Involvement of Proteins in Group I + II Intron Splicing

描述(申请人提供)：拟议的研究是一项关于蛋白质参与剪接第一组和第二组内含子的持续研究。这些内含子使用RNA催化的剪接机制，但需要蛋白质在体内进行有效剪接，以帮助将内含子RNA折叠到催化活性结构中。我们先前已经证明粗枝脉孢霉线粒体酪氨酸氨基-tRNA合成酶(CyT-18蛋白)识别I组内含子催化核心保守的tRNA样结构特征，并且其本身足以在体外促进不同I组内含子的剪接。在目前的资助期间，我们获得了一个剪接竞争的CyT-18蛋白的2.5 Angstrom晶体结构，并确定了一个与CyT-18协同作用的死盒蛋白(CyT-19)，它通过作为ATP依赖的RNA伴侣来促进I组内含子的剪接。在拟议的研究中，我们将继续对CyT-18及其与I组内含子RNA的复合物进行结构分析，并以CyT-19为模型系统，研究DExH/D-box蛋白如何介导RNA构象变化并获得其靶RNA的特异性。对于第二组内含子，我们开发了一个以可移动的乳酸乳球菌L1.LtrB内含子为中心的实验系统，该内含子编码一种逆转录酶(LTRA蛋白)，其功能是移动性和作为内含子特异性剪接因子(成熟酶)。在目前的授权期内，研究导致了一种模型，在该模型中，LTRA蛋白通过首先与内含子亚域DIVA中的高亲和力结合位点(LTRA编码区开始的一种特殊结构)结合来促进剪接，然后与催化核心的保守区进行额外的接触，将RNA折叠到催化活性结构中。在拟议的研究中，我们将继续研究成熟酶促进第II组内含子剪接的机制，探索DExH/D-box蛋白在第II组内含子剪接中的参与，并研究成熟酶是否可以进化为一般的第II组内含子剪接因子，这可能反映剪接体内含子进化的关键步骤。具体目标是：(1)继续对粗面拟青霉细胞色素T-18蛋白及其与I组内含子RNA的复合体进行结构分析。(2)继续研究Dead-box蛋白Cyt-19在第一组内含子剪接中的作用及其靶向依赖细胞色素T-18的第一组内含子。(3)继续探讨成熟酶促进第二组内含子剪接的机制。(4)探讨DExH/D-box蛋白在第二组内含子剪接中的作用。(5)探索一些成熟酶，包括叶绿体Matk蛋白，是否可以进化到剪接多个II组内含子的功能。这项研究旨在为蛋白质如何介导RNA折叠和RNA催化的反应提供新的信息，并深入了解内含子和剪接机制的进化，这对高等生物的基因表达至关重要。

DESCRIPTION (provided by applicant): The proposed research is a continued study of the involvement of proteins in splicing group I and group II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. We showed previously that the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) recognizes conserved tRNA-like structural features of the group I intron catalytic core and is by itself sufficient to promote the splicing of different group I introns in vitro. During the current grant period, we obtained a 2.5Angstrom crystal structure of a splicing-compete CYT-18 protein and identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing by acting as an ATPdependent RNA chaperone. In the proposed research, we would continue structural analysis of CYT-18 and its complexes with group I intron RNAs and use CYT-19 as a model system for studying how DExH/D-box proteins mediate RNA conformational changes and acquire specificity for their target RNAs. For group II introns, we have developed an experimental system centered around the mobile Lactococcus lactis L1.LtrB intron, which encodes a reverse transcriptase (LtrA protein) that functions in mobility and as an intron-specific splicing factor ("maturase"). Studies during the current grant period have led to a model in which the LtrA protein promotes splicing by binding first to a high affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding region, and then makes additional contacts with conserved regions of the catalytic core to fold the RNA into the catalytically active structure. In the proposed research, we would continue to study the mechanism of maturase-promoted group II intron splicing, explore the involvement of DExH/D-box proteins in group II intron splicing, and investigate whether maturases can evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns. Specific aims are: (1) To continue structural analysis of the N. crassa CYT-18 protein and its complexes with group I intron RNAs. (2) To continue to investigate how the DEAD-box protein CYT- 19 functions in group I intron splicing and its targeting to CYT-18-dependent group I introns. (3) To continue to investigate the mechanism of maturase-promoted group II intron splicing. (4) To explore the role of DExH/D-box proteins in group II intron splicing. (5) To explore whether some maturases, including chloroplast MatK proteins, can evolve to function in splicing multiple group II introns. This research is intended to provide novel information about how proteins mediate RNA folding and RNA-catalyzed reactions, as well as insight into the evolution of introns and splicing mechanisms, which are fundamentally important for gene expression in higher organisms.