本文主要研究内容
作者柴杨丽（2019）在《原生动物八肋游仆虫NMD途径UPF因子的功能分析》一文中研究指出：基因的准确表达对于机体细胞正常的生理活动至关重要,它受到了细胞中多种不同分子机制的调控。其中,无义介导的mRNA降解途径(nonsense-mediated mRNA decay,NMD),它作为一种真核生物基因表达质量控制机制,能够识别并降解那些含有提前终止密码子(premature termination codons,PTCs)的无义mRNA。mRNA降解依赖于细胞内蛋白质翻译过程,目前已知可以调节植物、动物、真菌和纤毛虫中转录物的表达水平。无义mRNA的识别是NMD机制的核心,UPF(up-frameshift)蛋白质是识别和降解无义mRNA的一类关键因子。前期研究表明,在八肋游仆虫(Euplotes octocarinatus)细胞中仅有UPF1和UPF2两种UPF蛋白质,没有发现存在于人和酵母等细胞中的UPF3。生物信息学分析显示,八肋游仆虫的UPF2(EoUPF2)包含三个串联的MIF4G(middle domain of translation initiation factor 4G)结构域和一个C末端UPF1的结合区域。大多数真核生物共享核心NMD因子,而NMD途径的分子机制在生物体之间却有所不同。本文中,首先对EoUPF1和其他真核生物UPF1共26个物种,以及EoUPF2和其他真核生物UPF2共24个物种的氨基酸序列进行比对,分别构建系统进化树,从它们的进化关系分析发现,EoUPF1与EoUPF2均与酿酒酵母(Saccharomyces cerevisiae)的进化关系较为接近。利用InterPro数据库分析,结果显示二者的核心结构域非常相似。其次,通过pull down实验验证了EoUPF1和EoUPF2之间、以及各自与第二类肽链释放因子eRF3(eukaryotic polypeptide release factor 3)之间,存在的相互作用关系,我们推测在八肋游仆虫细胞中,EoUPF1、EoUPF2与eRF3形成eRF3/EoUPF1/EoUPF2复合体。最后,我们进一步通过酵母双杂交和pull down实验证实,EoUPF2的MIF4G结构域与外显子连接复合体(exon-exon junction complex,EJC)组成因子Y14a/Y14b可以相互作用。在高等真核细胞中,UPF3介导UPF2与EJC核心因子Y14的相互作用,而八肋游仆虫细胞中没有UPF3因子。那么,EoUPF2在没有UPF3存在的情况下如何发挥功能呢?本研究证实,EoUPF2通过其MIF4G结构直接与EJC组成因子Y14a和Y14b相互作用。在β-半乳糖苷酶实验中,我们发现EoUPF2与Y14a的作用更强一些;而实时荧光定量PCR分析结果排除了EoUPF2与Y14a和Y14b相互作用强弱与Y14a和Y14b本身的拷贝数有关。由此,我们针对八肋游仆虫细胞的NMD途径启动机制提出假设:核糖体翻译过程中遇到提前终止密码子(PTC)时会停下来,eRF3与EoUPF1、EoUPF2相互作用,然后形成了eRF3/EoUPF1/EoUPF2复合体。而EoUPF2通过与EoUPF1和Y14相互作用,介导了eRF3/EoUPF1/EoUPF2复合体与EJC的耦联,启动了无义mRNA的识别过程。本文的实验结果,可以为进一步阐明八肋游仆虫NMD机制提供支持。
Abstract
ji yin de zhun que biao da dui yu ji ti xi bao zheng chang de sheng li huo dong zhi guan chong yao ,ta shou dao le xi bao zhong duo chong bu tong fen zi ji zhi de diao kong 。ji zhong ,mo yi jie dao de mRNAjiang jie tu jing (nonsense-mediated mRNA decay,NMD),ta zuo wei yi chong zhen he sheng wu ji yin biao da zhi liang kong zhi ji zhi ,neng gou shi bie bing jiang jie na xie han you di qian zhong zhi mi ma zi (premature termination codons,PTCs)de mo yi mRNA。mRNAjiang jie yi lai yu xi bao nei dan bai zhi fan yi guo cheng ,mu qian yi zhi ke yi diao jie zhi wu 、dong wu 、zhen jun he qian mao chong zhong zhuai lu wu de biao da shui ping 。mo yi mRNAde shi bie shi NMDji zhi de he xin ,UPF(up-frameshift)dan bai zhi shi shi bie he jiang jie mo yi mRNAde yi lei guan jian yin zi 。qian ji yan jiu biao ming ,zai ba le you pu chong (Euplotes octocarinatus)xi bao zhong jin you UPF1he UPF2liang chong UPFdan bai zhi ,mei you fa xian cun zai yu ren he jiao mu deng xi bao zhong de UPF3。sheng wu xin xi xue fen xi xian shi ,ba le you pu chong de UPF2(EoUPF2)bao han san ge chuan lian de MIF4G(middle domain of translation initiation factor 4G)jie gou yu he yi ge Cmo duan UPF1de jie ge ou yu 。