Structural Biology of Retrotransposition and pre-mRNA Splicing

项目摘要/摘要 非长末端重复序列(Non-LtR)反转录元件和剪接体内含子占整个基因组的70% 人类基因组。这两种遗传元素都被认为是从第二组内含子祖先进化而来的。 第二类内含子是催化RNA，既能进行逆转录转座，又能参与前mRNA剪接。 第二类内含子参与逆转录转座，并利用复制粘贴机制作为逆转录元件发挥作用 这允许使用逆转录酶(RT)和内含子RNA插入DNA基因组中的新位置 模板。尽管逆转录元件在真核生物基因组中普遍存在，但人们对逆转录元件的了解相对较少。 逆转座的精确分子机制。第二类内含子也是剪接体的祖先， 它负责催化真核生物中的前-信使核糖核酸剪接。这种进化的连锁性得到了 第二组内含子的活性部位与剪接体的活性部位是保守的。第二类内含子可以 催化自我剪接反应，导致内含子套索的切除和相邻外显子的连接。 关于前信使核糖核酸剪接的确切机制和 活性部位内高度保守的核苷酸的功能。第二类内含子由两个主要内含子组成 形成核糖核蛋白(RNP)复合体的成分：1)自剪接催化RNA和2)多- 具有RT活性的功能性成熟酶蛋白。我们已经分离出了一个耐热的II类内含子RNP 表现出高水平的逆转录转座和剪接活性。我们的第二类内含子复合体非常容易被 高分辨结构测定和体外生化研究。对两者都有机械的洞察 逆转录转位和前信使核糖核酸剪接，我们的目标是使用单粒子冷冻-EM，X射线结晶学， 遗传学和单分子方法来表征和捕捉不同阶段的催化 第二组内含子系统。从这些研究中获得的知识将提供对 在高等真核生物中发现的逆转录元件和剪接机制的机制。这项工作还奠定了 为今后哺乳动物逆转录因子的生化和结构研究奠定基础。在……里面 综上所述，这项提案的目标将进一步了解遗传元件的结构和功能 它们构成了人类基因组的大部分。

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.