【作者】 王芳；

【导师】 韩伟；

【作者基本信息】 中国科学技术大学 ， 生物物理学， 2019， 博士

【摘要】 RNA聚合酶Ⅲ在体内负责转录包括tRNA、5S rRNA和U6 snRNA在内的一系列小分子非编码RNA,这些转录产物在蛋白质合成、RNA加工、以及蛋白质转运等过程中都发挥了至关重要的调控作用。RNA聚合酶Ⅲ起始转录需要转录因子TFⅢB和TFⅢC的帮助,TFⅢB作为RNA聚合酶Ⅲ的转录起始复合物,对于介导RNA聚合酶Ⅲ和TFⅢC之间的相互作用以及RNA聚合酶Ⅲ正确定位到其靶基因的转录起始位点极为关键。已有研究表明,TFⅢB和TFⅢC的翻译后修饰,如磷酸化修饰,具有调控RNA聚合酶Ⅲ活性的作用。然而目前关于蛋白质的泛素化修饰是否以及如何调控肿瘤细胞RNA聚合酶Ⅲ的活性并不清楚。有趣的是,与正常细胞相比,肿瘤细胞中的RNA聚合酶Ⅲ呈现高度活化的现象。这种RNA聚合酶Ⅲ活性的增强被证实在正常细胞发生致癌性转化过程中发挥了关键的作用,可能主动地促进了肿瘤的发生发展。然而目前关于RNA聚合酶Ⅲ在肿瘤细胞中活性增强的机制仍不明确。本文研究发现,作为RNA聚合酶Ⅲ转录起始复合物TFⅢB的关键组分,BRF1在细胞内发生多聚泛素化修饰,这是由BRF1特异性的泛素连接酶RNF12介导的。RNF12作为一个新的BRF1相互作用蛋白,其N端260个氨基酸介导了与BRF1(206-409)的结合。体内外泛素化实验结果表明,RNF12能够结合BRF1、并催化BRF1形成Lys27和Lys33依赖的多聚泛素链。更为重要的是,在功能上,RNF12能够通过促进BRF1的多聚泛素化,阻止BRF1在RNA聚合酶Ⅲ靶基因启动子区域的定位,最终抑制RNA聚合酶Ⅲ的活性及肿瘤细胞的增殖。这些研究结果揭示了一种蛋白质泛素化修饰调控RNA聚合酶Ⅲ活性的新机制,并暗示RNF12的低表达可能是肿瘤细胞中RNA聚合酶Ⅲ异常活化的一个重要原因。因此,RNF12也可能作为一个新的干预肿瘤增殖的潜在分子靶标。更多还原

【Abstract】 As the largest of the eukaryotic DNA-dependent RNA polymerases,RNA polymerase Ⅲ(Pol Ⅲ)is responsible for the transcription of small non-coding RNAs,including tRNAs,5S rRNA,and U6 snRNA.These Pol Ⅲ transcripts control several fundamental metabolic processes such as protein translation and RNA processing,thereby dictating the growth rate of a cell.The accurate initiation of PolⅢ-dependent transcription requires at least two general transcription factors TFⅢB and TFⅢC.TFⅢC recognizes and binds to specific sequence elements in target gene promoters,thereafter allowing the recruitment of TFⅢB.The binding of TFIIIB to the promoter in turn precisely positions Pol Ⅲ at the transcription start site.It has been shown that the post-translational modification of TFⅢB and TFⅢC,such as phosphorylation,is involved in the regulation of Pol Ⅲ-dependent transcription.However,it remains unknown whether Pol Ⅲ-dependent transcription is also regulated by protein ubiquitination.Intriguingly,it has long been recognized that compared to normal cells,both transformed and tumor cells exhibit elevated Pol Ⅲ-dependent transcription.In addition,enhanced Pol Ⅲ-dependent transcription is required for oncogenic transformation of normal cells,indicating that enhanced Pol Ⅲ-dependent transcription not only allows cancer cells to meet their high demands for protein synthesis,but it is also actively involved in tumorigenesis.However,the molecular mechanisms underlying the dysregulation of RNA polymerase Ⅲ in cancer cells remain largely unclear.In this study,we show that RNF12,a RING domain-containing ubiquitin E3 ligase,physically interacts with BRF1.The results from domain mapping experiments suggest that RNF12(206-409)directly interacts with BRF1(1-260).Via direct interaction,RNF12 catalyzes Lys27-and Lys33-linked polyubiquitination of BRF1.Furthermore,RNF12 is able to negatively regulate Pol Ⅲ-dependent transcription and cell proliferation via BRF1.These findings uncover a novel mechanism for the regulation of BRF1 and reveal RNF12-mediated BRF1 ubiquitination plays an important role in the regulation of Pol Ⅲ-dependent transcription.更多还原