【作者】 邵洁；

【导师】 杨洁；

【作者基本信息】 天津医科大学 ， 免疫学， 2011， 博士

【摘要】 目的：人Tudor-SN蛋白是一种广泛表达于胞浆中的多功能RNA结合蛋白,它可以参与细胞内多种生物学过程。应激颗粒(Stress Granules, SGs)是存在于胞浆中的含有mRNA的颗粒结构,它是在细胞应答应激时聚集产生的。研究发现Tudor-SN蛋白在应激刺激下可以与G3BP蛋白产生细胞内共定位。因此本课题旨在研究Tudor-SN蛋白如何参与SGs以及胞浆中另一类RNA颗粒即加工小体(Processing-bodies, P-bodies)的聚集功能,从而探讨Tudor-SN蛋白在mRNA的转录后基因调控过程中的作用机制。方法：本课题实验分为四部分,第一部分是利用免疫荧光抗体染色技术研究Tudor-SN参与SGs的聚集并与TIA-1蛋白的细胞内定位；第二部分实验利用同样的实验方法研究Tudor-SN参与P-bodies的聚集并与Ago2蛋白和Dcp-1蛋白的细胞内定位；第三部分实验利用siRNA干扰技术和免疫荧光抗体染色技术研究RNAi后的Tudor-SN低表达对于SGs形成的影响。第四部分实验是利用免疫荧光抗体染色技术研究Tudor-SN蛋白在参与SGs聚集过程中的动态运输过程。结果：1.Tudor-SN蛋白在应激刺激下参与SGs的聚集并通过其SN domain与TIA-1蛋白存在细胞内共定位,但是Tudor-SN蛋白聚集到SGs的过程滞后于TIA-1。2.Tudor-N蛋白在应激刺激下参与P-bodies的聚集并与Ago2蛋白和Dcp1蛋白分别形成细胞内共定位。3.RNAi后的细胞内Tudor-SN蛋白低表达不会阻止SG聚集,但会影响SGs的致密程度和大小。4.Tudor-SN蛋白在参与SGs形成过程中依赖于微管蛋白(Microtubules)——α-Tubulin。结论：Tudor-SN蛋白作为一种RNA结合蛋白不仅可以参与SGs的聚集,也可以参与P-bodies的聚集,是被新发现的RNA颗粒共享蛋白组分,并在应答应激时可以与SGs的标志蛋白TIA-1及P-bodies的标志蛋白Dcp1产生细胞内共定位。但它并不是SGs聚集的启动蛋白组分,它在应激条件下聚集到SGs的过程依赖于微管蛋白α-Tubulin的支持和引导作用。更多还原

【Abstract】 Objectives:Human Tudor-SN protein is a ubiquitously expressed, multifunctional RNA-binding protein involved in various kinds of biological processes. SGs are mRNA containing cytoplasmic structures that are assembled in response to stress. Tudor-SN has been found to physiologically interact with G3BP in the same SGs in response to different stress stimuli.Our project aims at the study of Tudor-SN protein assembly onto SGs and Processing-bodies and its mechanism of post-transcriptional gene regulation.Methods:Our project includes four experimental parts. In Part I, we study the assembly of Tudor-SN onto SGs and its colocalization with TIA-1under stress condition by immunoflourescence staining. In Part II, we study the recruitment of Tudor-SN onto P-bodies and its colocalization with P-bodies components:Ago2and Dcpl. In Part III, we study whether knockdown of endogenous Tudor-SN protein could affect the formation of SGs by RNAi mechanism. In Part IV, we study the kinetic recruitment and transportation of Tudor-SN onto SGs by immunoflourescence.Results:1. The assembly of Tudor-SN protein onto SGs is mediated by its SN domain in response to stress. Recruitment, of Tudor-SN onto SGs lags behind TIA-1.2. Tudor-SN protein can assemble onto P-bodies and colocalizes with Ago2and Dcpl under same stress stimuli.3. Knockdown of endogenous Tudor-SN protein did not inhibit the formation of SGs but decrease the size and density of SGs.4. The kinetic recruitment of Tudor-SN protein onto SGs is dependent on a-Tubulin, a Microtubule protein.Conclusions:These findings identify Tudor-SN is a key component recruited onto SGs as well as to P-bodies. It can colocalize with TIA-1, Ago2and Dcpl separately under the same stress conditions. The assembly of Tudor-SN onto SGs is dependent on a-Tubulin. However, Tudor-SN may not be as essential as G3BP to initiate the formation of SGs but affect the aggregation of SGs.更多还原