Ty Element Retrotransposition in Saccharomyces cerevisiae

我们的研究涉及Ty元素在萌芽酵母中逆转录转座的机制和后果。TY元件由五个相关的长末端重复序列(LTR)反转录转座子家族组成，它们通过RNA中间体转座。Ty基因组包含两个基因，分别对应于逆转录病毒的&lt；i&gt；gag&lt；/i&gt；和&lt；i&gt；ol&lt；/i&gt；基因。反转录转座子被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蛋白的成熟和逆转录发生在TY病毒样颗粒(TY-VLP)内，这似乎是转座过程中必不可少的。尽管Ty-VLPs积累在细胞质中，但Ty预整合复合体必须通过核膜才能进入基因组，该复合体包含Ty cDNA、元件编码的整合酶(IN)和其他蛋白质。每一类Ty元件都是非随机整合的，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。所有可获得的证据表明，TY分子仍然存在于细胞内，不具传染性。因此，这些元件和它们的宿主进化出了控制机制，将转座和元件介导的基因组重排保持在较低水平，并进化出整合位点偏好，从而降低了导致有害突变的可能性。在过去的一年里，我们在鉴定调节Ty1逆转座子的宿主基因方面取得了进展。第一项研究涉及对4739个基因缺失突变的系统筛选，以确定那些增加Ty1流动性的突变(Ty1限制或&lt；i&gt；rtt&lt；/i&gt；基因)。在91个已鉴定的突变体中，80%编码与染色质结构和功能、DNA修复和重组以及转录等核过程有关的产物。然而，包括额外的Ty1和Ty3筛查的生物信息学分析表明，参与各种生物学过程的264个独特基因影响酵母中Ty的迁移率。对我们筛选中发现的33个&lt；i&gt；rtt&lt；/i&gt；突变体的进一步鉴定表明，5个突变体中Ty1 RNA水平上升，其余的在转录后影响迁移率。在转录延伸缺陷的突变体中，Ty1的RNA和cDNA水平保持不变，包括&lt；i&gt；ck b2&amp；#916；&lt；/i&gt；和&lt；i&gt；Elf1&amp；#916；&lt；/i&gt；，这表明Ty1整合在这些菌株中可能更有效。&lt；i&gt；CAN1&lt；/i&gt；基因座的插入位点偏好需要参与PAF复合亚单位基因泛素化组蛋白H2B的Ty1限制基因，以及&lt；i&gt；Bre1&gt；/i&gt；和&lt；i&gt；RAD6&lt；/i&gt；组蛋白H3被&lt；i&gt；RTT109&lt；/i&gt；和&lt；i&gt；ASF1&lt；/i&gt；转录延伸。I&gt；SPT5&lt；/I&gt；我们的结果表明，多条途径限制了Ty1的移动，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图景。&lt；BR&gt；&lt；BR&gt；&lt；BR&gt；&lt；BR&gt；Despite全序列差异，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，我们通过检测IN的保守的锌结合结构域(ZBD)来继续我们对Ty1蛋白功能组织的研究。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中间(X32)序列中的13个残基。用丙氨酸取代锌配位的组氨酸或半胱氨酸残基可以减少4000多倍的转座，并导致IN和逆转录酶(RT)的不稳定性以及蛋白质降解过程的低效。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在逆转录病毒中高度保守，尽管它们对整合或病毒感染性的影响尚未确定。与HHCC突变相比，所有X32突变体都表现出稳定的IN和RT，蛋白质的加工和cDNA的产生没有受到影响。然而，对选定的转座缺陷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在体外没有显示出大量的协同整合产物。我们的结果表明，组氨酸/半胱氨酸残基在整合前的转座步骤中起重要作用，而疏水残基在IN多聚体中起作用。

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.