Group II Intron and Related Reverse Transcriptases

项目摘要/摘要 拟议的研究涉及对第二组内含子和相关逆转录酶(RTS)的持续研究， 它们的生物学功能、反转录机制和rna-seq应用，包括分析。 用于RNA诊断和液体活组织检查的人细胞外囊泡和血浆RNA。第二类内含子RTS 由细菌反转录元件编码，称为可移动的第二组内含子，被认为是进化和... 高等生物体内含子和反转录元件的中心。第二类内含子逆转录酶在结构和功能上不同 并具有用于RNA-SEQ和其他生物技术应用的新的生化特性。 阳离子。细菌还编码与第二组内含子rts密切相关的各种其他rts，包括 在CRISPR-CAS系统中在RNA间隔区获取中起作用的RTS，以及多种类型的自由站立的 已获得细胞功能的ING RT。目前，人们对这些RT或它们的潜在作用知之甚少 用于生物技术应用。在以前的工作中，我们开发了表达第二组的一般方法 并将这些方法应用于芽孢杆菌第II类内含子RT-S。 RIAL嗜热者获得耐热的II族内含子RTS(TGIRT)。我们发现TGIRT具有更高的 比逆转录病毒RTS更高的保真度和处理能力，以及一种新的模板切换活动，可以轻松实现 RNA-seq适配器添加。利用这些性质，我们开发了基于TGIRT的方法 高通量RNA测序(TGIRT-SEQ)，支持困难或不可能的应用 与其他目前可用的RT，包括同时分析几乎所有的RNA生物型从小 起始材料的数量。我们证明了这些方法在人体细胞分析中的有效性。 LAR、胞外囊泡和血浆RNA，并已开始探索其临床应用。另外， 我们首次获得了全长TGIRT与模板-引发物的络合物的X-射线晶体结构。 即将到来的dNTP，为解决有关逆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 具有改进的用于生物技术应用的性能的RTS。在拟议的研究中，我们将调查 II组独特生化特性的逆转录机制和结构基础 内含子RTS，探索RTS的进化起源及其与RNA依赖的RNA聚合酶的关系， 继续研究CRISPR相关的RTS，并将这些研究扩展到新的细菌RTS类别。毛皮- 因此，我们将继续开发TGIRT-SEQ方法，并跟进我们之前TGIRT-SEQ的主要发现 人细胞、胞外囊泡和血浆RNA的分析，包括其机制和调节 胞外囊泡RNA的包装和分泌，新的小非编码RNA的特征，以及 短3‘tRNA片段的功能。最后，我们将继续开发TGIRT-seq的临床应用， 包括癌症和其他人类疾病的RNA诊断和液体活组织检查的新方法。

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.