【作者】 彭正羽；

【导师】 徐平；

【作者基本信息】 复旦大学 ， 生理学及生物物理学， 2006， 博士

【摘要】 NSSR1在NCAML1、GluR-B前体mRNA剪接和细胞凋亡中的作用及机制本课题组在以往的研究中,克隆了NSSR1基因并发现它在神经系统中的特异分布以及它可以调节NCAM L1和Trk C的前体mRNA可变剪接。本论文在此基础上研究了NSSR1基因在小鼠中枢神经系统发育过程中的表达分布,NSSR1蛋白及其去磷酸化对前体mRNA可变剪接的调控以及它在P19细胞凋亡及分化中的功能。我们首先系统的研究了NSSR1基因在小鼠中枢神经系统各组织在发育过程中的表达以及分布情况,不同发育时期的小鼠全脑组织Westem-blot结果显示,NSSR1蛋白随着胚胎发育其表达也不断增加,其中增加最明显的时间在E10-E12之间,在出生后NSSR1的表达趋于稳定。大脑不同部位组织的Western—blot分析显示NSSR1在中枢神经系统的各组分中都有表达。通过对新生小鼠和成年小鼠不同组织的IHC结果显示,NSSR1在新生小鼠大脑和成年鼠大脑中的表达分布有着较明显的不同。在海马中,新生小鼠大脑海马中的NSSR1表达较低而成鼠的表达显著增强。在成鼠小脑中,NSSR1广泛分布在小脑皮质包括浦肯野氏等神经元细胞中,而在新生鼠中主要在浦肯野氏细胞中表达。NSSR1在新生鼠和成鼠中枢神经系统中表达的差异与神经元的发育是密切相关的,海马颗粒细胞在发育时序上是最晚形成的神经元,因此在新生鼠海马颗粒细胞中几乎检测不到NSSR1的表达,而在成鼠海马中,NSSR1的表达非常强烈。而小脑浦肯野氏细胞是小脑中最早产生的神经元,因此在发育早期NSSR1就有明显的表达,然后随着神经元细胞的迁移,小脑皮层的其它神经元如颗粒细胞也开始表达NSSR1蛋白。与其它神经组织中NSSR1主要在细胞核内表达不同,视网膜中NSSR1主要表达在细胞质中。此外,NSSR1仅在新生鼠ONL中有少量表达,随着发育NSSR1蛋白开始在成鼠INL、GCL层表达。视网膜中神经元的发育属于调节型发育(regulative development),新生小鼠视网膜的神经网络比较简单,然后随着发育其复杂程度不断提高,NSSR1的表达与此一致。以往的研究(多数为体外剪接试验)显示NSSR1可以调节β-Globin等基因前体mRNA的可变剪接,本课题组也发现NSSR1能够调节两个在神经发育和功能中发挥重要作用的基因—内源性NCAM L1和Trk C的前体mRNA剪接,为了深入研究NSSR1在体内对前体mRNA剪接的调节作用,我们首先构建了NCAM L1小基因,然后研究了NSSR1对该小基因exon2的剪接调节作用。在内源表达NSSR1的Hela、PFSK细胞中,小基因被剪接产生两个亚型(含exon2与不含exon2),而无内源NSSR1的COS-1、R28细胞内则只有一个亚型(含exon2)。另外,过表达NSSR1或siRNA也已经证明可以影响内源性NCAM L1基因exon2的剪接。然而接下来我们过表达NSSR1及其siRNA的实验结果表明,NCAM L1小基因的可变剪接不受NSSR1的调控。我们的结果提示NSSR1在调节NCAMLl基因exon2的剪接时可能需要我们构建的NCAM L1小基因外的顺式元件的参与。在前体mRNA剪接过程中,SR蛋白的磷酸化与其功能有密切关系。Shin等人的研究发现热休克可以使NSSR1去磷酸化从而抑制β-Globin等基因前体mRNA的可变剪接。为了研究NSSR1的磷酸化状态对内源性NCAM L1基因可变剪接的影响,我们对PFSK细胞热休克处理,结果发现NSSR1蛋白的去磷酸化形式增加,同时热休克后,过表达NSSR1可以增加内源NCAM L1基因不含Exon2的剪接亚型的产生。我们的结果说明在热休克后,NSSR1也可以通过调节被剪接基因不同剪接亚型的产生来帮助细胞进入应激状态。接下来我们用非特异的激酶抑制剂K252a处理转染的PFSK细胞,结果发现K252a也可以促使NSSR1蛋白向去磷酸化状态转变,同时PFSK细胞在K252a处理后,过表达NSSR1可以增加内源NCAML1基因不含Exon2的剪接亚型的剪接,证明用非特异的激酶抑制剂K252a处理转染的PFSK细胞,其对NSSR1蛋白的影响与用热休克处理细胞一致。提示NSSR1的磷酸化状态可以影响前体mRNA的剪接。同时我们也研究了NSSR1对GluR-B小基因前体mRNA剪接的调节作用,发现NSSR1过表达可以促进GluR-B小基因FLIP亚型(含Exon 14)的剪接。而热休克以及K252a处理都促进GluR-B小基因Trruncate亚型(不含exon14、15)的剪接,说明NSSR1磷酸化状态的改变同样影响GluR-B小基因的剪接。为研究NSSR1各功能域在调节可变剪接中的功能,我们用分别缺失RRM、RS1、RS2、RS3等功能域的缺失突变克隆与GluR-B小基因共转染COS-1细胞,结果发现NSSR1的各功能域在调节前体mRNA的可变剪接中都有作用,与野生型NSSR1比,三个RS功能域的分别缺失都使GluR-B小基因FLIP亚型的剪接减少,而RRM功能域的缺失则使GluR-B小基因FLIP亚型的剪接增加。前体mRNA剪接在细胞的程序性死亡中发挥重要作用,作为一个有着重要功能的SR蛋白,NSSR1可能在其中发挥作用。我们的研究发现,NSSR1可以抑制由RA/BMP4联合诱导的P19细胞凋亡,但RA单独作用没有效果,进一步的研究发现,NSSR1可以促进由RA/BMP4联合诱导的P19向神经元方向的分化,并且促进其神经突起的生长。在调节细胞凋亡的过程中,NSSR1有可能通过调节凋亡相关蛋白的可变剪接来发挥作用,目前我们正在对此可能性进行验证。最后,我们建立了NSSR1的昆虫表达体系,在果蝇细胞系S2细胞中成功的表达了NSSR1蛋白,为将来进行体外剪接等研究打下基础。综合本文结果,我们通过小鼠中枢神经系统发育过程中的NSSR1蛋白表达特征、脑发育过程中的表达和分布变化、NSSR1蛋白去磷酸化后对前体mRNA剪接的调控以及NSSR1对细胞凋亡和分化的调控等方面的研究结果,提示了NSSR1在神经发育中及调节前体mRNA可变剪接和细胞凋亡中具有重要作用。更多还原

【Abstract】 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.更多还原