Involvement of Proteins in Group I + II Intron Splicing

描述（由申请人提供）：拟议的研究是对蛋白质参与剪接I组和II组内含子的持续研究。这些内含子使用RNA催化剪接机制，但需要在体内进行有效剪接的蛋白质来帮助将内含子RNA折叠成具有催化活性的结构。我们之前的研究表明，粗神经孢子虫线粒体酪氨酸- trna合成酶（CYT-18蛋白）识别I族内含子催化核心的保守trna样结构特征，并且其本身足以在体外促进不同I族内含子的剪接。在目前的资助期内，我们获得了剪接竞争CYT-18蛋白的2.5埃晶体结构，并鉴定了一个DEAD-box蛋白（CYT-19），该蛋白通过作为atp依赖的RNA伴侣，与CYT-18协同作用，促进I组内含子剪接。在拟开展的研究中，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，并将CYT-19作为模型系统，研究DExH/D-box蛋白如何介导RNA构象变化并获得对其靶RNA的特异性。对于II组内含子，我们开发了一个以移动乳酸乳球菌L1为中心的实验系统。LtrB内含子，它编码一种逆转录酶（LtrA蛋白），该酶具有移动性和作为内含子特异性剪接因子（“成熟酶”）的功能。当前资助期的研究已经建立了一个模型，其中LtrA蛋白首先结合到内含子亚结构域DIVa的高亲和力结合位点（LtrA编码区开始的特殊结构），然后与催化核心的保守区域进行额外接触，将RNA折叠成催化活性结构，从而促进剪接。在接下来的研究中，我们将继续研究成熟酶促进II组内含子剪接的机制，探索DExH/D-box蛋白在II组内含子剪接中的作用，并研究成熟酶是否可以进化成一般的II组内含子剪接因子，从而可能反映剪接体内含子进化的关键步骤。具体目的是：(1)继续对草苔CYT-18蛋白及其与I组内含子rna复合物进行结构分析。(2)继续研究DEAD-box蛋白CYT- 19在I组内含子剪接中的作用及其对依赖CYT-18的I组内含子的靶向作用。(3)继续研究成熟酶促进II组内含子剪接的机制。(4)探讨DExH/D-box蛋白在II组内含子剪接中的作用。(5)探讨包括叶绿体MatK蛋白在内的一些成熟酶是否可以进化到剪接多个II族内含子的功能。本研究旨在提供关于蛋白质如何介导RNA折叠和RNA催化反应的新信息，并深入了解内含子的进化和剪接机制，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.