1.SRSF10在体内的剪接调控网络与机制研究 2.BCLAF1在人结直肠癌中的功能研究
文献类型：学位论文
| 作者 | 周雪霞 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-04 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 冯英 |
| 关键词 | 第一部分：选择性剪接 SRSF10 剪接调控 转录组测序 第二部分：BCLAF1 结直肠癌 肿瘤发生 SRSF10 选择性剪接 |
| 其他题名 | 1. Investigation of the network and mechanisms for SRSF10 in splicing regulation in vivo 2. Investigation of the functions for BCLAF1 in human colorectal cancer |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 第一部分中文摘要 SRSF10在体内的剪接调控网络与机制研究 RNA剪接（RNA splicing）是指从信使RNA前体（pre-mRNA）中去除内含子、连接外显子形成成熟mRNA的过程，由于剪接位点的选择不同，选择性剪接控制着从单一前体mRNA生成多种成熟mRNA的过程，因此是真核生物中继转录后调控基因表达和决定蛋白质多样性的重要层次。 SRSF10（serine-arginine rich splicing factor 10）是SR（serine-arginine rich）蛋白家族的一员，已知其在体外是一个序列特异性的剪接促进蛋白，在小鼠胚胎发育，尤其是心脏与肝脏的发育中也扮演重要角色。但是作为一个剪接调控蛋白，其在体内的剪接底物尚不清楚，并且在体内的剪接调控机制也研究甚少。 本研究利用转录组测序技术结合自主研发的生物信息分析软件，在鸡细胞系DT40中寻找受SRSF10调节的体内剪接底物网络。研究发现，SRSF10参与调节5种基本的剪接事件类型，其中以调节“盒式”（cassette）外显子接入或去除事件为主；验证的结果也表明，SRSF10同时具有增强外显子接入和抑制外显子接入的双重功能；结合序列分析发现，外显子的接入与否与SRSF10的结合位点在调控外显子上的分布有关；在功能上，SRSF10所调控的剪接事件多为应激或凋亡相关的基因；与此一致的是，在细胞表型上，SRSF10敲除的DT40细胞对内质网应激诱导的凋亡更为敏感。这些结果提示着SRSF10可能通过调节应激相关基因的选择性剪接，控制着内质网应激下的细胞存活能力。以上研究结果加深了人们对于SRSF10在体内调节的剪接网络和机制的理解，同时也证明SRSF10在应激下的细胞存活能力中发挥重要作用。 第二部分中文摘要 BCLAF1在人结直肠癌中的功能研究 肿瘤细胞中通常存在异常的选择性剪接。例如，参与细胞增殖和浸润能力的许多基因具有不同的选择性剪接亚型，其中某些特定的剪接亚型出现高表达后，将更有利于肿瘤细胞的增殖和迁移，从而促使细胞转化。这些基因的异常选择性剪接发生通常起因于RNA结合蛋白的异常表达。SR（Serine-arginine-rich）蛋白是一类研究较多的RNA结合蛋白，作为剪接调控因子，SR蛋白表达量的改变将影响细胞内的剪接调控网络。 Bcl-2相关转录因子（Bcl-2-associated transcription factor，BCLAF1）已知可参与多个生理过程。该基因受到选择性剪接影响可生成多个转录产物，但关于该基因的剪接调控机制以及异常剪接发生是否与肿瘤发生发展相关目前还不清楚。 本研究中，我们首先以结直肠癌样本为研究对象，证明了BCLAF1的外显子5a（exon5a）区域在肿瘤组织中，相比正常组织，以高比例接入。特异性敲低BCLAF1全长蛋白（BCLAF1-L），即包含exon5a的转录产物，可抑制细胞生长和克隆形成能力，并且显著降低裸鼠成瘤能力。此外，过表达BCLAF1-L可以在一定程度提高细胞的克隆形成能力，进一步确证了其在结肠癌细胞中的促癌功能。在剪接调控机制上，实验结果表明剪接蛋白SRSF10可以特异性地与exon5a结合从而促进其接入，而RNAi方法介导的SRSF10敲低可显著抑制exon5a的接入。与BCLAF1-L敲低后的表型一致，SRSF10的缺失同样影响结肠癌细胞的生长、克隆形成和成瘤能力。经过促剪接改变反义寡聚核苷酸处理，以模拟SRSF10敲低后对BCLAF1 pre-mRNA的选择性剪接改变后，细胞呈现出相似的表型，即生长的抑制和克隆形成能力的下降，由此可见SRSF10在BCLAF1的剪接调控及促进结肠癌细胞生长和存活能力中的重要性。最后，我们在临床结直肠癌样本中同样发现SRSF10呈上调表达，其高表达与BCLAF1 exon5a的高接入比例为显著的正相关，且二者均与结直肠癌的高分级相关。 因此，我们的研究表明BCLAF1-L剪接亚型和SRSF10剪接调控蛋白在结直肠癌发生发展中具有重要的生物学意义，同时，我们鉴定出SRSF10作为BCLAF1 pre-mRNA的选择性剪接调控蛋白，在结直肠癌中也发挥着促癌作用。 |
| 索取号 | D2014-082 |
| 英文摘要 | Abstract of the first part Investigation of the network and mechanisms for SRSF10 in splicing regulation in vivo RNA splicing is a process by which introns are removed from the precursor messenger RNA (pre-mRNA), and constitutive exons are joined together to produce mature mRNA isoforms. By choosing different splice sites, alternative splicing (AS) produces a diverse array of mRNA isoforms from a single primary transcript. Hence this process is an important mechanism post transcription for controlling gene expression and generating protein diversity in higher eukaryotic cells. Splicing factor SRSF10 (serine-arginine rich splicing factor 10), an atypical SR protein, is known to function as a sequence-specific splicing activator. It impacts on mouse embryonic development, especially on liver and heart development. But as a splicing regulator, little is known about its downstream splicing targets and its splicing regulation mechanisms in vivo. In