Mammalian L1 retrotransposon replication

最新发现： ORF 1 p是一个多聚体，其单体具有三个结构域：一个功能未知的快速进化的氨基末端结构域;一个卷曲螺旋结构域，其是多聚体形成所需的，并且在经历了几次适应性进化后也快速进化;以及一个高度保守的结合核酸的羧基末端结构域。这些结构和进化特征是迄今为止研究的所有哺乳动物L1 ORF 1 p的典型特征。因此，ORF 1 p氨基末端的快速进化对于哺乳动物进化过程中L1活性的持久性必不可少。为了理解为什么，我们正在确定ORF 1 p进化对其结构、反转录转座以及一些对反转录转座至关重要的ORF 1 p特性的影响：例如，RNA结合、多聚体形成和核酸伴侣活性。(Also参见Z 01 DK 057811 -01 LMCB：哺乳动物L1反转录转座子-宿主相互作用）。我们构建了一个祖先版本的ORF 1 p（L1 Pa 5），早于现代版本（L1 Pa 1）的进化变化和马赛克ORF 1 p的，包含现代和祖先的地区。在证明ORF 1 p在大肠杆菌中表达后，我们用杆状病毒生产了ORF 1 p。由于ORF 1 p转录本翻译不当，不适合在大肠杆菌中表达。杆状病毒表达为生化和结构分析提供了充足的蛋白质。多螺旋结构预测程序和分析离心都表明，与小鼠ORF 1 p不同，现代人ORF 1 p要么是二聚体，要么是非常不稳定的三聚体。相比之下，multicoil预测，预适应的祖先卷曲螺旋将形成一个强大的三聚体。所有可用的哺乳动物非人ORF 1 p氨基酸序列的多螺旋预测表明，强大的三聚体形成的规则。在卷曲螺旋结构域的一个小区域中，仅用几个祖先氨基酸取代它们的现代对应物，就完全废除了反转录转座，并将多螺旋预测从二聚体/弱三聚体转换为强三聚体。但是，仅仅将这些祖先氨基酸的一个子集恢复到它们的现代对应物，就可以恢复逆转录转座和二聚体/弱三聚体的预测。系统发育分析的ORF 1 p在人类谱系和ORF 1 p从不同物种的进化前因表明，除了那些氨基酸进行适应性变化（正选择），卷曲螺旋结构域的这一地区，否则表现出非常小的进化变化。我们现在正在实施杆状病毒表达我们的各种ORF 1 p蛋白的生化和结构分析。我们还继续与NIDDK的结构生物学家Fred Dyda和Allison Hickman合作，以确定它们的结构。我们现在正在进行核酸结合实验，初步尝试将ORF 1 p与RNA或DNA共结晶，因为我们无法仅用蛋白质获得晶体。 一种新的逆转录转座试验的开发-逆转录转座试验通常依赖于检测剪接RNA转录本的DNA拷贝（报告基因）。RNA剪接的需要确保DNA拷贝已经通过RNA中间体，这是逆转录转座的标志。目前的逆转录转座测定存在三个主要问题：第一-剪接反应通过正常的剪接体途径发生，这是L1转录物通常不会遇到的途径，因为L1 RNA没有剪接。剪接的RNA结合该途径特有的蛋白质，并且具有与非剪接RNA显著不同的代谢命运。我们用一个自我剪接的内含子取代了剪接体内含子，从而纠正了这个问题。虽然这两个载体产生几乎相同的插入片段，但它们的反转录转座活性不同，当L1元件包含祖先ORF 1 p时，这种差异似乎大大增强。第二，L1 3' UTR RNA的完整性由于2kb报告基因的存在而严重受损。因此，存在于200 bp 3' UTR RNA中的高度保守序列和序列基序的任何作用都可能被其与2000 bp的外来RNA的并置所压倒。我们希望解决这个问题，取代报告基因与15 bp的序列分裂的自我剪接（自催化）内含子。因此，一旦转录本合成，内含子就会被剪切掉，产生与正常L1转录本仅相差15个核苷酸的L1转录本。通过在3' UTR的最不保守区域插入报告基因，我们希望将其对L1 RNA的影响降至最低。第三，本试验没有揭示L1反转录转座中中间步骤的信息;即，L1转录和cDNA水平。为了解决这个问题，我们将通过与适当的互补PNA序列（一种磷酸二酯骨架被酰胺键取代的核酸聚合物）杂交来分离各种逆转录转座中间体和产物，然后通过真实的时间（RT）PCR来定量产物。我们正在与丹尼尔阿佩拉博士（NIDDK）合作，他对PNA合成进行了修改，大大提高了其灵敏度和特异性。

RECENT FINDINGS: THE FUNCTION AND STRUCTURE OF ORF1p - ORF1p is a multimer, the monomer of which has three domains: A rapidly evolving amino terminal domain of unknown function; a coiled coil domain, which is required for multimer formation and which also evolves rapidly having undergone several episodes of adaptive evolution; and a highly conserved carboxy-terminal domain that binds nucleic acids. These structural and evolutionary characteristics are typical of all the mammalian L1 ORF1p's so far examined. Thus, rapid evolution of the amino terminal half of ORF1p must be essential for the persistence of L1 activity during the evolution of mammals. To understand why, we are determining the effects of ORF1p evolution on its structure, on retrotransposition, and on a number of ORF1p properties shown to be essential for retrotransposition: e.g., RNA-binding, multimer formation, and nucleic acid chaperone activity. (Also see Z01 DK057811-01 LMCB: Mammalian L1 Retrotransposon - host interaction). We constructed an ancestral version of ORF1p (L1Pa5) that predated the evolutionary changes of the modern version (L1Pa1) and mosaic ORF1p's that contain modern and ancestral regions. We implemented baculovirus production of ORF1p after having shown that expression in E. coli was unsuitable due to improper translation of the ORF1p transcript. Baculovirus expression provides ample protein for both biochemical and structural analysis. Both the multicoil structural prediction program and analytical centrifugation showed that unlike mouse ORF1p, the modern human ORF1p is either a dimer or a very labile trimer. In contrast, multicoil predicts that the pre-adapted ancestral coiled coil will form a strong trimer. Multicoil predictions of all available mammalian