全基因组RNAi筛选鉴定细胞蛇组装的新的调控因子

Intracellular compartments such as organelles are essential for cellular function as they carry out many biological processes that are critical for the cells to live. Defects in the organelles are known to underlie many human diseases. Within a cell, nucleotides play essential roles as building blocks of DNA and RNA, as cofactors of enzymatic reactions, and as energy carriers and metabolic regulators. Consequently, the synthesis of nucleotides is tightly regulated within the cell. However, little is known about the subcellular distribution of CTP synthase (CTPS), an essential metabolic enzyme involved in the production of CTP. In 2010, I discovered that CTPS is compartmentalised into a filamentous structure in Drosophila which I called the cytoophidium (“cellular serpent” in Greek). Subsequently studies reported that CTPS forms similar filaments in bacteria, budding yeast and human cells, suggesting that filament-formation is a highly conserved and very ancient property of CTPS. To search for the factors regulating cytoophidium assembly, we have conducted a high-throughput genome-wide RNAi screen which involved knocking down ~13,900 genes individually in Drosophila cells and identified 534 potential candidate genes that appear to regulate cytoophidium assembly. The aims of this proposal are: 1) to perform a secondary screen to validate the candidate genes that were identified in the primary screen; 2) to functionally characterise the validated candidates in regulating cytoophidium in vivo using RNAi and CRISPR/Cas9 technologies; and 3) to determine the effect of key signalling pathways on cytoophidium assembly. Taken together, this study will provide an insight into the significance and mechanisms of compartmentalisation of metabolic enzymes in cellular functions.

细胞内区域化对细胞的功能至关重要。很多细胞赖以生存的生物过程需要被区域化。因此，细胞器的缺陷可以引起许多类型的人类疾病。核苷酸在细胞中发挥重要作用，其合成在细胞内被精密调节。2010年，申请人首次报道发现合成核苷酸CTP的关键酶CTP合成酶（CTPS）在细胞内形成蛇状结构。之后，从细菌到人类的细胞中都发现了细胞蛇结构的存在，说明形成细胞蛇是CTPS的一个高度保守且非常古老的特性。为寻找CTPS在细胞中组装成细胞蛇的关键调控因子，我们成功完成了果蝇S2细胞中全基因组范围13,900个基因的高通量初级筛选并在初级筛选中获得的534个候选基因。本项目将在扎实的前期工作基础上，首次拟通过基于全基因组的范围高通量筛选，寻找调控CTPS细胞蛇动态组装的调节因子。我们还将利用高效的CRISPR/Cas9技术对内源基因进行一系列修改，以探索CTPS细胞蛇在组织发育中的尚待认知的基本生物学功能和调控机制。

本项目利用果蝇的生命周期具有明确的发育阶段的特征，在空间和时间上具有不同的代谢需求的细胞类型，分析比较CTP合成酶（CTP synthase, CTPS）区域化。成功构建了CTPS-mcherry 和CTPSH355A点突变敲入果蝇，探索发现CTPS细胞蛇在果蝇幼虫不同组织和细胞中的时空分布特征。探索发现CTPS细胞蛇在果蝇幼虫不同组织和细胞中的时空分布特征；利用冷冻电镜技术解析CTPS聚合体分子结构，发现底物和产物结合形式的不同聚合体构象；利用酵母双杂交的方法广泛筛选出CTPS相互作用的因子吡咯啉五羧酸合成酶P5CS，发现CTPS影响P5CS蛋白细胞蛇的形成和组装，通过2D分类发现两种细胞蛇的构象有明显的差异，P5CS细胞蛇是由一个个领结组成，而CTPS细胞蛇是由X形结构单元构成，表明二者虽然都形成多聚体，但形态和组装方式各不相同; 利用临近蛋白标记方法（Turbo-ID）鉴定卵泡细胞中CTPS相互作用蛋白84个，通过基因本体生物过程分析，分别为参与细胞质翻译、核糖体组装、细胞器组装、核糖核蛋白组装等的蛋白；阐明CTPS在卵泡细胞中的极性分布特点和功能，以及在果蝇肠道细胞稳态维持中的功能和作用。研究结果有助于全面了解代谢酶CTPS细胞蛇结构和动态组装在果蝇生长发育中的基本生物学功能和调控机制。