HuD调节cpg15表达及其机制的研究
发布时间：2019-02-17 10:53
【摘要】：HuD调节cpg15表达及其机制的研究 神经可塑性相关基因cpg15在促进神经细胞的突起生长和突触的发育成熟,调节突触回路的形成,神经细胞再生和可塑性等方面发挥重要作用。许多生理、病理刺激如光刺激、神经损伤或缺血缺氧都会诱导cpgl5表达。为研究cpgl5诱导表达的机制,我们首先对cpg15 mRNA序列进行分析,发现其3’非翻译区(3'UTR)存在AU富集的序列(AU-rich elements, AREs),后者是RNA结合蛋白(RNAbinding proteins, RBPs)发挥转录后调节的主要靶点。HuD蛋白是一种神经元特异性的RBPs,在前体mRNA选择性剪接、RNA转运、稳定性和翻译等多个转录后环节调控靶基因的表达,从而在神经元的发育和维持过程中发挥重要作用。 我们的前期研究发现小鼠短暂性全脑缺血再灌注后,海马组织中的cpg15mRNA和蛋白表达呈阶段性上升趋势,持续14天后下降,21天恢复正常水平。同时,HuD蛋白的表达也出现与cpg15同样模式的改变,提示HuD蛋白可能和cpgl5表达存在一定的联系。 针对HuD是否参与调节cpg15表达这一问题,我们在细胞培养体系中分析了HuD过量表达对cpg15表达的影响。结果发现在HuD过量表达的细胞中,cpg15mRNA和蛋白都出现表达上调,说明HuD和cpgl5的同步表达改变之间存在着调控与被调控的关系。但是,在HuD抑制表达的细胞中,cpgl5表达没有出现明显的改变,可能因为抑制HuD的效果被Hu蛋白家族其他成员代偿所致。 为了研究cpg15基因上的核苷酸序列ARE在HuD调节cpgl5表达中的作用,我们分析了HuD对含有cpg15调节序列ARE的报告基因表达的影响。结果发现,HuD过量表达显著升高含3'UTR的报告基因的表达,而不能增加缺失ARE序列的报告基因的表达。而且,ARE序列缺失后,cpg15 mRNA和蛋白表达都不受HuD过表达的影响。这些结果说明ARE在HuD调节cpgl5表达中起重要作用。 我们接着采用免疫沉淀研究了ARE序列在介导cpg15 mRNA与HuD蛋白结合中的重要性,结果发现ARE序列介导cpg15 mRNA与HuD蛋白结合,但不是唯一序列。 我们还初步探讨了HuD的结构域对cpgl5表达的影响。不论与mRNA稳定性相关的RRM1和RRM2 (RNA-recognition motifs)的缺失、还是与poly(A)结合及翻译起始有关的Hinge region与RRM3的缺失,都减弱了HuD增加GFP-CPG15表达的能力,而且Hinge region与RRM3缺失对GFP-CPG15诱导表达的抑制作用更明显,说明mRNA稳定性和翻译调节都参与cpg15表达调控。 综合以上结果,本论文结果说明HuD通过cpg15 mRNA3'UTR区的ARE序列调节cpg15基因表达,这一过程与HuD参与cpg15 mRNA稳定性和翻译调节有关。
[Abstract]:HuD regulates the expression of cpg15 and its mechanisms; cpg15 plays an important role in promoting neurite growth and synaptic development and maturation, regulating synaptic circuit formation, nerve cell regeneration and plasticity. Many physiological and pathological stimuli such as light stimulation, nerve injury or ischemia and hypoxia can induce cpgl5 expression. In order to study the mechanism of cpgl5 induced expression, we first analyzed the cpg15 mRNA sequence and found that its 3 'untranslated region (3'UTR) contained a AU enriched sequence (AU-rich elements, AREs), which is a RNA binding protein (RNAbinding proteins,). RBPs) plays a major role in posttranscriptional regulation. HuD protein is a neuron-specific RBPs, that regulates the expression of target genes in multiple post-transcriptional links, such as selective splicing of precursor mRNA, RNA transport, stability and translation. It plays an important role in the development and maintenance of neurons. Our previous study found that the expression of cpg15mRNA and protein in hippocampal tissue of mice increased gradually after transient global cerebral ischemia-reperfusion, decreased after 14 days, and returned to normal level at 21 days. At the same time, the expression of HuD protein changed the same pattern as cpg15, suggesting that HuD protein may be related to the expression of cpgl5. In view of whether HuD is involved in regulating the expression of cpg15, we analyzed the effect of overexpression of HuD on the expression of cpg15 in cell culture system. The results showed that the expression of cpg15mRNA and protein were up-regulated in the overexpression of HuD, indicating that there was a regulated and regulated relationship between the changes in the synchronous expression of HuD and cpgl5. However, there was no significant change in the expression of cpgl5 in the cells inhibited by HuD, which may be due to the compensatory effect of the inhibition of HuD by other members of the Hu protein family. In order to study the role of nucleotide sequence ARE on cpg15 gene in the regulation of cpgl5 expression by HuD, we analyzed the effect of HuD on the expression of reporter gene containing cpg15 regulatory sequence ARE. The results showed that overexpression of HuD significantly increased the expression of reporter gene containing 3'UTR, but could not increase the expression of reporter gene with missing ARE sequence. Moreover, the expression of cpg15 mRNA and protein was not affected by the overexpression of HuD after the deletion of ARE sequence. These results suggest that ARE plays an important role in the regulation of cpgl5 expression by HuD. We then used immunoprecipitation to study the importance of ARE sequence in mediating the binding of cpg15 mRNA to HuD protein. It was found that ARE sequence mediates the binding of cpg15 mRNA to HuD protein, but it is not the only sequence. We also discussed the effect of HuD domain on cpgl5 expression. Both the absence of RRM1 and RRM2 (RNA-recognition motifs) associated with mRNA stability and the absence of Hinge region and RRM3 associated with poly (A) binding and translation initiation weakened HuD's ability to increase GFP-CPG15 expression. Moreover, the inhibition of GFP-CPG15 expression induced by Hinge region and RRM3 deletion was more obvious, indicating that both mRNA stability and translation regulation were involved in the regulation of cpg15 expression. Combined with the above results, it is suggested that HuD regulates the expression of cpg15 gene through the ARE sequence of the cpg15 mRNA3'UTR region, which is related to the involvement of HuD in cpg15 mRNA stability and translation regulation.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
