细胞在面对DNA双链断裂损伤时，主要有两种途径修复损伤，分别是同源重组和非同源末端连接，而细胞周期决定了损伤修复通路的选择。在S/G2期，细胞主要通过同源重组途径修复损伤，其中BRCA1 (Breast-cancer susceptibility gene 1)分子起到至关重要的作用；在G0/G1期，主要通过53BP1 (Tumor protein p53 binding protein 1)主导的非同源末端连接途径修复损伤。除此之外，BRCA1还在保护复制叉方面发挥了重要作用。BRCA1主要通过它在DNA损伤修复以及维持复制叉稳定方面发挥的功能，抑制肿瘤的发生发展。BRCA1突变会诱导乳腺癌、卵巢癌等肿瘤的发生，并且对于这类BRCA1突变肿瘤，使用诱导DNA损伤的药物，如PARP(Poly(ADP-ribose) polymerase)抑制剂，能够有效地杀伤肿瘤。因此可以通过研究BRCA1的表达调控机制，找到靶向BRCA1杀伤肿瘤的治疗策略。
  BRCA1的蛋白表达水平是随着细胞周期改变的，在S/G2期达到顶峰值，随后进入G0/G1期又会迅速下降。BRCA1在S/G2期表达水平高，有利于BRCA1拮抗非同源末端连接修复途径，使得细胞通过同源重组方式修复DNA损伤。另外在G1期，BRCA1表达需要维持低水平状态，否则过多的BRCA1蛋白会招募过多RAD51定位到DNA损伤位点，反而会导致更多DNA损伤的产生。所以BRCA1表达周期性波动的特性，有利于它维持基因组稳定性，但是BRCA1周期性表达调控的具体机制并不清楚。
  本研究发现转录水平或是翻译后水平均不决定BRCA1在S期与G1期的表达差异，反而主要是剪接相关的NMD (Nonsense mediated mRNA decay)过程决定了BRCA1蛋白在S期与G1期的周期性波动。本研究通过RNA-IP (RNA-Immunoprecipitation)偶联质谱方法进行筛选，发现RBM10 (RNA-binding motif protein 10)这一剪接因子决定了BRCA1的周期性表达差异，并且RBM10调控BRCA1蛋白周期性表达的过程受到上游CDK2 (Cyclin-dependent kinase 2)激酶调控。在S期，CDK2磷酸化RBM10的S89位点，增强了RBM10的正常功能，RBM10调控BRCA1的可变剪接，得到更多保留全长、具有正常功能、不被降解的BRCA1亚型mRNA；相反在G1期，RBM10的磷酸化水平低下，使得截短的、功能不全、容易被降解的BRCA1亚型mRNA相对水平增加，最终表现为BRCA1蛋白在S期表达水平高于G1期。
  另外本研究通过分析RNA-Seq结果，发现剪接相关的NMD途径调控了包括BRCA1在内众多蛋白的周期性表达差异。RBM10调控的基因绝大多数存在G1期与S期的表达差异。敲低RBM10后，这些基因的周期性表达差异会被削弱，其中有14个BRCAness基因的表达下调。此外CDK2通过调控可变剪接影响基因表达的机制存在普遍性。
  综上所述，本研究揭示了CDK2通过调控RBM10磷酸化维持BRCA1周期性表达差异的机制，并且可变剪接相关的NMD途径广泛地调控着下游周期性波动基因的表达差异。在应用方面，本研究发现联用CDK2抑制剂与PARP抑制剂对于杀伤肿瘤具有很好的协同效果，并揭示了RBM10可以作为肿瘤治疗的潜在靶点。
 

