Involvement of Proteins in Splicing Group I and Group II Introns

拟议的研究是一个继续研究蛋白质参与剪接组I和II内含子。这些 内含子使用RNA催化的剪接机制，但需要蛋白质来帮助将内含子RNA折叠成催化的剪接位点。 活性结构。以前，我们发现链孢菌中剪接I组所需的一个关键蛋白， crassa线粒体是线粒体（mt）酪氨酰-tRNA合成酶（TyrRS; CYT-18蛋白）。结构研究 在目前的资助期内，I组内含子结合到TyrRS的核苷酸结合折叠的一侧， 与结合tRNATyr的相反，并使用由三个单独插入形成的新RNA结合表面， 相对于非剪接细菌TyrRS的其他结构适应。此外，这些结构性适应似乎 这是真菌家族的mt TyrRS的独特特征，包括重要的人类和植物病原体。在 在拟议的研究中，我们将继续研究CYT-18的作用机制，它是如何进化到发挥作用的 以及剪接活性真菌mt TyrRS是否可能成为抗真菌药物的靶点。第ii组 内含子，我们开发了一个实验系统的基础上，移动的乳酸乳球菌LI. LtrB内含子，它en- 编码一种逆转录酶（RT），其在内含子迁移中起作用，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，提出关于RT/成熟酶如何稳定活性的具体假设。 RNA的结构和进化功能的剪接。在拟议的研究中，我们将使用生化，遗传， 和结构的方法来测试这些假设，并获得第一个全面的图片，如何第二组在- tron RT在RNA剪接中起作用。除了稳定活性RNA结构的蛋白质外，我们发现， 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分子伴侣破坏稳定的、无活性的结构，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Mss116p酿酒酵母，也在其他RNA加工反应和mt 翻译.我们的研究结果表明，CYT-19和Mss116p可能是一类DExH/D- 盒蛋白在结构多样的RNA和RNA/蛋白质复合物上广泛充当RNA伴侣，以及 他们提出了一种可能性，即DExH/D-box蛋白与一般RNA分子伴侣的功能相似， 在所有生物体的RNA代谢中起重要作用。在拟议的研究中，我们将测试这些假设 并利用简便的Ⅰ组和Ⅱ组内含子剪接分析进一步研究和确定其结构和功能 DExH/D-box蛋白作为一般的RNA分子伴侣。最后，我们将继续一个酵母基因- 遗传学筛选，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! 5vand 通过将这些剪接因子与DEAD-box RNA分子伴侣结合，我们希望能够重建完整的 这些重要的模型II组内含子的拼接装置。这项研究旨在提供新的信息， 关于蛋白质如何介导RNA折叠和RNA催化反应，内含子和剪接机制的进化， nisms，以及氨酰-tRNA合成酶、逆转录酶和DExH/D-box的功能和进化 蛋白质，都与人类疾病有关。

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 intrpn 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 assaysto 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 ge- netic screen to identify novel group I and II intron splicing factors, particularly those for the group II introns a!5vand bll Bycombining these splicing factors with DEAD-box RNAchaperones, 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 mecha- nisms, and the function and evolution of aminoacyl-tRNA synthetases, reverse transcriptases, and DExH/D-box proteins, all relevant to human diseases.