Group II Intron and Related Reverse Transcriptases

项目总结/摘要 拟议的研究包括继续研究第二组内含子和相关的逆转录酶（RT）， 它们的生物学功能，逆转录机制和RNA-seq应用，包括分析 用于RNA诊断和液体活检的人细胞外囊泡和血浆RNA。第二组内含子RT 由称为移动的II组内含子的细菌逆转录元件编码，这被认为是进化和- 高等生物中内含子和逆转录元件的前体。组II内含子RT在结构和功能上不同 从逆转录病毒RTs，并具有新的生物化学特性，可用于RNA-seq和其他生物技术应用， 阳离子。细菌还编码多种与II组内含子RT密切相关的其他RT，包括 在CRISPR-Cas系统中的RNA间隔区获取中起作用的RT，以及多个类别的独立的 已经获得细胞功能的RT。目前，人们对这些RT或其潜在的 用于生物技术应用。在以前的工作中，我们开发了表达第II组的一般方法 内含子和相关RTs的方法，并将这些方法应用于来自细菌的第II组内含子RTs， 将所述聚合物转化为热稳定的第II组内含子RT（TGIRT）。我们发现TGIRT具有更高的 保真度和持续合成能力，以及一种新的模板转换活性， RNA-seq衔接子添加。利用这些特性，我们开发了基于TGIRT的方法， 高通量RNA测序（TGIRT-seq），实现了难以或不可能实现的应用 与其他目前可用的RT，包括几乎所有的RNA生物型的同时分析，从小 起始材料的量。我们证明了这些方法用于分析人类细胞的有效性。 更大、细胞外囊泡和血浆RNA，并已开始探索其临床应用。此外，本发明还 我们首次获得了与模板引物复合的全长TGIRT的X射线晶体结构， 传入的dNTP，为解决有关反向转录的主要问题提供了结构基础- 第二组内含子RTs的作用机制、RTs的进化起源和第二组内含子的工程改造 RT具有改进的生物技术应用性能。在拟议的研究中，我们将调查 逆转录机制和结构基础的独特的生化性质的第二组 内含子RTs，探索RTs的进化起源及其与RNA依赖性RNA聚合酶的关系， 继续研究CRISPR相关的RT，并将这些研究扩展到新类型的细菌RT。毛皮- 因此，我们将继续发展TGIRT-seq方法，并跟踪我们以前的TGIRT-seq的关键发现 人类细胞，细胞外囊泡和血浆RNA的分析，包括机制和调节， 细胞外囊泡RNA包装和分泌，新的小非编码RNA的表征，以及 3'端tRNA短片段的功能。最后，我们将继续开发TGIRT-seq的临床应用， 包括RNA诊断和癌症及其他人类疾病的液体活检的新方法。

PROJECT SUMMARY/ABSTRACT The proposed research involves continued studies of group II intron and related reverse transcriptases (RTs), their biological functions, reverse transcription mechanisms, and RNA-seq applications, including the analysis of human extracellular vesicle and plasma RNAs for RNA diagnostics and liquid biopsy. Group II intron RTs are encoded by bacterial retroelements called mobile group II introns, which are thought to be evolutionary an- cestors of introns and retroelements in higher organisms. Group II intron RTs differ structurally and functionally from retroviral RTs and have novel biochemical properties useful for RNA-seq and other biotechnological appli- cations. Bacteria also encode a variety of other RTs that are closely related to group II intron RTs, including RTs that function in RNA spacer acquisition in CRISPR-Cas systems, as well as multiple classes of free-stand- ing RTs that have acquired cellular functions. At present, little is known about these RTs or their potential for use in biotechnological applications. In previous work, we developed general methods for expressing group II intron and related RTs with high yield and activity and applied these methods to group II intron RTs from bacte- rial thermophiles to obtain Thermostable Group II Intron RTs (TGIRTs). We found that TGIRTs have higher fidelity and processivity than retroviral RTs as well as a novel template-switching activity that enables facile RNA-seq adapter addition. Taking advantage of these properties, we developed TGIRT-based methods for high-throughput RNA sequencing (TGIRT-seq) that enable applications that would be difficult or impossible with other currently available RTs, including the simultaneous profiling of nearly all RNA biotypes from small amounts of starting material. We demonstrated the efficacy of these methods for the analysis of human cellu- lar, extracellular vesicle, and plasma RNAs and have begun to explore their clinical applications. Additionally, we obtained a first-of-its kind X-ray crystal structure of a full-length TGIRT in complex with template-primer and incoming dNTP, providing a structural foundation for addressing major questions about the reverse transcrip- tion mechanism of group II intron RTs, the evolutionary origin of RTs, and the engineering of group II intron RTs with improved properties for biotechnological applications. In the proposed research, we will investigate the reverse transcription mechanism and structural basis for the distinctive biochemical properties of group II intron RTs, explore the evolutionary origin of RTs and their relationship to RNA-dependent RNA polymerases, continue studies of CRISPR-associated RTs, and extend these studies to new classes of bacterial RTs. Fur- ther, we will continue to develop TGIRT-seq methods and follow up key findings from our previous TGIRT-seq analyses of human cellular, extracellular vesicle, and plasma RNAs, including the mechanism and regulation of extracellular vesicle RNA packaging and secretion, the characterization of novel small non-coding RNAs, and the functions of short 3’ tRNA fragments. Finally, we will continue to develop clinical applications of TGIRT-seq, including new approaches for RNA diagnostics and liquid biopsy of cancer and other human diseases.