Ty Element Retrotransposition in Saccharomyces cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

基本信息

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
项目类别：
财政年份：
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

项目摘要

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TY元素逆转培训在萌芽的酵母菌糖疗法中的机制和后果。 TY元素包括长时间重复（LTR）逆转录子的五个相关家族，这些家族通过RNA中间体转载。 TY基因组包含两个对应于逆转录病毒的GAG和POL基因的基因。逆转录座子被转录为基因组长度RNA，这是通过元素编码的逆转录酶蛋白和翻译的逆转录模板。 Ty蛋白成熟和逆转录发生在Ty病毒样颗粒（TY-VLP）内，这对于转置过程似乎是必不可少的。尽管TY-VLP在细胞质中积聚，这是一种含有TY cDNA的TY预一体化复合物，但元素编码的积分酶（IN）和其他蛋白质也可能必须转移核膜才能进入基因组。每个TY元素类都集成非随机，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。所有可用的证据表明，TY元素仍然是细胞内且不感染的。因此，这些元素及其宿主具有发展的控制机制，以使换位和元素介导的基因组重排在低水平上，并积分位点的偏好减少了引起有害突变的可能性。在过去的一年中，我们在表征调节TY1逆转录位置的宿主基因方面取得了进展。第一项研究涉及4739个基因缺失突变体的系统筛选，以识别那些增加TY1迁移率（TY1限制或RTT基因）的筛选。在91个确定的突变体中，80％编码参与核过程的产品，例如染色质结构和功能，DNA修复和重组以及转录。然而，包含其他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。在我们的屏幕屏幕上鉴定出的33个RTT突变体的进一步表征表明，TY1 RNA水平在5个突变体中增加，其余的在转录后会影响迁移率。 TY1 RNA和cDNA水平在包括CKB2Delta和Elf1delta在内的突变体中保持不变，这表明在这些菌株中TY1的整合可能更有效。 CAN1基因座的插入位点偏好需要PAF复合体亚基基因以及BRE1和RAD6，RTT109和ASF1的组蛋白H3乙酰化以及SPT5的转录延长。我们的结果表明，多种途径限制了TY1迁移率和组蛋白修饰可能会保护编码区域免受插入诱变。由于这些基因也需要通过RNA聚合酶II有效转录，因此TY1插入的其他靶标可能会被停滞的转录复合物发现。正在进行的工作重点是定义TY1整合酶靶向结构域，并了解可用于野生型和靶向缺陷突变体换座事件的基因组景观。尽管总体序列发散，但某些基序在TY1和逆转录病毒蛋白之间仍然高度保守。在过去的一年中，我们通过研究IN的保守锌结合域（ZBD）来继续对TY1蛋白功能组织的研究。我们在IN-H22和C55之间的中间（X32）序列中突变了确定的组氨酸和半胱氨酸残基以及13个残基。用丙氨酸替换锌协调的组氨酸或半胱氨酸残基可减少4000倍以上，并导致IN和逆转录酶（RT）不稳定以及效率低下的蛋白水解处理。在X32区域中疏水残基I28，L32，I37和V45的丙氨酸取代减少了转座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，尽管尚未表征它们对整合或病毒感染性的影响。与HHCC突变相反，所有X32突变体在RT中均表现出稳定，并且蛋白质加工和cDNA的产生不受影响。然而，对选定的转置缺陷X32突变体的GST下拉和基因互补分析显示，相互作用减少了。此外，具有IN-L32a和IN-V45A突变的TY1 VLP在体外没有表现出大量的协同整合产物。我们的结果表明，组氨酸/半胱氨酸残基对于在整合之前的转置步骤很重要，而疏水残基在多聚化中起作用。