内含子剪接机制从酵母到拟南芥进化过程的研究

Intron splicing is an essential process in eukaryotic gene expression. Recent studies have revealed that splicing is not just removal of the intervening sequences in eukaryotic genes, rather it's tightly associated with almost every steps in gene expression including transcription, mRNA processing, mRNA stability, nuclear export, and translation. In addition, the events taken place in the nucleus including splicing are also linked to other cellular pathways such as ribosome biogenesis which will eventually lead to the control of cell growth. Although there has been a great advancement in our understanding about the factors and the machinery involved in intron splicing, the detailed mechanism of the biochemical reaction is still not well elucidated nor the intertwined network connecting splicing and other cellular processes. From our previous studies, we noticed that most plant introns cannot be effectively spliced in yeast cells. This observation prompts us to pursue a better understanding on the splicing mechanism from probing the differential requirements and functions of the cis- and trans-acting factors through evolution from yeast to plant. The information learned from this study will also help us appreciate the significance of the different strategies adopted by these organisms in intron splicing. In order to preserve the inherent structure and the physiological function of the plant target gene, we constructed a series of mutant constructs based on a simplified Arabidopsis RPL36B gene. The simplified Arabidopsis RPL36B gene is highly analogous to the natural Arabidopsis RPL36B gene except that the 2nd and the 3rd downstream introns were removed. In addition, the promoter and the 3' UTR were also replaced by the corresponding parts from yeast RPL36B gene. All the mutations were generated in the first intron derived from Arabidopsis RPL36B gene. We then transformed these mutant constructs to a yeast strain lacking both of the endogenous RPL36A and RPL36B genes but sustained by a RPL36B gene in a plasmid expressing uracil selection marker. After treatment with 5-FOA to evict the plasmid containing the yeast RPL36B gene, the Arabidopsis mutant construct becomes the only source of RPL36 proteins to the cells. Preliminary experiments have demonstrated a promising potential with this experimental design. We have identified the 5' splice site and the branch point as the critical components that differentiate the splicing mechanisms in these two organisms. We will further investigate the differences in the interacting factors pertinent to these two cis-elements. With the availability of this system, we are going to perform microarray analysis to screen for the coordinate changes in gene expression when cells strive to accommodate a mutant construct that cannot be efficiently spliced. This information will be very helpful to deduce the cellular processes that are coupled with intron splicing.

近年的研究让我们对内含子的功能与意义有了更深的认识，但也提出了新的问题。实验表明内含子可以影响转录、mRNA的修饰、稳定性、出核运输、和翻译效率等不同步骤的基因表达过程。同时内含子剪接反应也和细胞内的其他生化路径相互串联，如与核糖体的生成密切相关，进而影响细胞的生长。想要深入了解内含子的这些作用，或许要从较宽广的角度来认识内含子的剪接机制。我们注意到大部分植物的内含子不能在酵母细胞中剪接，希望藉着探索从酵母到植物进化过程中影响内含子剪接的順式元件和反式因子的相应变化，更深入地了解参与内含子剪接的要素。为了尽量保持待测目标基因的原有结构和生理功能，我们构建了一系列简化的拟南芥核糖蛋白RPL36B突变基因，转化至缺失内源酵母RPL36基因的细胞中，以便观察拟南芥突变基因在酵母细胞中的剪接效率及对细胞生长的效应。初步的实验结果肯定此检测系统的良好潜力，也有利于探索细胞内和内含子剪接相关联的反应。