Structural Biology of Retrotransposition and pre-mRNA Splicing

逆转录转座和前 mRNA 剪接的结构生物学

• 批准号: 10378727
• 负责人: Navtej Singh Toor
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378727
• 关键词: Active Sites; Biochemical; Catalysis; Catalytic RNA; Complex; Cryoelectron Microscopy; DNA; Defect; Development; Eukaryota; Excision; Exhibits; Exons; Foundations; Future; Genetic; Genome; Goals; Human Genome; In Vitro; Introns; Investigation; Knowledge; Ligation; Location; Molecular; Nucleotides; Paste substance; Prevalence; Proteins; RNA; RNA Splicing; RNA-Directed DNA Polymerase; Reaction; Research; Resolution; Retroelements; Retrotransposition; Ribonucleoproteins; Spliceosomes; Structure; System; Work; X-Ray Crystallography; genetic element; human disease; insight; mRNA Precursor; particle; programs; single molecule; structural biology; therapeutic RNA; thermostability

PROJECT SUMMARY/ABSTRACT Non-long terminal repeat (non-LTR) retroelements and spliceosomal introns comprise ~70% of the human genome. Both of these genetic elements are thought to have evolved from a group II intron ancestor. Group II introns are catalytic RNAs that are able to engage in both retrotransposition and pre-mRNA splicing. Group II introns engage in retrotransposition and function as retroelements using a copy-and-paste mechanism that allows insertion into new locations in DNA genomes using a reverse transcriptase (RT) and an intron RNA template. Despite the prevalence of retroelements in eukaryotic genomes, relatively little is known about the precise molecular mechanism of retrotransposition. Group II introns are also ancestral to the splicesome, which is responsible for catalyzing pre-mRNA splicing in eukaryotes. This evolutionary linkage is supported by the fact that the active site of the group II intron is conserved with that of the spliceosome. Group II introns can catalyze self-splicing reactions that results in the excision of intron lariat and ligation of the adjacent exons. There are still many unanswered questions regarding the precise mechanism of pre-mRNA splicing and the function of highly conserved nucleotides within the active site. Group II introns consist of two major components that form a ribonucleoprotein (RNP) complex: 1) a self-splicing catalytic RNA and 2) a multi- functional maturase protein that has RT activity. We have isolated a thermostable group II intron RNP that exhibits high levels of retrotransposition and splicing activity. Our group II intron complex is very amenable to high-resolution structure determination and in vitro biochemical studies. To gain mechanistic insight into both retrotransposition and pre-mRNA splicing, we aim to use single-particle cryo-EM, x-ray crystallography, genetics and single-molecule approaches to characterize and capture the different stages of catalysis in this group II intron system. The knowledge gained from these studies will provide direct insight into the mechanisms of both retroelements and the splicing machinery found in higher eukaryotes. This work also lays the foundation for the future biochemical and structural investigation of mammalian retroelements. In summary, the goals of this proposal will further knowledge of the structure and function of genetic elements that comprise a majority of the human genome.

项目总结/摘要 非长末端重复序列（non-LTR）逆转录元件和剪接体内含子构成了约70%的 人类基因组这两个遗传元件被认为是从II组内含子祖先进化而来的。 II组内含子是能够参与反转录转座和前mRNA剪接的催化RNA。 第二组内含子参与反转录转座，并作为反转录元件使用复制和粘贴机制 它允许使用逆转录酶（RT）和内含子RNA插入DNA基因组的新位置， template.尽管真核生物基因组中普遍存在逆转录因子，但对逆转录因子的作用知之甚少。 逆转录转座的精确分子机制。第二组内含子也是剪接体的祖先， 其负责催化真核生物中的前体mRNA剪接。这种进化联系得到了以下方面的支持： II组内含子的活性位点与剪接体的活性位点是保守的。II组内含子可以 催化自身剪接反应，导致内含子的切除和相邻外显子的连接。 关于前体mRNA剪接的精确机制和mRNA的表达， 活性位点内高度保守的核苷酸的功能。第二组内含子由两个主要的 形成核糖核蛋白（RNP）复合物的组分：1）自剪接催化RNA和2）多- 具有RT活性的功能性成熟酶蛋白。我们已经分离出一个热稳定的II组内含子RNP， 表现出高水平的反转录转座和剪接活性。我们的第二组内含子复合体非常适合 高分辨率结构测定和体外生物化学研究。为了获得对两者的机械洞察力， 反转录转座和前mRNA剪接，我们的目标是使用单粒子冷冻电镜，X射线晶体学， 遗传学和单分子方法来表征和捕获催化的不同阶段， II组内含子系统。从这些研究中获得的知识将直接洞察 在高等真核生物中发现的反转录元件和剪接机制。这项工作也奠定了 为将来哺乳动物逆转录因子的生物化学和结构研究奠定了基础。在 总之，本研究的目的是进一步了解遗传元件的结构和功能 构成了人类基因组的大部分。