多巴胺受体DRD1 mRNA 3’ UTR在DRD1转录后水平表达调控中的作用

【作者】 赵理；

【导师】 强伯勤； 袁建刚； 彭小忠；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 DRD1(dopamine receptor D1)是多巴胺受体的一种,是由七个跨膜区域(7-GM)组成的G蛋白偶联受体。DRD1广泛分布于神经系统,特别是尾壳核(Cpu),伏隔核(Act),视束(OT),脑皮层(Cx)和杏仁核。DRD1通过正调控cAMP,激活蛋白激酶A,从而激活下游信号通路和细胞膜离子通道。研究表明DRD1在神经前体细胞的增殖与分化中发挥重要作用,另外DRD1受体的表达异常与药物成瘾,帕金森综合症等多种神经系统疾病有密切关系。HuD是ELAVL(embryonic lethal abnormal visual like)/Hu家族中的一员,在神经系统中特异表达。HuD通过其蛋白含有的三个RRM结构域识别和结合靶mRNA 3’UTR中的ARE元件,增加靶mRNA稳定性,引导靶mRNA胞内运输和调控靶mRNA翻译。HuD在神经突起的生长与修复,神经干细胞和神经前体细胞的细胞周期调控等方面发挥重要作用。HuD表达异常与多种神经系统疾病相关。ARE元件是一种重要的顺式作用元件,富含A和U,参与介导mRNA的降解。Tala等人在ARED3.0数据库中汇集了所有NCBI收录的mRNA 3’UTR上含有ARE元件的基因。通过检索ARED3.0数据库,我们发现DRD1 mRNA的3’UTR上具有一个进化保守的ARE元件。通过Real-time PCR实验,我们发现RA诱导后向神经元方向分化的P19细胞中DRD1和HuD呈同时上升的趋势,这提示我们HuD有可能识别DRD1 mRNA 3’UTR的ARE元件,并由此调控DRD1基因的表达。我们首先应用RNA-chip方法进行了检测。利用HuD抗体在P19细胞全蛋白裂解液中捕获HuD蛋白-RNA复合体,然后通过链霉亲和素珠从细胞全蛋白裂解液中分离纯化出抗体-HuD蛋白-RNA复合体。经过酚氯仿抽提出其中的RNA后,通过逆转录得到相应的cDNA,最后利用特异性引物通过PCR扩增出特异条带。发现在P19细胞中HuD蛋白结合DRD1 mRNA。随后通过REMSA实验,证实HuD蛋白识别并结合DRD1 mRNA 3’UTR上的ARE元件。说明HuD通过识别和结合DRD1mRNA 3’UTR上的ARE元件来实现对DRD1 mRNA的调控。通过荧光素酶报告实验,我们发现HuD蛋白能够上调DRD1基因的表达。将野生型DRD1 3’UTR序列插入报告质粒荧光素酶基因编码区下游,用报告基因重组质粒和HuD真核瞬时表达载体共同转染293ET细胞,我们发现与转染空质粒的对照组相比,带有野生型DRD1 3’UTR序列的荧光报告素酶活性有明显的上升。Real-time PCR实验表明过表达HuD蛋白后,含有野生型DRD13’UTR的荧光报告素酶转录本数量明显高于对照组。在P19细胞中过表达HuD蛋白后,Real-time PCR实验表明DRD1 mRNA也出现了上升。这说明HuD蛋白识别和结合靶基因mRNA 3’UTR的ARE元件后增加DRD1 mRNA的稳定性,从而促使靶基因表达上调。最后我们还发现HuD蛋白影响miR-504对DRD1的上调作用。受到W.Huang等在HEK293细胞中观察到has-miR-504可以上调人源DRD1表达的报道的提示,我们在293ET细胞中观察到过表达mmu-miR-504可以上调小鼠源DRDl的表达。我们将miR-504瞬时表达载体和带有DRD1 3’UTR的荧光报告素酶表达质粒共同转染293E T细胞,24小时后荧光报告素酶表达出现显著上升;而如果将HuD瞬时表达载体与前二者同时转染293ET细胞,24小时后荧光报告素酶的上调程度会受到明显抑制。这是第一次发现HuD对靶基因的调控涉及miRNA。综上所述,我们发现DRD1 3’UTR上的ARE元件能够被HuD蛋白识别和结合,从而增加DRD1 mRNA的稳定性,提高DRD1在细胞中的表达。另外,miR-504可能影响HuD蛋白对于DRD1的表达调控。更多还原

