Group II Intron and Related Reverse Transcriptases

第二组内含子和相关逆转录酶

• 批准号: 10401772
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 86.83万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10401772
• 关键词: Address; Bacteria; Biochemical; Biological Process; Biotechnology; Cell physiology; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Crystallization; Development; Diagnostic; Engineering; Evolution; Foundations; High-Throughput RNA Sequencing; Human; Introns; Length; Malignant Neoplasms; Methods; Organism; Plasma; Property; RNA; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Regulation; Research; Retroelements; Retrotransposon; Retroviridae; Reverse Transcription; Roentgen Rays; Structure; System; Transfer RNA; Untranslated RNA; Work; base; clinical application; extracellular vesicles; follow-up; human disease; improved; liquid biopsy; next generation; novel; novel strategies; thermophilic organism; thermostability; transcriptome sequencing

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.

项目概要/摘要 拟议的研究涉及对 II 组内含子和相关逆转录酶 (RT) 的继续研究， 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。 由称为移动组 II 内含子的细菌逆向元件编码，这被认为是进化反 高等生物中的内含子和逆转录元件的突变体在结构和功能上有所不同。 来自逆转录病毒 RT，具有新颖的生化特性，可用于 RNA 测序和其他生物技术应用 细菌还编码与 II 组内含子 RT 密切相关的多种其他 RT，包括 在 CRISPR-Cas 系统中的 RNA 间隔区获取中发挥作用的 RT，以及多类独立的 RT 目前，人们对这些 RT 或其潜力知之甚少。 在之前的工作中，我们开发了表达第二组的通用方法。 内含子和相关 RT 具有高产率和活性，并将这些方法应用于来自细菌的 II 组内含子 RT 我们发现 TGIRT 具有更高的耐热性。 比逆转录病毒 RT 更保真度和持续性，以及一种新颖的模板转换活性，可以轻松实现 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) 使原本困难或不可能的应用成为可能 与其他当前可用的 RT 一起使用，包括同时分析来自小RNA的几乎所有生物型 我们证明了这些方法对于分析人类细胞的有效性。 lar、细胞外囊泡和血浆RNA，并已开始探索它们的临床应用。 我们首次获得了全长 TGIRT 与模板引物复合物的 X 射线晶体结构， 传入的 dNTP，为解决有关逆转录的主要问题提供了结构基础- II组内含子RT的作用机制、RT的进化起源以及II组内含子的工程 在拟议的研究中，我们将研究具有改进的生物技术应用特性的 RT。 II组独特生化特性的逆转录机制和结构基础 内含子 RT，探索 RT 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 RT，并将这些研究扩展到新类别的细菌 RT。 此后，我们将继续开发 TGIRT-seq 方法并跟进之前 TGIRT-seq 的主要发现 人类细胞、细胞外囊泡和血浆 RNA 的分析，包括其机制和调节 细胞外囊泡 RNA 包装和分泌、新型小非编码 RNA 的表征，以及 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 诊断和液体活检的新方法。