this study, we used RNA-seq coupled with our newly developed Java programs to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found SRSF10 is involved in five common modes of AS, with cassette exons being the most frequent targets for SRSF10 regulation; Validation results indicated SRSF10 could promote both exon inclusion and exclusion; Motif analysis revealed exon inclusion was partially determined by location of SRSF10 binding sequence around regulatory cassette exons; Functionally, many of SRSF10-verified alternative exons are linked to stress and apoptosis pathways; Consistently, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Therefore, we conclude that SRSF10 plays a critical role in cell survival after ER stress, perhaps reflecting its ability to regulate many stress- and apoptosis-related AS events. Taken together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo, and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions. Abstract of the second part Functional studies of the role for BCLAF1 in colorectal cancer Aberrant alternative splicing events are commonly observed in cancer cells and have been implicated in many types of cancer. For example, many genes that are involved in proliferation and invasiveness are frequently alternatively spliced, and their specific splice variants, which stimulate cell proliferation and migration and thus contribute to the transformed phenotype, are often up-regulated in tumors. These changes often involve dysregulated expression of RNA-binding proteins. SR proteins (Serine/arginine-rich proteins) are well-characterized RNA-binding proteins that play critical roles in splicing regulation, and changes in the expression of these proteins candramatically affect alternative splicing profiles in cells. Bcl-2-accociated transcription factor 1 (BCLAF1) has been shown to be involved in multiple biological processes. Transcript variants encoding different isoforms that are generated by alternative splicing have been found for this gene, but little is known about the mechanisms governing its splicing regulation and whether the misregulation is associated with cancer development. In this study, we demonstrated that inclusion of BCLAF1 alternative exon 5a was significantly increased in colorectal cancer (CRC) samples. Isoform-specific knockdown of BCLAF1 full-length protein (BCLAF1-L) derived from exon5a–containing transcript inhibited cell growth and colony formation in vitro, and significantly decreased tumor formation in nude mice. Moreover, overexpression of BCLAF1-L considerably increased colony forming efficiency of cells, further confirming its pro-tumorigenic capacity in human colon cancer cells. Mechanistic analysis revealed that splicing factor SRSF10 specifically interacts with exon5a and activates its inclusion, as RNAi-mediated knockdow |
| 语种 | 中文 |
| 公开日期 | 2015-12-24 |
| 源URL | [http://202.127.25.144/handle/331004/385]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_代谢性疾病的基因表达调控研究组 |
| 推荐引用方式 GB/T 7714 | 周雪霞. 1.SRSF10在体内的剪接调控网络与机制研究 2.BCLAF1在人结直肠癌中的功能研究[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2014. |
入库方式： OAI收割
来源：上海营养与健康研究所
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