Ty Element Retrotransposition in Saccharomyces cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 8175310
• 负责人: David J. Garfinkel
• 金额: $ 26.63万
• 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/8175310
• 关键词: AIDS/HIV problem; Acetylation; Affect; Alanine; Basic Science; Binding; Bioinformatics; Biological Process; Cancerous; Cells; Chromatin; Chromatin Structure; 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; pol genes; preference; tool; trafficking; transcription factor

Our research concerns the mechanism and consequences of Ty element retrotransposition in the budding yeast Saccharomyces. 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 Gag and Pol 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. 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 RTT 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 rtt 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 ckb2delta and elf1delta, suggesting Ty1 integration may be more efficient in these strains. Insertion site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6, histone H3 acetylation by RTT109 and ASF1, and transcription elongation by SPT5. 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. 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.

我们的研究涉及芽殖酵母中 Ty 元件逆转座的机制和后果。 Ty 元件包含五个相关的长末端重复 (LTR) 反转录转座子家族，这些家族通过 RNA 中间体进行转座。 Ty 基因组包含两个基因，分别对应逆转录病毒的 Gag 和 Pol 基因。逆转录转座子被转录成基因组长度的RNA，它是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，这似乎对于转座过程至关重要。尽管 Ty-VLP 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 预整合复合物必须穿过核膜才能进入基因组。每个 Ty 元件类别都非随机整合，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。所有现有证据都表明 Ty 元件保留在细胞内并且不具有传染性。因此，这些元件及其宿主已经进化出控制机制，以将转座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的可能性的整合位点偏好。 在过去的一年里，我们在表征调节 Ty1 逆转录转座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 限制性基因或 RTT 基因）。在已鉴定的 91 个突变体中，80% 编码涉及核过程的产物，例如染色质结构和功能、DNA 修复和重组以及转录。然而，包含额外 Ty1 和 Ty3 筛选的生物信息学分析表明，参与多种生物过程的 264 个独特基因会影响酵母中的 Ty 迁移率。对我们筛选中确定的 33 个 rtt 突变体的进一步表征表明，5 个突变体中的 Ty1 RNA 水平增加，其余的影响转录后的移动性。在转录延伸缺陷突变体（包括 ckb2delta 和 elf1delta）中，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。 CAN1 位点的插入位点偏好需要 Ty1 限制性基因参与 Paf 复合体亚基基因以及 BRE1 和 RAD6 的组蛋白 H2B 泛素化、RTT109 和 ASF1 的组蛋白 H3 乙酰化以及 SPT5 的转录延伸。我们的结果表明多种途径限制 Ty1 的移动性，组蛋白修饰可能保护编码区免受插入突变的影响。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物可能会发现 Ty1 插入的其他靶标。正在进行的工作重点是定义 Ty1 整合酶靶向结构域并了解野生型和靶向缺陷突变体中可用于转座事件的基因组景观。 尽管整体序列存在差异，但某些基序在 Ty1 和逆转录病毒蛋白之间高度保守。在过去的一年里，我们通过检查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 IN-H22 和 IN-C55 之间的最终组氨酸和半胱氨酸残基以及间插 (X32) 序列中的 13 个残基。用丙氨酸替换锌配位的组氨酸或半胱氨酸残基可将转座减少 4000 倍以上，并导致 IN 和逆转录酶 (RT) 不稳定以及低效的蛋白水解加工。 X32区域中疏水性残基I28、L32、I37和V45的丙氨酸取代使转座减少85-688倍。其中三个残基 L32、I37 和 V45 在逆转录病毒中高度保守，尽管它们对整合或病毒感染性的影响尚未得到表征。与 HHCC 突变相比，所有 X32 突变体均表现出稳定的 IN 和 RT，并且蛋白质加工和 cDNA 生产不受影响。然而，对选定的转座缺陷型 X32 突变体的 GST 下拉和基因内互补分析显示 IN-IN 相互作用减少。此外，具有 in-L32A 和 in-V45A 突变的 Ty1 VLP 在体外没有表现出高水平的协同整合产物。我们的结果表明，组氨酸/半胱氨酸残基对于整合之前的转座步骤很重要，而疏水残基在 IN 多聚化中起作用。

项目成果

Functional analysis of N-terminal residues of ty1 integrase.

ty1整合酶N端残基的功能分析。

• DOI: 10.1128/jvi.00159-09
• 发表时间: 2009
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Moore,SharonP;Garfinkel,DavidJ
• 通讯作者: Garfinkel,DavidJ