HuD調節(jié)cpg15表達及其機制的研究
發(fā)布時間：2019-02-17 10:53
【摘要】：HuD調節(jié)cpg15表達及其機制的研究 神經可塑性相關基因cpg15在促進神經細胞的突起生長和突觸的發(fā)育成熟,調節(jié)突觸回路的形成,神經細胞再生和可塑性等方面發(fā)揮重要作用。許多生理、病理刺激如光刺激、神經損傷或缺血缺氧都會誘導cpgl5表達。為研究cpgl5誘導表達的機制,我們首先對cpg15 mRNA序列進行分析,發(fā)現其3’非翻譯區(qū)(3'UTR)存在AU富集的序列(AU-rich elements, AREs),后者是RNA結合蛋白(RNAbinding proteins, RBPs)發(fā)揮轉錄后調節(jié)的主要靶點。HuD蛋白是一種神經元特異性的RBPs,在前體mRNA選擇性剪接、RNA轉運、穩(wěn)定性和翻譯等多個轉錄后環(huán)節(jié)調控靶基因的表達,從而在神經元的發(fā)育和維持過程中發(fā)揮重要作用。 我們的前期研究發(fā)現小鼠短暫性全腦缺血再灌注后,海馬組織中的cpg15mRNA和蛋白表達呈階段性上升趨勢,持續(xù)14天后下降,21天恢復正常水平。同時,HuD蛋白的表達也出現與cpg15同樣模式的改變,提示HuD蛋白可能和cpgl5表達存在一定的聯系。 針對HuD是否參與調節(jié)cpg15表達這一問題,我們在細胞培養(yǎng)體系中分析了HuD過量表達對cpg15表達的影響。結果發(fā)現在HuD過量表達的細胞中,cpg15mRNA和蛋白都出現表達上調,說明HuD和cpgl5的同步表達改變之間存在著調控與被調控的關系。但是,在HuD抑制表達的細胞中,cpgl5表達沒有出現明顯的改變,可能因為抑制HuD的效果被Hu蛋白家族其他成員代償所致。 為了研究cpg15基因上的核苷酸序列ARE在HuD調節(jié)cpgl5表達中的作用,我們分析了HuD對含有cpg15調節(jié)序列ARE的報告基因表達的影響。結果發(fā)現,HuD過量表達顯著升高含3'UTR的報告基因的表達,而不能增加缺失ARE序列的報告基因的表達。而且,ARE序列缺失后,cpg15 mRNA和蛋白表達都不受HuD過表達的影響。這些結果說明ARE在HuD調節(jié)cpgl5表達中起重要作用。 我們接著采用免疫沉淀研究了ARE序列在介導cpg15 mRNA與HuD蛋白結合中的重要性,結果發(fā)現ARE序列介導cpg15 mRNA與HuD蛋白結合,但不是唯一序列。 我們還初步探討了HuD的結構域對cpgl5表達的影響。不論與mRNA穩(wěn)定性相關的RRM1和RRM2 (RNA-recognition motifs)的缺失、還是與poly(A)結合及翻譯起始有關的Hinge region與RRM3的缺失,都減弱了HuD增加GFP-CPG15表達的能力,而且Hinge region與RRM3缺失對GFP-CPG15誘導表達的抑制作用更明顯,說明mRNA穩(wěn)定性和翻譯調節(jié)都參與cpg15表達調控。 綜合以上結果,本論文結果說明HuD通過cpg15 mRNA3'UTR區(qū)的ARE序列調節(jié)cpg15基因表達,這一過程與HuD參與cpg15 mRNA穩(wěn)定性和翻譯調節(jié)有關。
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363
【共引文獻】
相關期刊論文 前4條
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;張雋;衛(wèi)國;劉媛媛;王海昌;;TNF-α刺激下RNA結合蛋白HuR在大鼠心臟成纖維細胞中的表達變化[J];心臟雜志;2011年06期
4 孫淑娜;桂永浩;蔣t，
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