DFFA和PSMC3在RNA干扰功能中的调控作用

发布时间：2018-05-14 11:22

  本文选题：Argonaute2/Ago2 + DFFA　； 参考：《天津医科大学》2008年硕士论文

【摘要】：目的： RNA干扰是一种由siRNA或dsRNA介导的基因沉默表达的方式。通过与靶mRNAde特异性结合,这些小RNA与Argonaute (AGO)蛋白及相关因子形成RNA诱导的沉默复合体(RISC),后者完成对靶mRNA的剪切或者翻译抑制。前面通过酵母双杂交筛选出与人类Argonaute2 (Ago2)相关蛋白——DFFA和PSMC3,本实验在此基础上,进一步通过免疫共沉淀验证它们与Ago2相互作用的特异性,通过荧光实验,我们推测这两种蛋白在体内miRNA或者siRNA介导的mRNA剪切中发挥着重要功能。 方法： 利用免疫共沉淀的方法在细胞内验证DFFA、PSMC3同人类Ago2蛋白之间相互作用的特异性,通过GST pull-down验证DFFA同Argonaute2 PIWI结构域(hAgo2 PIWI)体外结合作用。构建内源性miR-21介导的GFP阳性Read-Out报告系统,通过该系统验证在分别敲除DFFA和PSMC3的情况下,被抑制的GFP表达效果是否得以增强；利用siRNA-GFP和表达GFP的质粒作为研究siRNA介导的GFP表达抑制,观察在分别敲除DFFA和PSMC3的情况下GFP的表达强度变化,间接推测两者在由小RNA介导的RNA干扰过程中的功能。 结果： 1.免疫共沉淀证实外源性DFFA和PSMC3同转染的hAgo2 PIWI存在着特异的相互作用。进一步的实验证明,DFFA和PSMC3 Flag融合蛋白的N端结构域可能与全长的hAgo2蛋白相互结合。 2.通过GST-pull down体外结合实验证实DFFA同hAgo2 PIWI的相互作用。 3.敲除了DFFA、PSMC3对miR-21介导的内源封闭报告系统产生的积极地作用。 4.敲除了DFFA、PSMC3对si-GFP介导的GFP抑制产生了逆转的效果。 结论： DFFA在体内、体外均同hAgo2 PIWI存在着相互作用,PSMC3在体内同hAgo2 PIWI存在着相互作用；DFFA和PSMC3 Flag融合蛋白的N端结构域可能与全长的hAgo2蛋白相互结合。敲除实验证实DFFA、PSMC3均参与了由miRNA或者siRNA介导的mRNA剪切过程,推测两者可能是新的RISC组件。
[Abstract]:Objective: RNA interference is a gene silencing expression mediated by siRNA or dsRNA. By binding specifically to the target mRNAde, these small RNA proteins and related factors form RNA induced silencing complex RISC, which inhibits the splicing or translation of the target mRNA. The proteins associated with human Argonaute2 Ago2 (DFFA and PSMC3) were screened by yeast two-hybrid. On this basis, the specificity of their interaction with Ago2 was further verified by immunoprecipitation, and fluorescence assay was used. We speculate that these two proteins play an important role in miRNA or siRNA mediated mRNA cleavage in vivo. Methods: The specificity of the interaction between DFFA-PSMC3 and human Ago2 protein was verified by immunoprecipitation in vitro, and the binding of DFFA to Argonaute2 PIWI domain hAgo2 PIWI was confirmed by GST pull-down in vitro. An endogenous miR-21 mediated GFP positive Read-Out reporting system was constructed to verify whether the inhibition of GFP expression was enhanced when DFFA and PSMC3 were knocked out respectively. SiRNA-GFP and GFP expression plasmids were used to study the inhibition of GFP expression mediated by siRNA. The changes of GFP expression intensity were observed when DFFA and PSMC3 were knocked out, respectively, and the function of GFP in RNA interference mediated by small RNA was inferred indirectly. Results: 1. Immunoprecipitation confirmed that exogenous DFFA and PSMC3 had specific interactions with transfected hAgo2 PIWI. Further experiments showed that the N-terminal domain of DFFA and PSMC3 Flag fusion protein may interact with the full-length hAgo2 protein. 2. The interaction between DFFA and hAgo2 PIWI was confirmed by GST-pull down binding assay in vitro. 3. Knock out the positive role of DFFAP PSMC3 in miR-21-mediated closed reporting systems. 4. Knockout of DFFAA PSMC3 reversed the inhibition of GFP mediated by si-GFP. Conclusion: DFFA has interaction with hAgo2 PIWI in vivo and in vitro. PSMC3 interacts with hAgo2 PIWI in vivo and the N-terminal domain of PSMC3 Flag fusion protein may bind to the full-length hAgo2 protein. Knockout experiments confirmed that both DFFA-PSMC3 were involved in the mRNA shearing process mediated by miRNA or siRNA, which suggested that both of them might be new RISC components.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【共引文献】

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1 王帅;对虾抗病毒相关蛋白的分子克隆与功能研究[D];山东大学;2010年

相关硕士学位论文 前3条

1 刘宁;对虾血细胞T7噬菌体展示文库构建及功能基因淘选[D];山东大学;2006年

2 韩涛;TRBP在RNA诱导的沉默复合体（RISC）中的作用研究[D];第四军医大学;2009年

3 孔鹏洲;AGO2相互作用蛋白PSMC3、DFFA对RNAi功能的影响[D];天津医科大学;2007年

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