Ty Element Retrotransposition in Saccharomyces cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 7965270
• 负责人: David J. Garfinkel
• 金额: $ 126.86万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7965270
• 关键词: AIDS/HIV problem; Acetylation; Affect; Alanine; Basic Science; Binding; Bioinformatics; Biological Process; Cancerous; Cells; Chromatin; Chromatin Structure; Classification; Code; Complementary DNA; Complex; Coupled; Cysteine; Cytoplasm; DNA Repair; DNA Sequence Rearrangement; Defect; Disease; Elements; Event; Exhibits; Family; Gagging; Gene Deletion; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; HIV; Histidine; Histone H2B; Histone H3; Human; Human Genome; In Vitro; Insertional Mutagenesis; Integrase; Laboratories; Learning; Length; Long Terminal Repeats; Malignant Neoplasms; Mediating; Mobile Genetic Elements; Modeling; Mutate; Mutation; Nuclear; Nuclear Envelope; Pathway interactions; Predisposition; Process; Production; Proteins; Proteolytic Processing; RNA; RNA Polymerase II; RNA Polymerase III; RNA-Directed DNA Polymerase; Research; Research Project Grants; Rest; Retroelements; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Short Interspersed Nucleotide Elements; Site; Transcription Elongation; Translations; Ubiquitination; Viral; Virus-like particle; Work; Yeasts; Zinc; carcinogenesis; functional genomics; genome sequencing; histone modification; interest; mammalian genome; mutant; particle; preference; tool; trafficking; transcription factor

Our research concerns the mechanism and consequences of Ty element retrotransposition in the budding yeast <I>Saccharomyces</i>. Ty elements comprise five related families of long terminal repeat (LTR) retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes that correspond to the <I>Gag</i> and <I>Pol</i> genes of retroviruses. The retrotransposon is transcribed into a genome-length RNA, which is the template for reverse transcription by an element-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase (IN) and perhaps other proteins must transit the nuclear membrane to gain access to the genome. Each Ty element class integrates nonrandomly and possesses distinctive targeting mechanisms that are influenced by the chromatin state or RNA polymerase III transcription factors. All available evidence suggests that Ty elements remain intracellular and are not infectious. Therefore, these elements and their host have evolved control mechanisms to keep transposition and element mediated genome rearrangements at a low level, and integration site preferences that reduce the possibility of causing deleterious mutations. <BR><BR><BR><BR>Over the past year, we have made progress on characterizing host genes that modulate Ty1 retrotransposition. The first study involved a systematic screen of 4739 gene-deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction or <I>RTT</i> genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the <I>rtt</i> mutants identified in our screen show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility posttranscriptionally. Ty1 RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including <I>ckb2Δ</i> and <I>elf1Δ</i>, suggesting Ty1 integration may be more efficient in these strains. Insertion site preference at the <I>CAN1</i> locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as <I>BRE1</i> and <I>RAD6</i>, histone H3 acetylation by <I>RTT109</i> and <I>ASF1</i>, and transcription elongation by <I>SPT5</i>. Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis. Since these genes are also required for efficient transcription by RNA polymerase II, additional targets for Ty1 insertion maybe uncovered by stalled transcription complexes. Ongoing work is focused on defining the Ty1 integrase targeting domain and understanding the genomic landscape available for transposition events in wild type and targeting-defective mutants. <BR><BR><BR><BR>Despite overall sequence divergence, certain motifs are highly conserved between Ty1 and retroviral proteins. Over the past year, we have continued our studies on the functional organization of Ty1 proteins by examining the conserved zinc-binding domain (ZBD) of IN. We mutated the definitive histidine and cysteine residues and thirteen residues in the intervening (X32) sequence between IN-H22 and IN-C55. Replacing the zinc-coordinating histidine or cysteine residues with alanine reduced transposition by more than 4000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45, in the X32 region reduced transposition 85- 688-fold. Three of these residues, L32, I37 and V45 are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutations, all the X32 mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, GST pull-downs and intragenic complementation analysis of selected transposition-defective X32 mutants revealed decreased IN-IN interactions. Furthermore, Ty1 VLPs with in-L32A and in-V45A mutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration while the hydrophobic residues function in IN multimerization.

