Function And Mechanism Of NSSR1 In Regulating NCAM L1 And GLUR-B Pre-mRNA Splicing And Apoptosis

Function and mechanism of NSSR1 in regulating NCAM L1 and GluR-B pre-mRNA splicing and apoptosisWe have previously cloned the NSSR1 cDNA and demonstrated its neural-specific expression and its potential roles in the alternative splicing of NCAM L1 and TrkC pre-mRNAs.The goal of this thesis is to continue our studies by focusing on the expression and distribution of NSSR1 during the development of central nerve system and functions of NSSR1 and its phosphorylated forms in regulating the alternative splicing of NCAM L1 and GluR-B pre-mRNAs,apoptosis and cell differentiation.We first systemically investigated the expression and distribution of NSSR1 in various tissues of mouse central nervou system at different developmental stages by Western blot analysis.The studies with whole-brain samples showed that the expression of NSSR1 proteins increases continuously during embryogenesis most significantly between E10 to E12.The expression became stable after the birth.The analysis also showed that NSSR1 expression is ubiquitous in various regions of mouse CNS.The immunohistochemical(IHC)analysis showed that in E12 embyos,NSSR1 is specifically distributed in the marginal and mantle layers but no signal in the ependymal layer observed.In addition,the expression of NSSR1 is different in neonatal and adult mouse neural tissues,for example,NSSR1 expression is very low in neonatal hippocampus but stong in adult.In cerebellar cortex,NSSR1 is widely expressed in adult purkinje and granule cell,but mainly expressed in Purkinje cell in neonatal.The observed difference of the expression and distribution of NSSR1 in neonatal and adult mouse neural tissues indicates that the expression may be closely related to the development of neurons.Such as,in granule cells in the hippocampus that develop at the latest,compared to the other brain regions,the expression of NSSR1 is barely detected in the neonates,but significant in the adults.In comparison,in the cerebellar Purkinje cells that are the ealiest formed neuron in the brain,NSSR1 expression occurs at the early stages of the development.In comparison,in the other neurons that develop later such as cerebellar granular cells,NSSR1 begins to be expressed at the stage when the cerebellar Purkinje cells are migrated to the other region of the cerebellum.In the retina,NSSR1 is cytosolic, but not nuclear as in other neural tissues.Moreover,it is only weakly expressed in ONL but much higher in adult GCL,INL and ONL.It is known that the development of retinal neurons is regulative one.The neonatal mouse retinal network is not fully developed in relative to that in aldult.We observed that in the neonatal retina,the expression of NSSR1 is extremely low and only occurs in the outnuclear layer(ONL).However,in the adult retina,NSSR1 is expressed highly in the inner nuclear layer(INL)and granular cell layer (GCL).Those results strongly suggest that NSSR1 expression is closely associated with neuronal maturation,indicating its roles in neuronal functions.Previous in vitro studies have shown that NSSR1 regulate the alternative splicing of pre-mRNA,such asβ-Globin pre-mRNA.Our studies have also demonstrated that NSSR1 moldulates the alternative splicing of endogenous NCAM L1 and Trk C pre-mRNAs,two genes important in neural development and function.In this thesis,in order to further study the neuronal function of NSSR1 in terms of regulating the alternative pre-mRNA splicing in vivo,we constructed a NCAML1 minigene.Using this minigene,we studied the function of NSSR1 in the splicing of the exon2.The result showed that in the cells expressing NSSR1 endogenously(Hela and PFSK),two spliced products(With or without the exon 2)are generated from the NCAM L1,but only one product containing the exon 2 in the cells containing no endogenous NSSR1(COS-1 and R28).Moreover,the studies with overexpression or silence of NSSR1 demonstrated that the splicing pattern of the exon 2 was altered for endogenous NCAM L1,but not for minigenes.All together,the results suggest that NSSR1 may regulate the splicing of the exon 2 via mechanisms associated with cis-elements located outsides the minigene.It means that this effect may require the cis-elements that are not included in the minigene.It is known that the function of SR proteins in regulating the alternative splicing of pre-mRNA is closely associated with their phosphorylation status.Shin et al demonstrated that heat shock causes depnosphorylation of NSSR1 and thereby inhibits the alternative splicing ofβ-Globn pre-mRNA.In order to investigate if phosphorylation status of NSSR1 alters the alternative splicing of NCAM L1 pre-mRNA,we used a similar strategy to heat shock the cells and found that the dephosphorylation of NSSR1 and exon 2 exclusion of endogenous NCAM L1 were increased or enhanced.We also further demonstated that K252a,a non-specific kinase inhibitor promotes NSSR1 overexpression-caused exon 2 exlusion of NCAM L1,a result consistent with that observed in heat shock treatment.The results suggest that NSSR1-regulated splicing of the exon 2 of NCAM L1 pre-mRNA is associated with phosphorylation-associated mechanisms.We salso tudied the function of NSSR1 in regulating the splicing of GluR-B minigene and found that NSSR1 overexpression promotes inclusion of the exon 14 (FLIP).Moreover,heat shock and K252a treatments both enhance the exon 14 inclusion in the splicing of the minigene.The results indicate that alteration of NSSR1 phosphorylation can elicit changes in splicing pattern of the exon 14.NSSR1 contains one N-terminal RNP-type RNA binding domain(RBD)and three C-terminal arginine-/serine-rich domain(RS)of various lengths and compositions.In order to understand the function of each domain in NSSR1-regulated pre-mRNA splicing, we prepared four DNA constructs with RRM,RS1,RS2 or RS3 deletion.The co-transfection studies with GluR-B minigene in COS-1 show that four domains are functionally important.In comparison with the wild-type NSSR1,the deletion of RS domains reduces the exon 14 inclusion.But,a deletion of the RRM domain increases the exon 14 inclusion,an opposite effect to RS domains.The results suggest that both protein-protein and protein-mRNA interactions are required for NSSR1 to regulate the exon 14 splicing of GluR-B minigene.The importance of alternative pre-mRNA splicing in regulating apoptosis has been recognized,such as that SC35 and ASF/SF2 has been proved to control caspase-2 pre-mRNA splicing.In the present studies,our study showed that NSSR1 reduces the apoptosis of RA/BMP4 co-induced P19 embryonal carcinoma cells but have no effect on P19 cells induced by RA only.In addition,we demonstrated that NSSR1 promotes the neural differentiation of RA/BMP4 co-induced P19 embryonal carcinoma cells and growth of neuritis.These results lead to the possibility that that NSSR1 may regulate the pre-mRNA splicing of apoptosis-assiociated genes.Currently,we are identifying the several candidate genes potentially regulated by NSSR1.In conclusion,by analyzing the expression and distribution of NSSR1 proteins during brain development,examining the effects of NSSR1 and phosphorylation on alternative pre-mRNA splicing and its structure-function relation and apoptosis,we began to demonstrate the molecular basis important in understanding the neuronal roles of NSSR1.