细胞核内actin聚合促进聚合酶RNA Pol II相分离调控转录过程的机制研究

Transcription controls essential cellular processes including selective expression of genes and cell differentiation. A variety of proteins are involved in the regulation of RNA Pol II transcription. Nuclear actin, as one of these regulators, is critical for the transcription of RNA Pol II, but the molecular mechanisms remain unknown. The phase separation regulation mechanism during transcription has been uncovered in the last two years, revealing the important role of phase separation in maintaining chromatin structure and regulating transcription. This project intends to study the regulation of nuclear actin on RNA Pol II from the perspective of phase separation. We propose that nuclear actin is involved in the regulation of transcriptional processes by controlling RNA Pol II phase separation through interacting with multiple proteins. Signaling such as serum stimulation causes polymerization of nuclear actin, enhances the interaction between unstructured regions of RNA Pol II molecules, promotes RNA Pol II phase separation, and initiates transcription. The project will use a variety of technologies for in-depth research. It is proposed to establish a live cell imaging system to control the phase separation process of RNA Pol II in real time, establish a sensitive and interference-free nuclear actin labeling method, determine the effect of multi-protein interaction on the phase separation process of RNA Pol II, and finally reveal the molecular mechanism of nuclear actin regulating RNA Pol II transcription. Using the new phase separation concept to explain the long-conflicted nuclear actin regulation of transcription, will open up a new perspective for the study of nuclear actin function, increase our understanding of transcriptional regulation, and guide us to understand more phenomena in the cell from the view of phase separation.

多种蛋白参与调控RNA聚合酶II的转录过程，细胞核内微丝对于聚合酶II的转录至关重要，但具体分子机制仍未知。转录过程中的相分离调控近两年被发现可以维持染色质结构、调节转录功能。本项目拟从相分离的角度研究微丝对聚合酶II的调节作用。我们提出微丝通过与多个蛋白相互作用控制聚合酶II相分离，参与调节转录过程。血清刺激等信号转导引起微丝的聚合，增强含有无结构区域的聚合酶II分子之间相互作用，促进聚合酶II相分离，引发转录起始。本项目将综合使用多种技术进行深入研究。拟建立活细胞成像系统实时控制聚合酶II相分离过程，建立灵敏的微丝无干扰标记方法，确定多蛋白相互作用对聚合酶II相分离过程的影响，最终揭示微丝调控聚合酶 II分子机制。利用全新的相分离概念解释困惑已久的微丝调控转录问题，将为研究细胞核内微丝的功能开启崭新视角，增加我们对于转录调控更深层次的认识，指导我们从相分离角度理解细胞内更多现象。

多种蛋白参与调控RNA聚合酶II的转录过程，细胞核内微丝对于聚合酶II的转录至关重要。受限于细胞核内肌动蛋白的含量和结构状态多样等因素，细胞核内肌动蛋白对转录调控的分子机制一直不明确。本研究从相分离出发，综合使用多种技术进行了深入研究。我们首先建立了“RNA-Pol II-PSF-NonO-N-WASP”的共相分离系统，并且验证了该相分离体系会招募Arp2/3和G-actin，并进一步促进G-actin的聚合。同时，通过干扰G-actin聚合等方法，本研究发现肌动蛋白的聚合会反过来促进RNA-Pol II的相分离，进而调控转录。该结果提出了核肌动蛋白在转录调控中的新机制，并为转录工厂的形成提供了分子基础。最后，本论文通过一系列N-WASP突变体发现N-WASP的酸性末端会破坏RNA-Pol II的相分离，这为转录起始向转录延伸的过渡提供了一个新的可能途径。利用全新的相分离概念解释细胞核内微丝调控RNA Pol II转录问题，为研究细胞核内微丝的功能开启崭新视角，增加我们对于转录调控更深层次的认识，指导我们从相分离角度理解细胞内更多现象。