da duo shu zhen he sheng wu gong xiang he xin NMDyin zi ,er NMDtu jing de fen zi ji zhi zai sheng wu ti zhi jian que you suo bu tong 。ben wen zhong ,shou xian dui EoUPF1he ji ta zhen he sheng wu UPF1gong 26ge wu chong ,yi ji EoUPF2he ji ta zhen he sheng wu UPF2gong 24ge wu chong de an ji suan xu lie jin hang bi dui ,fen bie gou jian ji tong jin hua shu ,cong ta men de jin hua guan ji fen xi fa xian ,EoUPF1yu EoUPF2jun yu niang jiu jiao mu (Saccharomyces cerevisiae)de jin hua guan ji jiao wei jie jin 。li yong InterProshu ju ku fen xi ,jie guo xian shi er zhe de he xin jie gou yu fei chang xiang shi 。ji ci ,tong guo pull downshi yan yan zheng le EoUPF1he EoUPF2zhi jian 、yi ji ge zi yu di er lei tai lian shi fang yin zi eRF3(eukaryotic polypeptide release factor 3)zhi jian ,cun zai de xiang hu zuo yong guan ji ,wo men tui ce zai ba le you pu chong xi bao zhong ,EoUPF1、EoUPF2yu eRF3xing cheng eRF3/EoUPF1/EoUPF2fu ge ti 。zui hou ,wo men jin yi bu tong guo jiao mu shuang za jiao he pull downshi yan zheng shi ,EoUPF2de MIF4Gjie gou yu yu wai xian zi lian jie fu ge ti (exon-exon junction complex,EJC)zu cheng yin zi Y14a/Y14bke yi xiang hu zuo yong 。zai gao deng zhen he xi bao zhong ,UPF3jie dao UPF2yu EJChe xin yin zi Y14de xiang hu zuo yong ,er ba le you pu chong xi bao zhong mei you UPF3yin zi 。na me ,EoUPF2zai mei you UPF3cun zai de qing kuang xia ru he fa hui gong neng ne ?ben yan jiu zheng shi ,EoUPF2tong guo ji MIF4Gjie gou zhi jie yu EJCzu cheng yin zi Y14ahe Y14bxiang hu zuo yong 。zai β-ban ru tang gan mei shi yan zhong ,wo men fa xian EoUPF2yu Y14ade zuo yong geng jiang yi xie ;er shi shi ying guang ding liang PCRfen xi jie guo pai chu le EoUPF2yu Y14ahe Y14bxiang hu zuo yong jiang ruo yu Y14ahe Y14bben shen de kao bei shu you guan 。you ci ,wo men zhen dui ba le you pu chong xi bao de NMDtu jing qi dong ji zhi di chu jia she :he tang ti fan yi guo cheng zhong yu dao di qian zhong zhi mi ma zi (PTC)shi hui ting xia lai ,eRF3yu EoUPF1、EoUPF2xiang hu zuo yong ,ran hou xing cheng le eRF3/EoUPF1/EoUPF2fu ge ti 。er EoUPF2tong guo yu EoUPF1he Y14xiang hu zuo yong ,jie dao le eRF3/EoUPF1/EoUPF2fu ge ti yu EJCde ou lian ,qi dong le mo yi mRNAde shi bie guo cheng 。ben wen de shi yan jie guo ,ke yi wei jin yi bu chan ming ba le you pu chong NMDji zhi di gong zhi chi 。
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论文作者分别是来自山西大学的柴杨丽，发表于刊物山西大学2019-11-12论文，是一篇关于无义介导的降解论文,提前终止密码子论文,上游移码蛋白论文,外显子连接复合体论文,肽链释放因子论文,山西大学2019-11-12论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自山西大学2019-11-12论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：无义介导的降解论文; 提前终止密码子论文; 上游移码蛋白论文; 外显子连接复合体论文; 肽链释放因子论文; 山西大学2019-11-12论文;