Factor-Dependent Splicing of Chloroplast Group II Introns

Barken II组内含子存在于细菌和细胞器基因组中，是一种核酶，通过与核pre-mRNA剪接密切相关的机制催化其自身的切除。与其他含有催化RNA片段的酶类似，蛋白质是体内有效的II族剪接所必需的。然而，只有两种II族剪接促进子的身份被报道，它们的生化作用尚不清楚。高等植物叶绿体基因组中含有大量II族内含子，为核编码剪接因子提供了潜在的靶点，本实验室之前的工作已经在玉米中发现了两个核突变，crs1和crs2，它们阻断了叶绿体中II族内含子的剪接。初步结果表明，crs1突变只阻断了一个II族内含子的剪接，而没有阻断I族内含子的剪接。相反，crs2阻断叶绿体中大多数II族内含子的剪接。这是第一个被描述的突变，阻断了叶绿体中II族顺式剪接。本研究的四个目标是：(1)进一步表征突变体表型，包括完整分析玉米叶绿体基因组中所有5‘和3’-剪接连接，单个假定的叶绿体“成熟酶”的表达，鉴定其他等位基因，双突变分析和寻找受crs1和crs2基因功能调节的rna结合活性；(2)克隆crs1和crs2基因；(3)分析与靶内含子剪接相关的crs1和crs2表达的发育和遗传调控；(4)鉴定叶绿体中调控II族内含子剪接的新核基因。这项研究的结果可能对剪接体剪接的机制和进化的理解以及rna催化的蛋白质促进反应具有启示意义。此外，该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. ***