Phosphorylation-dependent regulation of splicing and transport of mRNA

mRNA 剪接和运输的磷酸化依赖性调节

基本信息

批准号：
14035102
负责人：
HAGIWARA Masatoshi
金额：
$ 46.14万
依托单位：
Tokyo University of Agriculture and Technology (2006)Tokyo Medical and Dental University (2002-2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

项目摘要

1) RNA export factor, REF is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. To analyze the effect of transcription on the EJC formation, we developed a coupled in vitro system for examining transcription/splicing reactions. Analysis of the RNA-binding complexes revealed that REF associated with β-globin mRNA at regions other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and further analysis show that the deposition of REF apart from the EJC is dependent on 5' cap structure, but not splicing. Excess amounts of m7GpppG cap analog reduced REF binding to intronless mRNA, and a co-immunoprecipitation experiment revealed that REF interacts with the cap-binding protein CBP20. The export of Cy3-labeled intronless β-globin mRNA from nuclei of HeLa cells was enhanced by co-injection of CBP20 and REF. Thus, REF recruited by CBP20 may play a stimulatory role to e … More xport the capped intronless mRNAs.2) As many as two thirds of human genes have alternative mRNA isoforms. What is a spatiotemporal expression profile of each isoform? How are so many genes regulated in vivo? Regulation mechanisms of alternative splicing, however, have been studied mostly in vitro or in cultured cells.We have developed a transgenic alternative splicing reporter system that visualizes expression profiles of mutually exclusive alternative exons of a nematode C. elegans at a single cell level in vivo (nature methods, 2006). With this reporter system, we have visualized spatiotemporal profiles of tissue-specific and developmentally regulated alternative splicing events in living worms. By isolating and analyzing mutant worms defective in the color profiles, we have identified transacting factors and cis-elements involved in the splicing regulation (Mol Cell Biol, 2007; Genes Dev, 2008). Through these studies, we are coming to realize that molecular mechanisms of the alternative splicing regulation are conserved throughout metazoan evolution. Our reporter system will further elucidate expression profiles and regulation mechanisms of alternative splicing in vivo.3) To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we isolated and characterized exon-skipping mutants in fission yeast. The results showed that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF^<59>-U2AF^<23> complex is essential for ordered exon joining in constitutive splicing. In addition, the analyses of the mRNA export mutants revealed that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus. Our results contributed elucidation of molecular mechanisms of pre-mRNA splicing and nuclear mRNA export. Less

1）RNA输出因子REF是外显子连接络合物（EJC）的组成部分，以剪接依赖性方式沉积在mRNA上，并靶向拼接的mRNA进行导出。为了分析转录对EJC形成的影响，我们开发了一个耦合的体外系统，用于检查转录/剪接反应。对RNA结合复合物的分析表明，除EJC沉积位点以外的其他区域，REF与β-珠蛋白mRNA相关。 RNA聚合酶II和T7转录和进一步分析之间的比较表明，EJC与EJC分开的沉积取决于5'CAP结构，但不取决于剪接。过量的M7GPPPG CAP模拟降低了REF与内在的mRNA的结合，并且共免疫沉淀实验表明，REF与帽结合蛋白CBP20相互作用。通过共同注射CBP20和参考，从HeLa细胞核中的Cy3标记的无内在的无β-珠蛋白mRNA导出。这是CBP20招募的参考，可能会发挥刺激作用。每种同工型的时空表达曲线是什么？这么多基因在体内如何调节？但是，替代剪接的调节机制主要是在体外或培养的细胞中研究的。我们已经开发了一种转基因替代剪接报告基因系统，该系统可视化秀丽隐杆线虫在体内单个细胞水平上的互联物C. exemans的互斥外显子的表达曲线（自然方法，2006年）。借助此记者系统，我们可以看到生命蠕虫中组织特异性和开发的替代剪接事件的时空谱。通过分离和分析颜色曲线中有缺陷的突变蠕虫，我们已经确定了与剪接调节有关的交易因子和顺式元素（Mol Cell Biol，2007; Genes Dev，2008）。通过这些研究，我们开始意识到替代剪接调节的分子机制在整个后代进化过程中均得到保存。我们的报告基因系统将进一步阐明体内替代剪接的表达谱和调控机制。3）确定与该机制相关的基因，以确保在组成型剪接的前MRNA中订购5'至3'外显子，我们分离并表征了腐蚀性酵母中的外显子 - 屈服突变体。结果表明，SF1-U2AF^<59> -U2AF^<23>复合物对新生pre-mRNA的共透明识别对于在本构剪接中连接的有序外显子至关重要。此外，对mRNA输出突变体的分析表明，酵母中mRNA的子集通过与核仁的短暂关联导出了酵母中的mRNA。我们的结果促进了MRNA剪接和核mRNA输出的分子机制。较少的