Factor-Dependent Splicing of Chloroplast Group II Introns

在细菌和细胞器基因组中发现的Barken Group II内含子是通过与核前mRNA剪接密切相关的机制催化其自身切除的核酶。类似于含有催化RNA部分的其他酶，蛋白质是体内有效II组剪接所需的。然而，只有两个第二组剪接促进剂的身份已被报道，他们的生化作用还不清楚。高等植物的叶绿体基因组中含有大量的II组内含子，为核编码的剪接因子提供了潜在的靶点。本实验室以前的工作已经确定了玉米中的两个核突变，crs1和crs2，它们阻断了叶绿体中II组内含子的剪接。初步结果表明，crs1突变阻断了一个II组内含子的剪接，而没有I组内含子。相反，crs2阻断叶绿体中大多数II组内含子的剪接。这些是第一个描述的突变，阻止组II顺式剪接叶绿体。本研究的主要目的是：（1）进一步鉴定突变体的表型，包括对玉米叶绿体基因组中所有5'和3'剪接点的完整分析，单一叶绿体“成熟酶”的表达，额外等位基因的鉴定，双突变体的分析以及受crs1和crs2基因功能调控的RNA结合活性的研究，（2）crs1和crs2基因的克隆，（3）crs1和crs2表达与其靶内含子剪接相关的发育和遗传控制的分析，和（4）调节叶绿体中II组内含子剪接的新核基因的鉴定。 这项研究的结果可能对理解剪接体剪接的机制和演变以及RNA催化的蛋白质促进的反应具有普遍意义。此外，本项目将提高我们对调控RNA剪接在叶绿体发育中的作用和机制的理解。 ***

9630213 Barken Group II introns, found in bacterial and organelle genomes, are ribozymes that catalyze their own excision via a mechanism that is closely related to that of nuclear pre-mRNA splicing. In analogy to other enzymes containing catalytic RNA moieties, proteins are required for efficient group II splicing in vivo. However the identities of only two facilitators of group II splicing have been reported and their biochemical roles are not understood. Chloroplast genomes of higher plants contain numerous group II introns, providing potential targets for nuclear-encoded splicing factors, Previous work from this laboratory has identified two nuclear mutations in maize, crs1 and crs2, that block the splicing of group II introns in the chloroplast. Preliminary results suggest that the crs1 mutation blocks the splicing of just one group II intron and of no group I introns. In contrast, crs2 blocks the splicing of most group II introns in the chloroplast. These are the first described mutations that block group II cis-splicing in chloroplasts. Four aims of this work are,(1) Further characterization of the mutant phenotypes, including a complete analysis of all 5' and 3'-splice junctions in the maize chloroplast genome, the expression of the single putative chloroplast "maturase", identification of additional alleles, double-mutant analysis and a search for RNA-binding activities that are modulated by crs1 and crs2 gene function, (2) Cloning of the crs1 and crs2 genes, (3) Analysis of the developmental and genetic control of crs1 and crs2 expression correlated with the splicing of their target introns, and (4) Identification of new nuclear genes that modulate the splicing of group II introns in the chloroplast. %%% The results of the study may have implications for the understanding of the mechanisms and evolution of spliceosomal splicing and for RNA-catalyzed, protein-facilitated reactions in general. In addition, this project will improve our understanding of the role and mechanisms of regu lated RNA splicing in chloroplast development. ***