Involvement of Proteins in Splicing Group I and Group II Introns

这项拟议的研究是对蛋白质参与剪接I和II组内含子的继续研究。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折叠到催化的 Cally Active结构。以前，我们发现在脉孢子虫中剪接I组内含子所需的一个关键蛋白 粗面线粒体是线粒体酪氨酰-tRNA合成酶(TyrRS；Cyt-18蛋白)。结构研究 在当前的授权期内，显示I组内含子与TyrRS的核苷酸结合折叠在一侧 与结合tRNatyr并使用由三个单独的插入形成的新的RNA结合表面的相反 与非剪接细菌TyrRs相关的其他结构适应。此外，这些结构性调整出现了 包括重要的人类和植物病原体在内的真菌家族中的Mt TyrRS的独特特征。在……里面 在提出的研究中，我们将继续研究细胞色素T-18的作用机制，它是如何进化到功能的 在RNA剪接中，以及剪接活性的真菌mt TyrRS是否可能成为抗真菌药物的靶点。对于第II组 LtrB内含子的基础上，我们开发了一套实验系统，该系统能够增强Lactostres LI.LtrB的功能。 编码逆转录酶(RT)，在内含子移动性和内含子特异性剪接因子中都起作用 (“成熟”)。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，这 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结构，并进化为剪接功能。在拟议的研究中，我们将使用生化、遗传、 和结构性方法来检验这些假设，并获得第一个全面的图景，说明第二组是如何- Tron RT在RNA剪接中发挥作用。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，并获得它们功能的证据 RNA伴侣破坏稳定的、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，细胞色素T-19 在粗枝杆菌和酿酒酵母Mss 116P中，也在其他RNA加工反应中起作用，在Mt. 翻译。我们的发现提示，Cyt-19和Mss116p可能是DExH/D-D型的创始成员。 盒蛋白，在结构不同的RNA和RNA/蛋白质复合体上广泛充当RNA伴侣，以及 它们增加了DExH/D-box蛋白存在并发挥作用的可能性，DExH/D-box蛋白的功能与普通RNA伴侣相似 在所有生物体的RNA新陈代谢中起着重要作用。在拟议的研究中，我们将检验这些假设 并利用易化组I和组II内含子剪接实验进一步研究和确定其结构和功能 作为一般RNA伴侣的DExH/D-box蛋白的特性。最后，我们将继续进行酵母菌- 磁性筛选鉴定新的I组和II组内含子剪接因子，特别是II组内含子a！5vand Bll通过将这些剪接因子与死盒RNA伴侣结合起来，我们希望重建完整的 这些重要的模式II内含子的剪接装置。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催化的反应，内含子的进化和剪接机制- 以及氨基酰-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.