核仁蛋白 Bms1l 调控肝脏细胞增殖的分子机制研究

Cell proliferation is one of the basic cellular activities during organ development. DNA replication is the prerequisite for cell proliferation. During DNA replication, replication forks will inevitably encounter the ongoing gene transcription on the same template. In this case, there will be a physical conflict between DNA replication and gene transcription. In high eukaryotes, rRNA genes are tandemly repeated in the genome to form a gene cluster (rDNA region). rRNA is highly transcribed even during DNA replication. Therefore, with the conflict between DNA replication and gene transcription a prominent feature in the rDNA region especially during organogenesis, organisms must have developed a mechanism to circumvent this clash. However, we have limited knowledge regarding the nucleolar factors involved in this process. Bms1 is a nucleolar protein. Our previous studies have shown that loss-of-function of the zebrafish Bms1l led to a small liver phenotype and that Bms1l and Rcl1 form a complex that is essential for the processing and maturation of 18S rRNA. Recently, we surprisingly found that the bms1l-/- mutant displayed abnormal DNA replication and increased number of nucleoli. We also found that p53 and its downstream genes were significantly up-regulated and proteins related to DNA replication fork progression (e.g RPA2 and Rad51) were drastically up-regulated in the bms1l-/- mutant. Moreover, p53 and RPA2 are enriched in the nucleolus in the bms1l-/- mutant. Based on these data, we propose that Bms1l, in addition to its role in 18S rRNA processing and maturation, serves as a regulator of DNA replication by resolving the conflict between replication and transcription. The proposed research plan will try to unravel the possible role(s) of Bms1l in DNA replication, with specific focus on studying the role of Bms1l in the formation of the replication fork barrier (RFB) within the rDNA gene cluster. This will allow us to understand the molecular mechanism of Bsm1l in promoting liver development. So far, there is no report on the function of Bms1 in DNA replication. The outcome of our research will extend our understanding of the biological function of Bms1 in organogenesis.

细胞增殖是器官发育的必要条件，而DNA顺利完成复制是细胞增殖的前提。DNA复制时必然会经过基因转录活跃区，这时复制和转录彼此间就会互相干扰。在真核细胞增殖期间，rRNA基因属于高转录基因，加之rRNA基因串联重复形成基因簇，因此在器官发育时rDNA区域的DNA复制必须克服与rRNA转录之间的矛盾，但目前尚不清楚有哪些核仁因子参与协调该过程。Bms1是一个核仁蛋白，我们之前报道过斑马鱼Bms1l失活导致小肝脏表型、Bms1与Rcl1形成复合体参与18S rRNA的加工成熟。我们最近意外发现bms1l 突变体中DNA复制发生异常、核仁数目增加、复制相关蛋白p53及RPA2等的表达显著上调并富集于核仁中。据此我们推测Bms1l不仅参与18S rRNA加工成熟，还可能在细胞增殖期间在调和rDNA复制与rRNA转录之间的矛盾中发挥重要功能。本研究旨在阐明Bms1l的新功能以揭示肝脏发育调控的新机制。

核仁蛋白Bms1和Rcl1均为核糖体小亚基组装复合体的核心成员。Bms1与Rcl1组成复合体，该复合体对18S rRNA加工成熟十分重要，但Bms1是否还行使其他生物学功能却无任何报道。.在本项目研究中利用斑马鱼遗传模型和人培养细胞模型，我们发现核仁中的Bms1除了能与Rcl1互作外，还能与rRNA转录终止因子TTF1互作。我们发现Bms1l通过与Ttf1互作，能够借助Bms1自身的GTPase酶活将Ttf1从RFB位点解离，进而调控细胞周期进程。发现在bms1l突变的斑马鱼体内，rDNA转录水平上升、复制叉行进受阻，最终使得细胞周期在S/G2转换过程中受到阻滞，但是整个基因组DNA的复制却保持异常活跃的状态。rDNA转录标志因子ubf、tif-IA和taf1b，DNA损伤反应标志因子Chk2、Rad51和p53，以及复制叉阻滞因子Rpa2、PCNA、Fen1和Ttf1均在bms1l突变体中显著上升，且Ttf1滞留在RFB上，最终导致细胞周期受阻在S期。根据以上研究结果，我们认为核仁蛋白Bms1l通过分别与Rcl1和Ttf1互作，在平衡S期rDNA的转录和复制过程中发挥重要作用，并通过平衡rDNA复制-转录发挥S期检验点的功能。.在本项目研究中，我们对斑马鱼18S rRNA加工的精确酶切位点进行了研究，确定了A’，A0，A1，E，2五个切割位点的精确位置。同时，我们对Bms1的互作蛋白Rcl1的开展了研究，发现核仁蛋白Rcl1是斑马鱼消化器官发育所必需、Rcl1的缺失影响pre-rRNA在A1位点的加工过程、Rcl1的缺失导致一系列核糖体生物合成基因表达上调。.在本项目研究中，我们还对斑马鱼rDNA的种类及其表达进行了分析，发现斑马鱼基因组中有8种18S DNA，其中7种可以定位到对应的染色体，但我们发现其中只有M-型（母源型）和S-型（体细胞型）表达，其中M-型主要在卵子中表达，而S-型在体细胞中表达。.总结而言，在过去四年里我们超额完成了研究任务，发表了数篇高水平研究论文，在核仁因子调控细胞周期领域取得重要进展，同时明确了斑马鱼18S rRNA加工成熟过程，并确定了Rcl1在18S rRNA加工过程中的功能。S期检验点概念的提出拓展了细胞周期调控的内涵，18S rRNA酶切位点的确定为研究斑马鱼核糖体小亚基组装提供了基础。