  When DNA double-strand breaks (DSBs) happens, there are two different pathways to repair damaged DNA: homologous recombination (HR) and non-homologous end-joining (NHEJ). The choice of DSB repair pathway relies on cell cycle phase. In S/G2 phase, homologous recombination is the major pathway to repair DSB. However, in G0/G1 phase, DNA damage is repaired by NHEJ pathway. Here, BRCA1 (Breast-cancer susceptibility gene 1) is essential for homologous recombination repair. BRCA1 also plays important roles in protecting replication fork. BRCA1 acts as a tumor suppressor gene through its functions in HR repair and replication fork protection. Mutation of BRCA1 increases risk of cancers especially like breast and ovarian cancer. As for these cancers with BRCA1 loss-of-function mutation, drugs that cause DNA damage just like PARP (Poly (ADP-ribose) polymerase) inhibitor, are always available to kill these cancers. Therefore, it's an excellent strategy to find out a treatment method that targets BRCA1 through uncovering the mechanism of BRCA1 expression.
The expression of BRCA1 changes along cell cycle. The level of BRCA1 proteins is higher in S/G2 than G0/G1 phases. BRCA1 could compete with 53BP1 (Tumor protein p53 binding protein 1) favoring HR pathway instead of NHEJ pathway in S/G2 phase because of its higher expression level. On the other hand, it's necessary to keep BRCA1 protein level lower in G1 phase. Too much BRCA1 protein will recruit more RAD51 into the DSB sites in G1 phase, which causes more DNA damage. In conclusion, the cyclical fluctuation of BRCA1 protein is important for its function of maintaining genomic stability. However, the mechanism of BRCA1 cyclical fluctuation expression is not clear until now.
This study finds out that neither transcription or post-translation level decides the difference of BRCA1 expression between G1 and S phase. Instead, alternative splicing mediated NMD (Nonsense mediated mRNA decay) pathway impacts BRCA1 cyclical fluctuation expression. Next, this study screens out that RBM10 (RNA-binding motif protein 10) as a splicing factor decides the difference of BRCA1 expression along cell cycle by RNA-Immunoprecipitation coupled mass spectrum. CDK2 (Cyclin-dependent kinase 2) influences RBM10's splicing function in the upstream to increase BRCA1 expression. in S phase, CDK2 phosphorylates RBM10 at Ser89 site, which enhances the normal function of RBM10, then BRCA1 is alternatively spliced to retain more full-length isoforms with normal function that cannot be degraded by NMD pathway. On the contrary, the hypo-phosphorylated RBM10 changes splicing of BRCA1 in G1 phase, making more truncated isoforms with weaker function that be degraded easily. Therefore, the level of BRCA1 protein is lower in G1 than S phase.
On the other hand, this study uncovers that alternative splicing mediated NMD pathway widely controls genes’ cyclical fluctuation expression according to the results of RNA sequencing. As for the genes regulated by RBM10, there are always difference between their expression in G1 or S phase. Knocking down RBM10 tends to reduce their expression differences. 14 BRCAness genes expression reduce after knockdown of RBM10. Besides, CDK2 influences genes expression by alternative splicing universally.
In conclusion, this study discovered that RBM10 phosphorylated by CDK2 decides the cyclical expression of BRCA1, and alternative splicing mediated NMD pathway generally impacts cyclical genes expression. In the aspect of clinical application, this study discovers that there are synergistic effects towards killing cancer cells by combination of CDK2 inhibitor and PARP inhibitor, and RBM10 is also an efficient target for cancer therapy.
 

缩略词表 1
第一章 引言 3
第二章 综述 6
2.1 BRCA1相关研究 6
2.1.1 DNA损伤修复途径 6
2.1.2 BRCA1结构与功能 8
2.1.3 BRCA1的表达调控 10
2.1.4 BRCA1与肿瘤 12
2.2 可变剪接相关研究 14
2.2.1 剪接的过程 14
2.2.2 剪接因子分类及功能 17
2.2.3 可变剪接相关的NMD通路 19
2.2.4 BRCA1的可变剪接 21
2.3 RBM10相关研究 24
2.3.1 RBM10的功能 24
2.3.2 RBM10相关的疾病 27
第三章 材料与方法 30
3.1 实验材料 30
3.1.1 细胞系 30
3.1.2 试剂 30
3.2 实验方法 36
3.2.1 细胞培养与基因敲除细胞株的构建 36
3.2.2 质粒构建、转化与提取 38
3.2.3 质粒转染 43
3.2.4 RNA干扰实验（RNAi） 44
3.2.5 慢病毒感染 44
3.2.6 蛋白印迹实验（Western blot） 45
3.2.7 RNA提取与逆转录PCR 46
3.2.8 实时荧光定量PCR（qPCR） 48
3.2.9 细胞免疫荧光 51
3.2.10 细胞周期同步化与细胞周期检测 51
3.2.11 蛋白免疫共沉淀（Co-IP） 52
3.2.12 染色质免疫共沉淀（ChIP） 53
3.2.12 RNA pull-down偶联质谱 54
3.2.13 RNA免疫共沉淀（RNA-IP） 56
3.2.14 可变剪接微基因报告系统与cDNA PCR法 57
3.2.16 蛋白磷酸化质谱 60
3.2.17 单克隆形成实验 60
3.2.18 免疫组化 61
3.2.19 HR效率实验 62
3.2.20 DNA fiber实验 62
3.2.21 裸鼠移植瘤实验 63
3.2.22 RNA-Seq及数据分析 63
第四章 结果 64
4.1 RNA剪接相关的NMD途径决定BRCA1周期波动 64
4.2 RBM10作为S期BRCA1的剪接因子 68
4.3 RBM10决定BRCA1的周期表达差异 70
4.4 RBM10通过可变剪接促进BRCA1表达 74
4.5 CDK2通过调节RBM10磷酸化影响BRCA1周期波动表达 77
4.6 RBM10通过调控BRCA1周期性表达以维持基因组稳定性 88
4.7 RBM10影响肿瘤形成并作为肿瘤治疗靶点 92
第五章 讨论 97
第六章 结论 103
参考文献 105
学位论文答辩委员会名单 112
个人简历、在学期间发表的学术论文与研究成果 113
致谢 114
北京大学学位论文原创性声明和使用授权说明 116

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