【摘要】：乙肝病毒的轉(zhuǎn)錄后調(diào)控元件(PRE)是乙肝病毒中所有的轉(zhuǎn)錄本都具有的—段重要調(diào)控元件。目前為止發(fā)現(xiàn)它的最主要功能是幫助不剪接的HBV轉(zhuǎn)錄本preS/S轉(zhuǎn)運到核外,并且近年來也發(fā)現(xiàn)PRE具有外顯子剪接增強子(ESE)的功能,可以增強HBV pgRNA的剪接。本文繼發(fā)現(xiàn)PRE的ESE功能后第一次發(fā)現(xiàn)PRE可能是一個潛在的內(nèi)含子剪接沉默子(ISS),并且其功能可能與自身局部的二級結(jié)構(gòu)和所處的位置有關(guān)。 最初通過我室以前構(gòu)造的ISS報告基因pZW8-SMN1我們發(fā)現(xiàn)乙肝病毒的PRE具有很強的抑制SMN1基因外顯子7剪接的作用。通過進一步將其序列打斷并逐漸恢復(fù)長度發(fā)現(xiàn)起ISS功能的最短序列是位于PRE3'端的一段長105堿基的片段(pre2bcd)。進一步的實驗發(fā)現(xiàn)該片段的ISS的功能具有很強的位置依賴性,并且在SMN1基因內(nèi)含子6距外顯子7的3'剪接位點82個堿基處具有最強的效果。通過序列比對我們發(fā)現(xiàn)pre2bcd上包含了已經(jīng)發(fā)現(xiàn)的PRE結(jié)合蛋白PTB所有的2個結(jié)合位點,但兩個位點的缺失以及PTB的過表達和下調(diào)對pre2bcd在SMN1中的沉默功能沒有明顯的作用。pre2bcd和其上的兩個PTB結(jié)合位點的缺失對HBV pgRNA的剪接都沒有明顯作用。核酸內(nèi)切酶印跡法(RNase footprinting assay)實驗發(fā)現(xiàn)pre2bcd可以形成比較有序的二級結(jié)構(gòu),其主要的結(jié)構(gòu)特征是兩個莖環(huán)SL1和SL2。本文的這些結(jié)果暗示pre2bcd這一功能片段很可能不同于一般的剪接順式調(diào)控元件的機制,而是在通過自身的結(jié)構(gòu)和所處的位置發(fā)揮作用。 我們的研究結(jié)果證明乙肝病毒的轉(zhuǎn)錄后調(diào)控元件(PRE)含有可變剪接抑制元件。該剪接元件抑制附近的可變剪接位點的使用。乙肝病毒在該剪接元件具有潛在的3'剪接位點(AG)。我們的結(jié)果提示乙肝病毒可能是通過抑制臨近可變剪接位點的選擇而促進遠端剪接位點選擇的。
[Abstract]:The posttranscriptional regulatory element (PRE) of hepatitis B virus is an important regulatory element in all transcripts of hepatitis B virus. Up to now, it has been found that its main function is to help the non-splicing HBV transcripts to transport to the outside nucleus. In recent years, it has also been found that PRE has the function of exon splicing enhancer (ESE), which can enhance the splicing of HBV pgRNA. After discovering the ESE function of PRE, this paper finds for the first time that PRE may be a potential intron splicing silencer (ISS), and its function may be related to its local secondary structure and location. Through the ISS reporter gene pZW8-SMN1 constructed in our laboratory we found that HBV PRE could inhibit the splicing of exon 7 of SMN1 gene. By further interrupting its sequence and gradually restoring its length, it was found that the shortest sequence of ISS function was a 105-base fragment (pre2bcd) located at the end of pre 3'. Further experiments showed that the ISS function of the fragment was highly location-dependent and had the strongest effect at the 82-base site of 3'splicing site between intron 6 and exon 7 of the SMN1 gene. By sequence comparison, we found that pre2bcd contains all the two binding sites of the PRE binding protein PTB. However, the deletion of two sites and the overexpression and down-regulation of PTB had no obvious effect on the silencing function of pre2bcd in SMN1. Neither the deletion of two PTB binding sites nor the deletion of two PTB binding sites on pre2bcd had any significant effect on the splicing of HBV pgRNA. Nucleic acid endonuclease imprinting (RNase footprinting assay) assay showed that pre2bcd could form an orderly secondary structure, the main structural characteristics of which were SL1 and SL2 of two stem rings. These results suggest that the functional fragment of pre2bcd is probably different from the conventional mechanism of splicing cis-regulating elements, but it is acting through its own structure and location. Our results suggest that (PRE), a posttranscriptional regulator of hepatitis B virus, contains variable splicing suppressor elements. The splicing element suppresses the use of nearby variable splicing sites. Hepatitis B virus has a potential 3'splicing site (AG). In the splicing element Our results suggest that HBV may promote the selection of distal splicing sites by inhibiting the selection of adjacent variable splicing sites.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R346

【共引文獻】

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1 李毅;磷酸甘油醛脫氫酶（GAPDH）對乙型肝炎病毒表面抗原表達的影響[D];重慶醫(yī)科大學;2009年

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