我们的研究关注了Ty元素在出芽酵母中逆转录的机制和后果。Ty元件包括五个相关的长末端重复（LTR）逆转录转座子家族，它们通过RNA中间体转座。Ty基因组包含两个与逆转录病毒的&lt；I>Gag</ I&gt；和&lt；I>Pol</ I&gt；基因相对应的基因。逆转录转座子转录成基因组长度的RNA，该RNA是由元件编码的逆转录酶蛋白进行逆转录和翻译的模板。Ty蛋白成熟和逆转录发生在Ty病毒样颗粒（Ty- vlp）内，这似乎是转位过程所必需的。尽管Ty- vlps在细胞质中积累，但含有Ty cDNA、元件编码整合酶（in）和其他蛋白质的Ty预整合复合体必须通过核膜进入基因组。每个Ty元件类别非随机整合，并具有受染色质状态或RNA聚合酶III转录因子影响的独特靶向机制。所有现有的证据表明，Ty元素留在细胞内，不具有传染性。因此，这些元件及其宿主已经进化出控制机制，将转位和元件介导的基因组重排保持在较低水平，以及降低引起有害突变可能性的整合位点偏好。在过去的一年中，我们在表征调节Ty1逆转录的宿主基因方面取得了进展。第一项研究涉及对4739个基因缺失突变体进行系统筛选，以确定那些增加Ty1流动性的突变体（Ty1限制或&lt；I>RTT</ I&gt；基因）。在鉴定的91个突变体中，80%编码的产物涉及染色质结构和功能、DNA修复和重组以及转录等核过程。然而，包含额外的Ty1和Ty3筛选的生物信息学分析表明，264个独特的基因参与各种生物过程，影响酵母中Ty的流动性。对我们筛选到的33个&lt；I>rtt</ I&gt；突变体的进一步表征表明，5个突变体中Ty1 RNA水平升高，其余突变体影响转录后的流动性。在转录延伸缺陷突变体中，包括&lt；I>ckb2Δ</ I&gt；和&lt；I>elf1Δ</ I>， Ty1 RNA和cDNA水平保持不变，表明Ty1整合在这些菌株中可能更有效。在&lt；I>CAN1</ I&gt；位点上的插入位点偏好需要Ty1限制性基因参与组蛋白H2B被Paf复合物亚基基因泛素化，以及&lt；I>BRE1</ I&gt；和&lt；I>RAD6</ I&gt；组蛋白H3被&lt；I>RTT109</ I&gt；和&lt；I>ASF1</ I&gt；转录延伸。我们的研究结果表明，多种途径限制Ty1的迁移，组蛋白修饰可能保护编码区免受插入突变。由于这些基因也是RNA聚合酶II有效转录所必需的，因此停滞的转录复合体可能会发现Ty1插入的其他靶点。正在进行的工作集中在定义Ty1整合酶靶向区域和了解野生型和靶向缺陷突变体中转座事件的基因组景观。<BR><BR><BR><BR> &lt；尽管总体序列存在差异，但Ty1和逆转录病毒蛋白之间的某些基序是高度保守的。在过去的一年里，我们通过检测IN的保守锌结合域（ZBD）继续对Ty1蛋白的功能组织进行研究。我们突变了最终的组氨酸和半胱氨酸残基以及in - h22和in - c55之间的中间（X32）序列中的13个残基。用丙氨酸取代锌配位组氨酸或半胱氨酸残基减少了4000倍以上的转位，并导致IN和逆转录酶（RT）不稳定以及低效率的蛋白水解加工。X32区疏水残基I28、L32、I37和V45的丙氨酸取代使转位减少85- 688倍。其中三个残基，L32， I37和V45在逆转录病毒中是高度保守的，尽管它们对整合或病毒传染性的影响尚未被表征。与HHCC突变相比，所有X32突变体均表现出稳定的In和RT，蛋白质加工和cDNA产生不受影响。然而，GST下拉和基因内互补分析显示，选定的转位缺陷X32突变体的IN-IN相互作用减少。此外，具有in- l32a和in- v45a突变的Ty1 VLPs在体外没有表现出大量的协同整合产物。我们的研究结果表明，组氨酸/半胱氨酸残基在整合之前的转位步骤中很重要，而疏水残基在in多聚化中起作用。