Involvement of Proteins in Splicing Group I and Group II Introns

描述(申请人提供)：拟议的研究是一项关于蛋白质参与剪接I和II组内含子的持续研究。这些内含子使用RNA催化的剪接机制，但需要蛋白质帮助将内含子RNA折叠成催化活性结构。此前，我们发现粗毛脉孢菌线粒体I组内含子剪接所需的一个关键蛋白是线粒体(Mt)酪氨酰-tRNA合成酶(TyrRS；Cyt-18蛋白)。在本授权期内的结构研究表明，I组内含子与TyrRS的核苷酸结合折叠在与tRNatyr结合的一侧，并使用一个新的RNA结合面，该表面由三个单独的插入和相对于非剪接细菌TyrRS的其他结构适配形成。此外，这些结构适应似乎是包括重要的人类和植物病原体的真菌家族Mt TyrRSs的独特特征。在拟议的研究中，我们将继续研究Cyt-18的作用机制，它是如何进化到RNA剪接中的，以及剪接活性的真菌mt TyrRSs是否可能成为抗真菌药物的靶点。对于第二组内含子，我们开发了一个基于可移动的乳酸乳球菌LI.LtrB内含子的实验系统，该内含子编码一种逆转录酶(RT)，该逆转录酶既起内含子移动作用，又作为内含子特异性剪接因子(成熟酶)发挥作用。在目前的授权期内，我们描绘了蛋白质和内含子RNA的相互作用区域，结合结构模型，提出了关于RT/成熟酶如何稳定活性RNA结构并进化为剪接功能的具体假设。在拟议的研究中，我们将使用生化、遗传和结构方法来检验这些假设，并获得第一张关于第二组内含子RT如何在RNA剪接中发挥作用的全面图片。除了稳定活性RNA结构的蛋白质外，我们还发现mt组I和II内含子的有效剪接需要死盒蛋白质，并获得证据表明它们作为RNA伴侣来破坏稳定的、不活跃的结构，这些结构是RNA折叠中的“动力学陷阱”。这些蛋白质，在粗粒酵母和酿酒酵母Mss 116p中的Cyt-19，也在其他RNA加工反应和mt翻译中发挥作用。我们的发现表明，Cyt-19和Mss 116p可能是一类DExH/D-box蛋白的创始成员，这些蛋白广泛地作为结构不同的RNA和RNA/蛋白质复合体上的RNA伴侣发挥作用，并提出了在所有生物体中存在功能类似于一般RNA伴侣的DExH/D-box蛋白并在RNA代谢中发挥重要作用的可能性。在拟议的研究中，我们将检验这些假设，并使用简易组I和组II内含子剪接试验来进一步研究和定义作为一般RNA伴侣的DExH/D-box蛋白的结构和功能特征。最后，我们将继续进行酵母遗传筛选，以寻找新的第I组和第II组内含子剪接因子，特别是第II组内含子α~5V和BL1的剪接因子。通过将这些剪接因子与死盒RNA伴侣相结合，我们希望重建这些重要的模型第II组内含子的完整剪接装置。这项研究旨在为蛋白质如何介导RNA折叠和RNA催化的反应、内含子和剪接机制的进化以及与人类疾病相关的氨基酰-tRNA合成酶、逆转录酶和DExH/D-box蛋白质的功能和进化提供新的信息。

DESCRIPTION (provided by applicant): The proposed research is a continued study of the involvement of proteins in splicing group I and II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins to help fold the intron RNA into the catalyti- cally active structure. Previously, we found that a key protein required for splicing group I introris in Neurospora crassa mitochondria is the mitochondrial (mt) tyrosyl-tRNA synthetase (TyrRS; CYT-18 protein). Structural studies during the current grant period showed that group I introns bind to the TyrRS's nucleotide-binding fold on the side opposite that which binds tRNATyr and use a new RNA-binding surface formed by three separate insertions and other structural adaptations relative to non-splicing bacterial TyrRSs. Moreover, these structural adaptations appear uniquely characteristic of the mt TyrRSs of a family of fungi that includes important human and plant pathogens. In the proposed research, we would continue to study the mechanism of action of CYT-18, how it evolved to function in RNA splicing, and whether splicing-active fungal mt TyrRSs might be a target for antifungal drugs. For group II introns, we developed an experimental system based on the mobile Lactococcus lactis LI.LtrB intron, which en- codes a reverse transcriptase (RT) that functions both in intron mobility and as an intron-specific splicing factor ("maturase"). During the current grant period, we delineated interacting regions of the protein and intron RNA, which in conjunction with structural models, suggest specific hypotheses about how RT/maturases stabilize the active RNA structure and evolved to function in splicing. In the proposed research, we would use biochemical, genetic, and structural approaches to test these hypotheses and obtain the first comprehensive picture of how a group II in- tron RT functions in RNA splicing. In addition to proteins that stabilize the active RNA structure, we found that the efficient splicing of mt group I and II introns requires DEAD-box proteins and obtained evidence that they function as RNA chaperones to disrupt stable, inactive structures that are "kinetic traps" in RNA folding. These proteins, CYT-19 in N. crassa and Mss116p Saccharomyces cerevisiae, also function in other RNA processing reactions and in mt translation. Our findings suggest that CYT-19 and Mss116p may be the founding members of a class of DExH/D- box proteins that act broadly as RNA chaperones on structurally diverse RNAs and RNA/protein complexes, and they raise the possibility that DExH/D-box proteins that function similarly as general RNA chaperones exist and play an important role in RNA metabolism in all organisms. In the proposed research, we would test these hypotheses and use the facile group I and group II intron splicing assays to further study and define the structural and functional characteristics of DExH/D-box proteins that act as general RNA chaperones. Finally, we will continue a yeast genetic screen to identify novel group I and II intron splicing factors, particularly those for the group II introns a!5v and bll By combining these splicing factors with DEAD-box RNA chaperones, we hope to reconstitute the complete splicing apparatus for these important model group II introns. This research is intended to provide novel information about how proteins mediate RNA folding and RNA-catalyzed reactions, the evolution of introns and splicing mechanisms, and the function and evolution of aminoacyl-tRNA synthetases, reverse transcriptases, and DExH/D-box proteins, all relevant to human diseases.