Factor-Dependent Splicing of Chloroplast Group II Introns

许多基本的生物学过程涉及结构复杂的RNA，这些RNA与核糖核蛋白颗粒(RNPs)中的蛋白质复合。RNPs中RNA和蛋白质成分的贡献是不同的。在某些情况下，RNA亚基具有催化活性，蛋白质用于增强活性RNA结构的形成或稳定性。在其他方面，RNA为催化活性蛋白质提供了组装支架。已知RNA和蛋白质分子协同作用形成底物结合表面，原则上它们可以协同作用形成活性部位。该项目使用涉及第二组内含子“自剪接”的RNPs作为工具，探索RNPs中RNA和蛋白质之间的合作模式。第二组内含子是高度结构的催化RNA，需要蛋白质在体内有效地发挥作用。第二组内含子RNPs是剖析RNA和蛋白质组分在RNP组装和功能中作用的优秀模型，因为它们相对简单，而且促进内含子功能的蛋白质可以从基因上鉴定出来，然后在体外进行研究。该项目的重点是三个核编码蛋白，CRS2，CAF1和CAF2，它们是叶绿体中第二组内含子剪接所必需的。CRS2与肽基-tRNA水解酶相关。CAF1和CAF2相互之间关系密切，属于一个不同的植物蛋白家族，该家族还包括第二组内含子剪接因子CRS1。CAF1/CAF2/CRS1家族的定义特征是一个以前未被识别的古老起源的RNA结合结构域，最近被命名为CRM结构域。先前的数据表明，CRS2/CAF2和CRS2/CAF1复合体介导叶绿体中不同内含子集的剪接，并在体内与其基因定义的内含子靶标结合。以下工作模型已从先前的工作中产生并将在本项目过程中进行测试：(A)CRM域因子CAF1和CAF2具有内含子特异性结合活性并影响内含子折叠；(B)CRS2通过与CAF的相互作用被招募到特定的内含子；(C)CRS2的S保守的肽-tRNA水解酶活性部位可能在剪接催化过程中贡献功能基团。不同但互补的实验方法利用了缺乏三种因子中每一种的突变菌株的可用性，三种蛋白质中每一种的抗血清，以及针对每种蛋白质的成熟的重组表达系统。

Many essential biological processes involve intricately structured RNAs that are complexed with proteins in ribonucleoprotein particles (RNPs). The contributions of the RNA and protein components in RNPs are varied. In some instances, RNA subunits harbor catalytic activity and the proteins serve to enhance the formation or stability of the active RNA structure. In others, RNAs provide an assembly scaffold for catalytically-active proteins. RNA and protein molecules are known to cooperate to form substrate binding surfaces and, in principle, they could cooperate to form an active site. This project uses RNPs involving "self-splicing" group II introns as tools to explore modes of cooperation between RNA and protein in RNPs. Group II introns are highly structured catalytic RNAs that require proteins to function efficiently in vivo. Group II intron RNPs are excellent models for dissecting the roles of RNA and protein components in RNP assembly and function because they are relatively simple, and because proteins that facilitate intron function can be identified genetically and then studied in vitro. This project focuses on three nucleus-encoded proteins, CRS2, CAF1, and CAF2, which are required for the splicing of group II introns in the chloroplast. CRS2 is related to peptidyl-tRNA hydrolase enzymes. CAF1 and CAF2 are closely related to one another, and belong to a diverse family of plant proteins that also includes the group II intron splicing factor CRS1. The defining feature of the CAF1/CAF2/CRS1 family is a previously unrecognized RNA binding domain of ancient origin, recently named the CRM domain. Prior data indicate that CRS2/CAF2 and CRS2/CAF1 complexes mediate the splicing of different intron sets in the chloroplast and are bound in vivo to their genetically-defined intron targets. The following working model has emerged from prior work and will be tested during the course of this project: (A) the CRM domain factors CAF1 and CAF2 harbor intron-specific binding activity and influence intron folding; (B) CRS2 is recruited to specific introns via interaction with a CAF; (C) CRS2's conserved peptidyl-tRNA hydrolase active site may contribute functional groups during splicing catalysis. The varied but complementary experimental approaches take advantage of the availability of mutant strains lacking each of the three factors, antisera to each of the three proteins, and proven recombinant expression systems for each protein.