ40-myc-RBM3 were used as controls.3.Detection of effects of RBM3 mutations on its neuroprotective functionRBM3 mutant plasmids were transfected into SH-SY5Y cells for 48 h,respectively.Apoptosis was induced by the all-trans retinoic acid(RA).Western blotting was used to detect the protein expression level of apoptic marker,cleaved PARP,in order to determine the effect of RBM3 mutations on its neuroprotective function.Results1.Eight different RBM3 mutants were successfully constructed2.RBM3 protein with RRM deletion or RGG deletion was mainly localized in the cytoplasm.In addtion,RBM3 with potential arginine methylation site deletion(AA87/90or AA99/105)was also increased in the distribution of cytoplasm.In contrast,mutations in potential phosphorylation sites of Ser102?Tyr129?Ser147?Tyr155 did not affect the subcellular distrubtion of RBM3 protein.3.Deletions in either RRM domain(N)or RGG domain(C)of RBM3 impaired the neuroprotective effects of RBM3 against RA-induced apoptosis in SH-SY5Y cells.In contrast,Arg99/105 replacement by Ala completely abrogated the neuroprotective role of RBM3.Surprisingly,Arg87/90 replacement by Ala even boosted the neuroprotective role of RBM3.Conclusion1.Both RRM and RGG domain of RBM3 contain nuclear localization signals of RBM3 protein.2.Potential arginine methylation sites AA87/90 and AA99/105 cooperatively determine the nuclear localization of RBM3.3.Both RRM and RGG motif are required for the neuroprotective effect of RBM3protein.Arg99/105 sites are also involved in the neuroprotective role of RBM3,while Arg87/90 may have inhibitory effects on neuroprotective effect of RBM3.Our finding that Arg87/90 mutation boosts the neuroprotective capacity of RBM3 protein,provides important clues for further revealing the molecular mechanism underlying RBM3-conferred neuroprotection.