Ty Element Retrotransposition in Saccharomyces cerevisiae

我们的研究关注Ty元件在芽殖酵母中的反转录转座的机制和后果。Ty元件包括通过RNA中间体转座的长末端重复（LTR）反转录转座子的五个相关家族。Ty基因组含有两个基因，对应于逆转录病毒的Gag和Pol基因。逆转录转座子被转录成基因组长度的RNA，其是通过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，这似乎是转座过程所必需的。虽然Ty-VLP在细胞质中积累，但含有Ty cDNA、元件编码的整合酶（IN）和可能的其他蛋白质的Ty预整合复合物必须穿过核膜以进入基因组。每个Ty元件类整合非随机和具有独特的靶向机制，受染色质状态或RNA聚合酶III转录因子的影响。所有可用的证据表明，Ty元素保持在细胞内，不具有感染性。因此，这些元件和它们的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低水平，并且整合位点偏好降低了引起有害突变的可能性。 在过去的一年中，我们已经取得了进展，表征宿主基因，调节Ty1逆转录转座。第一项研究涉及4739个基因缺失突变体的系统筛选，以确定那些增加Ty1流动性（Ty1限制或RTT基因）。在所鉴定的91个突变体中，80%编码涉及核过程的产物，如染色质结构和功能、DNA修复和重组以及转录。然而，生物信息学分析，包括额外的Ty1和Ty3屏幕表明，264个独特的基因参与各种生物过程中影响Ty在酵母中的流动性。在我们的筛选中鉴定的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，其余的影响转录后的流动性。Ty1 RNA和cDNA水平保持不变的突变体在转录延长缺陷，包括ckb2 δ和elf1 δ，表明Ty1整合可能是更有效的，在这些菌株。CAN1位点的插入位点偏好需要Ty1限制性基因参与组蛋白H2B通过Paf复合物亚基基因的泛素化，以及BRE1和RAD 6，组蛋白H3通过RTT109和ASF 1的乙酰化，以及通过SPT 5的转录延伸。我们的研究结果表明，多种途径限制Ty1的流动性和组蛋白修饰可能会保护编码区的插入突变。由于这些基因也是RNA聚合酶II有效转录所需的，因此Ty1插入的其他靶点可能会被停滞的转录复合物所发现。正在进行的工作集中在定义Ty1整合酶靶向结构域和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。 尽管总体序列差异，但某些基序在Ty1和逆转录病毒蛋白之间高度保守。在过去的一年中，我们继续我们的研究Ty1蛋白的功能组织通过检查保守的锌结合结构域（ZBD）的IN。我们突变了确定的组氨酸和半胱氨酸残基以及IN-H22和IN-C55之间的间插（X32）序列中的13个残基。用丙氨酸替换锌配位组氨酸或半胱氨酸残基使转座减少了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。在X32区域中疏水残基I28、L32、I37和V45的丙氨酸取代使转座减少85 - 688倍。这些残基中的三个，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感染性的影响尚未被表征。与HHCC突变相反，所有X32突变体都表现出稳定的IN和RT，蛋白质加工和cDNA产生不受影响。然而，GST下拉和选择的转座缺陷型X32突变体的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在体外没有表现出实质水平的协同整合产物。我们的研究结果表明，组氨酸/半胱氨酸残基是重要的转座前整合的步骤，而疏水性残基功能IN多聚化。

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.