Involvement of Proteins in Splicing Group I and Group II Introns

描述（由申请人提供）：拟议的研究是对蛋白质参与剪接组I和II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。以前，我们发现，拼接粗糙脉孢菌线粒体中I组introris所需的关键蛋白质是线粒体（mt）酪氨酰-tRNA合成酶（TyrRS; CYT-18蛋白）。在目前的授权期间的结构研究表明，I组内含子结合到TyrRS的核苷酸结合折叠的相对侧，结合tRNATyr，并使用一个新的RNA结合表面形成的三个独立的插入和其他结构适应相对于非剪接细菌TyrRS。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的mt TyrRS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，它如何进化到在RNA剪接中发挥作用，以及剪接活性真菌mt TyrRS是否可能成为抗真菌药物的靶点。对于II组内含子，我们开发了一个基于移动的乳酸乳球菌LI.LtrB内含子的实验系统，该内含子编码逆转录酶（RT），该逆转录酶在内含子移动中起作用并作为内含子特异性剪接因子（“成熟酶”）。在目前的资助期间，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，我们将使用生物化学，遗传学和结构的方法来测试这些假设，并获得第一个全面的图片，如何组II的内含子RT功能的RNA剪接。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DEAD盒蛋白，并获得了它们作为RNA伴侣蛋白破坏稳定的非活性结构的证据，这些结构是RNA折叠中的“动力学陷阱”。这些蛋白质，CYT-19在N. crassa和Mss 116 p酿酒酵母也在其它RNA加工反应和mt翻译中起作用。我们的研究结果表明，CYT-19和Mss 116 p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多样的RNA和RNA/蛋白质复合物上的RNA伴侣，并且它们提高了DExH/D-box蛋白作为一般RNA伴侣的功能类似地存在并在所有生物体中的RNA代谢中发挥重要作用的可能性。在拟议的研究中，我们将测试这些假设，并使用简单的组I和组II内含子剪接测定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，我们将继续酵母遗传筛选，以确定新的第一和第二组内含子剪接因子，特别是那些第二组内含子a！5v和bll通过将这些剪接因子与DEAD盒RNA伴侣结合，我们希望重建这些重要的模型II组内含子的完整剪接装置。这项研究旨在提供有关蛋白质如何介导RNA折叠和RNA催化反应，内含子和剪接机制的演变，以及氨酰-tRNA合成酶，逆转录酶和DExH/D-box蛋白的功能和演变的新信息，所有这些都与人类疾病有关。

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.