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.

项目总结/文摘