Phosphorylation-dependent regulation of splicing and transport of mRNA

1)RNA输出因子REF是外显子连接复合物（EJC）的一种组分，以剪接依赖性方式沉积在mRNA上，并靶向剪接的mRNA用于输出。为了分析转录对EJC形成的影响，我们开发了一种用于检测转录/剪接反应的体外偶联系统。对RNA结合复合物的分析表明，REF与EJC沉积位点以外的区域的β-珠蛋白mRNA相关。RNA聚合酶II和T7转录之间的比较和进一步的分析表明，REF的沉积除了EJC是依赖于5'帽结构，而不是剪接。过量的m7 GpppG帽类似物减少REF与无内含子mRNA的结合，并且免疫共沉淀实验揭示REF与帽结合蛋白CBP 20相互作用。共注射CBP 20和REF可促进Cy 3标记的无内含子β珠蛋白mRNA从HeLa细胞核的输出，因此，CBP 20募集的REF可能对HeLa细胞的凋亡起刺激作用。 ...更多信息 2）多达三分之二的人类基因具有替代的mRNA同种型。每个亚型的时空表达谱是什么？那么多基因在体内是如何调控的呢？选择性剪接的调控机制主要是在体外或培养细胞中研究的，我们已经开发了一个转基因选择性剪接报告系统，该系统可以显示线虫C. elegans在体内单细胞水平（nature methods，2006）。有了这个报告系统，我们已经可视化的时空分布的组织特异性和发育调节的选择性剪接活蠕虫事件。通过分离和分析在颜色谱中有缺陷的突变蠕虫，我们已经鉴定了参与剪接调节的反式作用因子和顺式元件（Mol Cell Biol，2007; Genes Dev，2008）。通过这些研究，我们逐渐认识到选择性剪接调控的分子机制在后生动物的进化过程中是保守的。我们的报告系统将进一步阐明体内选择性剪接的表达谱和调控机制。3）为了鉴定参与确保组成性剪接前体mRNA中5'至3'外显子有序连接的机制的基因，我们分离并表征了裂殖酵母中的外显子跳跃突变体。结果表明，SF 1-U2 AF ^ -U2 AF ^复合物对新生前mRNA的共转录识别<59><23>对于组成性剪接中有序的外显子连接是必不可少的。此外，mRNA输出突变体的分析表明，酵母中的mRNA的一个子集是通过与核仁的瞬时关联从细胞核输出的。我们的研究结果有助于阐明前体mRNA剪接和核mRNA输出的分子机制。少

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