[Abstract]:HuD regulates the expression of cpg15 and its mechanisms; cpg15 plays an important role in promoting neurite growth and synaptic development and maturation, regulating synaptic circuit formation, nerve cell regeneration and plasticity. Many physiological and pathological stimuli such as light stimulation, nerve injury or ischemia and hypoxia can induce cpgl5 expression. In order to study the mechanism of cpgl5 induced expression, we first analyzed the cpg15 mRNA sequence and found that its 3 'untranslated region (3'UTR) contained a AU enriched sequence (AU-rich elements, AREs), which is a RNA binding protein (RNAbinding proteins,). RBPs) plays a major role in posttranscriptional regulation. HuD protein is a neuron-specific RBPs, that regulates the expression of target genes in multiple post-transcriptional links, such as selective splicing of precursor mRNA, RNA transport, stability and translation. It plays an important role in the development and maintenance of neurons. Our previous study found that the expression of cpg15mRNA and protein in hippocampal tissue of mice increased gradually after transient global cerebral ischemia-reperfusion, decreased after 14 days, and returned to normal level at 21 days. At the same time, the expression of HuD protein changed the same pattern as cpg15, suggesting that HuD protein may be related to the expression of cpgl5. In view of whether HuD is involved in regulating the expression of cpg15, we analyzed the effect of overexpression of HuD on the expression of cpg15 in cell culture system. The results showed that the expression of cpg15mRNA and protein were up-regulated in the overexpression of HuD, indicating that there was a regulated and regulated relationship between the changes in the synchronous expression of HuD and cpgl5. However, there was no significant change in the expression of cpgl5 in the cells inhibited by HuD, which may be due to the compensatory effect of the inhibition of HuD by other members of the Hu protein family. In order to study the role of nucleotide sequence ARE on cpg15 gene in the regulation of cpgl5 expression by HuD, we analyzed the effect of HuD on the expression of reporter gene containing cpg15 regulatory sequence ARE. The results showed that overexpression of HuD significantly increased the expression of reporter gene containing 3'UTR, but could not increase the expression of reporter gene with missing ARE sequence. Moreover, the expression of cpg15 mRNA and protein was not affected by the overexpression of HuD after the deletion of ARE sequence. These results suggest that ARE plays an important role in the regulation of cpgl5 expression by HuD. We then used immunoprecipitation to study the importance of ARE sequence in mediating the binding of cpg15 mRNA to HuD protein. It was found that ARE sequence mediates the binding of cpg15 mRNA to HuD protein, but it is not the only sequence. We also discussed the effect of HuD domain on cpgl5 expression. Both the absence of RRM1 and RRM2 (RNA-recognition motifs) associated with mRNA stability and the absence of Hinge region and RRM3 associated with poly (A) binding and translation initiation weakened HuD's ability to increase GFP-CPG15 expression. Moreover, the inhibition of GFP-CPG15 expression induced by Hinge region and RRM3 deletion was more obvious, indicating that both mRNA stability and translation regulation were involved in the regulation of cpg15 expression. Combined with the above results, it is suggested that HuD regulates the expression of cpg15 gene through the ARE sequence of the cpg15 mRNA3'UTR region, which is related to the involvement of HuD in cpg15 mRNA stability and translation regulation.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
【共引文献】
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1 ZHOU HuaLin;MANGELSDORF Marie;LIU JiangHong;ZHU Li;WU Jane Y;;RNA-binding proteins in neurological diseases[J];Science China(Life Sciences);2014年04期
2 Andrii Vislovukh;Thaiz Rivera Vargas;Anna Polesskaya;Irina Groisman;;Role of 3'-untranslated region translational control in cancer development, diagnostics and treatment[J];World Journal of Biological Chemistry;2014年01期
3 柏丹娜;高群;高延;张隽;卫国;刘媛媛;王海昌;;TNF-α刺激下RNA结合蛋白HuR在大鼠心脏成纤维细胞中的表达变化[J];心脏杂志;2011年06期
4 孙淑娜;桂永浩;蒋t，
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