Regulation And Function Of Retroelements

逆转录因子的调控和功能

• 批准号: 7333935
• 负责人: Henry L. LEVIN
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7333935
• 关键词: Regulation; Function; Retroelements

Diseases caused by retroviruses such as AIDS and leukemia have intensified the need to understand the mechanisms of retrovirus replication. Our primary objectives are to understand how reverse transcription of viral mRNA occurs and how the cDNA products are integrated into the genome of infected cells. Owing to their similarity to retroviruses, LTR-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. During the synthesis of cDNA, reverse transcriptase (RT) generates a series of highly specific intermediates. We identified residues of Tf1 RT that recognize specific intermediates of cDNA by screening large numbers of RT mutants. A combination of genetic assays and physical analyses identified a set of 35 mutations that inhibited integration without reducing reverse transcription. Our experiments focused on a cluster of mutations in ribonuclease H (RNase H) that included a region with five single amino acid substitutions in a five?amino acid segment. Surprisingly, these mutations in RNase H did not reduce the levels of full-length double stranded cDNA. Crystallographic studies by Dr. Edward Arnold and colleagues indicated that the corresponding residues of HIV-1 RT interact directly with the PPT. This observation led us to test whether the mutations in the RNase H of Tf1 were defective for the cleavages on either side of the PPT that generate the primer, or the cleavage that removes the PPT after it has primed plus strand synthesis. Defects in the position these cleavages would alter the sequences at the 3' end of the minus strand and as a result have a drastic impact on the ability of IN to catalyze strand transfer. The sequence from the 3? ends of the minus strand cDNA from Tf1 particles was determined using ligation mediated PCR. The mutations clearly increased the levels of cDNA that retained the PPT at the 3? end. In order to determine whether this added sequence resulted from a defect in the cleavages that generate the PPT or the cleavage that removes the PPT after synthesis of plus strand DNA, the 5? end of the plus strand DNA was analyzed. The results of primer extension provided strong evidence that the mutations in RNase H specifically inhibited the removal of the PPT RNA from the 5? end of the plus strand. As a result, our data identified a cluster of conserved amino acids in RNase H that have the specific function of removing the PPT. In addition to removing the PPT primer from the plus strand, RNase H is known to remove the primer from the 5? end of the minus strand DNA. Tf1 uses a unique mechanism of self-priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus strand synthesis with an 11 nucleotide RNA removed from the 5? end of its own transcript. We tested whether the self-primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self-primer. This indicates that integration of the Tf1 cDNA relies on mechanisms other than reverse transcription to remove the primer. Our analysis of the genome sequence of S. pombe revealed a strong clustering of pre-existing LTRs associated with the 5? end of ORFs. Experiments based on the production of new integration events revealed that the association of Tf1 with LTRs was the result of integration preference. To define the determinants of the target sites we developed an in vivo assay for integration using a plasmid that contained ade6 as the target and a plasmid with Tf1 that induced transposition. The version of Tf1 we expressed contained a neo gene to cause target plasmids with insertions to gain resistance to kanamycin. When Tf1-neo was expressed, the plasmid with ade6 served as an efficient target for integration. We isolated 50 separate insertions in the intact target plasmid and found ninety-five percent occurred within a 160 nt region in the ade6 promoter. To determine which sequences of Ade6 were required for efficient integration, we created a series of 10 deletions within the target plasmid. This analysis revealed that the 160 nt region of the promoter was the only sequence that was required for efficient integration. We asked whether promoter activity was required for integration by measuring transcript levels of ade6. Deletions of sequence on either side of the 160 nt region caused five to ten-fold reductions in ade6 mRNA. Nevertheless, the deletions caused no reduction in integration efficiency. These results indicated that transcription was not important for target site activity. We next considered whether transcription factors themselves were directing the integration of Tf1. To identify positions where factors bind in the promoter of Ade6, we used micrococcal nuclease mapping. We observed a strong correlation between micrococcal sensitive sites and the position of the prominent insertion sites. This suggested transcription factors played a role in directing Tf1 integration. Hoffman and colleagues showed previously that the transcription factor Atf1p binds to and activates the promoter of fbp1. We tested whether the promoter of fbp1 is a target of Tf1 integration using the target plasmid assay. We found that the fbp1 promoter was a target for Tf1 insertion and that the majority of the insertions occurred 40 nt from the position where Atf1p binds. A mutation that blocks the binding of Aft1p caused a significant reduction in Tf1 integration at the promoter of fbp1. These data indicate that Atf1p is responsible for targeting Tf1 to specific insertion sites in the fbp1 promoter.