non-human ORF1p amino acid sequences showed that strong trimer formation is the rule. Substitution of just a few ancestral amino acids for their modern counterparts in a small region of the coiled coil domain completely abolishes retrotransposition and converts the multicoil prediction from dimer / weak trimer to strong trimer. But restoration of just a subset of these ancestral amino acids to their modern counterparts restores retrotransposition and the prediction of dimer / weak trimer. Phylogenetic analysis of both the evolutionary antecedents of ORF1p in the human lineage and of ORF1p from different species showed that, aside from those amino acids that underwent adaptive change (positive selection), this region of the coiled coil domain has otherwise exhibited very little evolutionary change. We are now implementing baculovirus expression of our various ORF1p proteins for biochemical and structural analysis. We are also continuing our collaboration with Fred Dyda and Allison Hickman, structural biologists in NIDDK to determine their structures. We are now carrying out nucleic acid binding experiments preliminary to attempting to co-crystallizing ORF1p with RNA or DNA, as we could not obtain crystals with just the protein. DEVELOPMENT OF A NEW RETROTRANSPOSITION ASSAY - Retrotransposition assays generally rely on the detection of a DNA copy (reporter gene) of a spliced RNA transcript. The requirement for RNA splicing ensures that DNA copy has gone through an RNA intermediate, a hallmark of retrotransposition. There are three major problems with the current retrotransposition assay: First - The splicing reaction occurs via the normal splicesomal pathway, a pathway that L1 transcripts do not normally encounter as L1 RNA is not spliced. Spliced RNAs bind proteins unique to this pathway and have dramatically different metabolic fates from non-spliced RNAs. We rectified this problem by substituting a self-splicing intron for the splicesomal intron. Although the two vectors generate nearly identical inserts, they differ in their retrotransposition activity, a difference that seemingly is greatly enhanced when the L1 element contains the ancestral ORF1p. Second - The integrity of the L1 3' UTR RNA is severely compromised by the presence of the 2 kb reporter gene. Thus, any role of the highly conserved sequences and sequence motifs present in the 200 bp 3' UTR RNA could be overwhelmed by its juxtaposition to 2000 bp of extraneous RNA. We hope to address this problem by replacing the reporter gene with a 15 bp sequence split by the self-splicing (autocatalytic) intron. Thus, as soon as the transcript is synthesized, the intron is spliced out generating an L1 transcript that differs from a normal L1 transcript by only 15 nucleotides. And by inserting the reporter in the least conserved region of the 3' UTR we hope to minimize its effect on L1 RNA. Third, the present assay does not reveal information on the intermediate steps in L1 retrotransposition; i.e., L1 transcript and cDNA levels. To address this issue we will isolate the various retrotransposition intermediates and products by hybridization with the appropriate complementary PNA sequence (a nucleic acid polymer in which the phoshpodiester backbone is replace by amide bonds) and then quantify the products by real time (RT) PCR. We are collaborating in this effort with Dr. Daniel Appella (NIDDK) who has developed modifications to PNA synthesis that greatly increase both its sensitivity and specificity.