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.

项目摘要/摘要 拟议的研究涉及对II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，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类细胞外囊泡和血浆RNA。 II组内含子RTS 由称为移动II组内含子的细菌编码，被认为是进化的 较高生物体中内含子和恢复元素的果子。 II组内含子RTS在结构和功能上不同 从逆转录病毒RT中，具有对RNA-SEQ和其他生物技术应用有用的新型生化特性 阳离子。细菌还编码与II组内含子RT密切相关的各种其他RT，包括 RT在CRISPR-CAS系统中的RNA垫片中起作用，以及多种自由台的RT- 获得了细胞函数的RT。目前，对这些RT或它们的潜力知之甚少 用于生物技术应用。在以前的工作中，我们开发了表达II组的一般方法 内含子和相关的RT具有高收率和活性，并将这些方法应用于Bacte-的II组内含子RT rial嗜热剂获得可热稳定的II组内含子RT（TGIRTS）。我们发现Tgirts具有更高的 比逆转录病毒RT以及一种新颖的模板开关活动，使您能够轻松 RNA-seq适配器添加。利用这些属性，我们开发了基于TGIRT的方法 高通量RNA测序（tgirt-seq），启用了困难或不可能的应用程序 与其他目前可用的RT相关，包括几乎所有来自小的RNA生物型的简单分析 起始材料的数量。我们证明了这些方法在分析人类cellu-的效率 LAR，细胞外囊泡和血浆RNA，并开始探索其临床应用。此外， 我们获得了与模板播种机的复合物中的全长TGIRT的首个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性基础 - II组内含子RTS的特征机制，RT的进化起源和II组内含子的工程 具有改进生物技术应用特性的RT。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相关的RT，并将这些研究扩展到新的细菌RT。毛皮- 因此，我们将继续开发TGIRT-seq方法，并跟进以前的TGIRT-SEQ的关键发现 人类细胞，细胞外囊泡和血浆RNA的分析，包括机理和调节 细胞外囊泡RNA包装和分泌，新型小型非编码RNA的表征以及 短3'tRNA片段的功能。最后，我们将继续开发Tgirt-seq的临床应用， 包括用于RNA诊断的新方法和癌症和其他人类疾病的液体活检。