由逆转录病毒（例如艾滋病和白血病）引起的疾病加剧了了解逆转录病毒复制机制的需求。我们的主要目标是了解病毒mRNA的逆转录以及如何整合到感染细胞的基因组中。由于它们与逆转录病毒的相似性，LTR-返回转座子是逆转录病毒复制的重要模型。在我们的实验室研究的逆转录座子是裂变酵母菌酵母酸酯POMBE的TF1元素。在合成cDNA期间，逆转录酶（RT）会产生一系列高度特定的中间体。我们通过筛选大量的RT突变体鉴定了TF1 RT的残基，该残基识别cDNA的特定中间体。遗传测定和物理分析的结合确定了一组35个突变，这些突变抑制了整合而不减少逆转录。我们的实验集中在核糖核酸酶H（RNase H）中的一系列突变，其中包括一个在五个氨基酸段中具有五个单个氨基酸取代的区域。令人惊讶的是，RNase H中的这些突变并未降低全长双链cDNA的水平。 Edward Arnold博士及其同事的晶体学研究表明，HIV-1 RT的相应残基与PPT直接相互作用。该观察结果使我们测试了TF1的RNase H中的突变是否有缺陷，因为PPT的任何一侧的裂解产生了底漆，还是在其启动启动加和链合成后去除PPT的裂解。这些裂解的位置缺陷会改变负链3'端的序列，因此对IN到催化链转移的能力产生了巨大影响。来自3的序列？使用连接介导的PCR测定TF1颗粒的负链cDNA的末端。突变清楚地增加了将PPT保留在3的cDNA的水平？结尾。为了确定该添加序列是由产生PPT的裂解中的缺陷而导致的，或者是在合成Plus strand dna（5？）后去除PPT的裂解的5？分析了加链DNA的末端。底漆延伸的结果提供了有力的证据，表明RNase H中的突变特异性抑制了从5中去除PPT RNA？加号的结尾。结果，我们的数据确定了RNase H中的一组保守氨基酸簇，该氨基酸具有去除PPT的特定功能。 除了从Plus链中取出PPT底漆外，RNase H还已知可以从5中去除底漆？减去链DNA的末端。 TF1使用独特的自我宣传机制来启动逆转录。从5？自己的成绩单的结尾。我们测试了TF1的自启示器是否与RNase H从cDNA中去除的TRNA引物相似。我们对从病毒样颗粒中提取的TF1 cDNA的分析揭示了令人惊讶的观察结果，即主体cDNA保留了自primer。这表明TF1 cDNA的整合依赖于逆转录以外的机制去除引物。 我们对S. pombe的基因组序列的分析表明，与5个相关的LTR的强烈聚类？ ORF的结尾。基于新整合事件的生产的实验表明，TF1与LTR的关联是整合偏好的结果。为了定义目标位点的决定因素，我们开发了一种体内测定，使用包含ADE6作为靶标的质粒和具有TF1的质粒的质粒进行整合，该质粒带有诱导换位的TF1。我们表达的TF1版本包含一个新基因，可引起带有插入的靶质粒，以获得对卡纳米霉素的抗性。当表达TF1-NEO时，具有ADE6的质粒是有效整合的靶标。我们在完整的靶质粒中分离了50个单独的插入，发现百分之九十五发生在ADE6启动子中的160 nt区域内。为了确定ADE6的有效整合需要哪些序列，我们在目标质粒中创建了一系列10个缺失。该分析表明，启动子的160 NT区域是有效整合所需的唯一序列。我们询问是否需要通过测量ADE6的转录水平进行集成的启动子活动。 160 NT区域两侧的序列缺失导致ADE6 mRNA降低了5至十倍。然而，删除并没有降低整合效率。这些结果表明，转录对于目标位点活性并不重要。接下来，我们考虑转录因子本身是否指导TF1的整合。为了识别因子在ADE6启动子中结合的位置，我们使用了微球菌核酸酶映射。我们观察到微球菌敏感位点与突出插入位点的位置之间存在很强的相关性。这表明转录因子在指导TF1集成中发挥了作用。霍夫曼及其同事以前表明，转录因子ATF1P与FBP1的启动子结合并激活。我们测试了FBP1的启动子是否是使用靶质粒测定的TF1积分的靶标。我们发现FBP1启动子是TF1插入的靶标，并且大多数插入发生在ATF1P结合的位置的40 nt。阻断AFT1P结合的突变导致FBP1启动子的TF1积分显着降低。这些数据表明ATF1P负责将TF1靶向FBP1启动子中的特定插入位点。