【Abstract】 DRD1 (dopamine receptor D1) is one of dopamine receptors, and is G-protein-coupled receptor constituted by 7 membrane spaning domain. DRD1 widely expresses in nerve sysytem, especially in dorsal caudate putamen, nucleus accumbens septi, optic lemniscus, cortex and amygdaloid nucleus. When DRD1 is actived , it upregulates cAMP ,which then actives protein kinase A, and downstream signal pathways and ion channels. DRD1 has been reported to be important for nerve system’s functions, such as movement, study and memory. Abnormal expression of DRD1 has been reported to be associated with many never system diseases, such as drug addiction and Parkinson disease.HuD is a member of ELAVL(embryonic lethal abnormal visual like )/Hu family, which specially express in neuronal system. HuD protein recognises and bind to ARE element by its three RRM motifs, and increases target mRNA stability, involves in transport and translation of target mRNA. HuD plays important roles in outgrow and repair of neurite, regulation of cell cycles of neuron stem cell and neuron precursor cell.ARE elements are important cis-acting elements located in mRNA 3’UTR, which are rich of A and U, and are determinant of RNA instability. According to records of NCBI, Tala et al have assembled all genes which have ARE elements in their 3’UTR to ARED 3.0 database. And we found there was a typical ARE element in DRD1 mRNA’s 3’UTR and this ARE is highly conservative in evolution. We found that DRD1 mRNA and HuD mRNA both increased during the differentation of RA induced P19 cell by Real-time PCR. It means HuD protein may recognize the ARE element of DRD1 mRNA 3’UTR and regulat the expression of DRD1.At first, we proved our hypothesis by RNA-chip experiment. We utilized antibody of HuD to capture ’HuD protein-RNA’ complex in cytoplasm lysate, and utilized beads with streptavidin to pure this complex, then extracted RNA by guanidnium-Phenol Chloroform, finally we get cDNAs by RT-PCR. We used special PCR primer to amplify special band, and confirm HuD protein bind to DRD1 mRNA in P19 cell. Then we comfirm HuD protein recognize and bind ARE element of DRD1 mRNA 3’UTR by REMSA. This indicated HuD protein bind to DRD1 mRNA by recognize ARE element of DRD1 mRNA 3’UTR.In the dual-luciferase reporter gene experiment in 293ET cell, we found HuD can upregulate DRD1 expression. We inserted wild DRD1 3’UTR to the downstream of the CDS of luciferase. Overexpressed it with HuD protein in 293ET cell, we found the actitivation of luciferase increased. And Real-time PCR revealed the transcripts of wild DRD1 3’UTR increased in the same time. It indicated HuD increase the stability of DRD1 mRNA by recognizing and binding to the ARE element of DRD1 3’UTR, and increase its expression.Finally we found HuD affect the regulation of miR-504 to DRD1. As W.Huang observed in HEK293 cell that has-miR-504 can increase the DRD1, we observed mmu-miR-504 increase DRD1 in 293ET. When we overexpressed miR-504 or HuD alone, the activiation of luciferase increased, but when we overexpressed both HuD protein and miR-504 in 293ET cell, the upregulation of activiation of luciferase was depressed obviously. This is the firs report about the effect between HuD and microRNA.As described above, we found there is an ARE element located in DRD1 3’UTR. HuD protein recognizes and binds to the ARE element, increase the stability of DRD1 mRNA, and upregulate the expression of DRD1. Besids, we found HuD effects the regulation of miR-504 to the mRNA 3’UTR of DRD1.更多还原