乙肝病毒转录后调控元件PRE的结构与剪接调控功能的研究
发布时间：2018-08-17 17:04
【摘要】：乙肝病毒的转录后调控元件(PRE)是乙肝病毒中所有的转录本都具有的—段重要调控元件。目前为止发现它的最主要功能是帮助不剪接的HBV转录本preS/S转运到核外,并且近年来也发现PRE具有外显子剪接增强子(ESE)的功能,可以增强HBV pgRNA的剪接。本文继发现PRE的ESE功能后第一次发现PRE可能是一个潜在的内含子剪接沉默子(ISS),并且其功能可能与自身局部的二级结构和所处的位置有关。 最初通过我室以前构造的ISS报告基因pZW8-SMN1我们发现乙肝病毒的PRE具有很强的抑制SMN1基因外显子7剪接的作用。通过进一步将其序列打断并逐渐恢复长度发现起ISS功能的最短序列是位于PRE3'端的一段长105碱基的片段(pre2bcd)。进一步的实验发现该片段的ISS的功能具有很强的位置依赖性,并且在SMN1基因内含子6距外显子7的3'剪接位点82个碱基处具有最强的效果。通过序列比对我们发现pre2bcd上包含了已经发现的PRE结合蛋白PTB所有的2个结合位点,但两个位点的缺失以及PTB的过表达和下调对pre2bcd在SMN1中的沉默功能没有明显的作用。pre2bcd和其上的两个PTB结合位点的缺失对HBV pgRNA的剪接都没有明显作用。核酸内切酶印迹法(RNase footprinting assay)实验发现pre2bcd可以形成比较有序的二级结构,其主要的结构特征是两个茎环SL1和SL2。本文的这些结果暗示pre2bcd这一功能片段很可能不同于一般的剪接顺式调控元件的机制,而是在通过自身的结构和所处的位置发挥作用。 我们的研究结果证明乙肝病毒的转录后调控元件(PRE)含有可变剪接抑制元件。该剪接元件抑制附近的可变剪接位点的使用。乙肝病毒在该剪接元件具有潜在的3'剪接位点(AG)。我们的结果提示乙肝病毒可能是通过抑制临近可变剪接位点的选择而促进远端剪接位点选择的。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
本文编号：2188314
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
【共引文献】
相关硕士学位论文 前1条
1 李毅;磷酸甘油醛脱氢酶（GAPDH）对乙型肝炎病毒表面抗原表达的影响[D];重庆医科大学;2009年
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