细胞骨架细丝蛋白A在RNA聚合酶III指导的基因转录中的作用机制研究

Cytoskeletal protein filamin A(FLNA) plays diverse roles in human cell including cell adhension and migration,cell signalling, transcription and cytoskeleton maintenance. Our previous work showed that FLNA is localised to nucleolus and represses ribosomal RNA（rRNA） gene transcription and cell proliferation. Since rRNAs including 28S,18S and 5.8S rRNA are important components of ribosome and transcribed by RNA polymerase I(pol I); whereas 5S rRNA, another component of ribosome, is transcribed by RNA polymerase III(pol III). It raises a hypothesis that FLNA could also regulate gene transcripion by RNA pol III. Indeed, our latest data show that FLNA siRNA is able to stimulate 5S rRNA and two tRNA genes expression in Saos2 cell. Using immunofluorescence staining immunoprecipitation methods we demonstrated that FLNA associates with transcription factor TFIIIC2. Together, these data indicate that FLNA is invovled in the regulation of gene trnscription by RNA polymerase III. In this project we'll further investigate the role of FLNA and its regulatory mechanism in gene transcription by RNA pol III through performing the following work：1）determine what pol III-controlled genes are modulated by FLNA. 2) determine whether filamin A directly involves pol III-directed transcription. 3)understand whether FLNA binds RNA pol III transcription machinery. 4) uncover the mechanism by which FLNA modulate pol III-directed transcription.The data from the work will extend our understanding to the role of FLNA in gene transcription and cell proliferation.

细丝蛋白A（FLNA）在细胞中起着多种作用，我们前期的研究表明FLNA位于细胞的核仁区，并抑制核糖体RNA基因转录和细胞增殖。由于核糖体RNA（28S，18S，5.8S rRNA）由RNA聚合酶I转录，而核糖体5S rRNA由RNA聚合酶III转录，由此假设：FLNA也能调节RNA聚合酶III(pol III)指导的转录。我们最近的研究结果表明FLNA siRNA 能增强5S rRNA 和2个tRNA 基因的表达；荧光染色和免疫沉淀证明FLNA能与转录因子TFIIIC2结合。本项目将继续研究FLNA在RNA聚合酶III指导的转录中的作用及其机制，包括：1.FLNA调节哪些pol III控制的基因？2.FLNA是否直接参与此过程的调节？3.FLNA是否结合pol III 转录装置？4.FLNA如何调节pol III指导的转录机制。本研究将阐明FLNA在转录和细胞增殖中作用机制。

细胞骨架细丝蛋白A（FLNA）是非常重要的蛋白，参与细胞中的许多生物过程。以前的研究表明，FLNA能促进或者抑制癌症的生长和发展；然而, 其中作用机制并不完全了解。我们这里的研究表明，在293T和SaOS2细胞中，除了一部分tRNA基因外， FLNA敲低显著增加RNA聚合酶III指导的基因转录。反之，在FLNA缺陷的细胞系A7中重新表达FLNA则抑制所有pol III指导的基因转录，提示FLNA是通过细胞特异性的方式抑制pol III转录的。染色质沉淀实验表明，pol III 基因转录的抑制与pol III 转录器械在其基因的启动子上招募降低相关。免疫荧光显微镜技术和免疫共沉淀实验表明，FLNA在细胞核中通过其肌动蛋白结合域与pol III 转录器械结合。对FLNA作用机制研究表明，FLNA是通过限制pol III 转录器械因子在启动子上的招募而抑制pol III的基因转录，特别是对FLNA表达变化敏感的那些基因更是如此。这些发现不仅拓展了解FLNA在细胞中的功能，也为FLNA如何抑制细胞增殖的机制提供崭新视角。