Factor-Dependent Splicing of Chloroplast Group II Introns

许多重要的生物过程涉及结构复杂的rna，这些rna与核糖核蛋白颗粒（RNPs）中的蛋白质复合物。RNA和蛋白质组分在RNPs中的作用是多种多样的。在某些情况下，RNA亚基具有催化活性，蛋白质有助于增强活性RNA结构的形成或稳定性。在其他情况下，rna为具有催化活性的蛋白质提供组装支架。众所周知，RNA和蛋白质分子合作形成底物结合表面，原则上，它们可以合作形成活性位点。本项目以包含“自剪接”II组内含子的RNPs为工具，探索RNPs中RNA和蛋白质之间的合作模式。II组内含子是高度结构化的催化rna，需要蛋白质在体内有效地发挥作用。II组内含子RNPs是解剖RNA和蛋白质组分在RNP组装和功能中的作用的极好模型，因为它们相对简单，并且因为促进内含子功能的蛋白质可以通过基因鉴定然后在体外研究。本项目重点研究叶绿体II族内含子剪接所需的三种核编码蛋白CRS2、CAF1和CAF2。CRS2与肽基trna水解酶有关。CAF1和CAF2彼此密切相关，属于一个不同的植物蛋白家族，该家族还包括II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复合物介导叶绿体中不同内含子组的剪接，并在体内结合到其遗传定义的内含子靶点上。以下工作模型是从先前的工作中产生的，并将在本项目的过程中进行测试：(A) CRM结构域因子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上；(C) CRS2保守的肽基trna水解酶活性位点可能在剪接催化过程中贡献官能团。不同但互补的实验方法利用了缺乏三种因子中的每一种的突变株的可用性，三种蛋白质的抗血清，以及每种蛋白质的经过验证的重组表达系统。

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.