Ty Element Retrotransposition in S. cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 7052636
• 负责人: David J. Garfinkel
• 金额: --
• 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/7052636
• 关键词: Saccharomyces cerevisiae; chemical stability; complementary DNA; functional /structural genomics; fungal genetics; gene mutation; genetic transcription; genetic translation; genome; helicase; microorganism culture; nuclear membrane; nuclease; posttranscriptional RNA processing; transposon /insertion element

Our research concerns the mechanism and consequences of Ty (Transposon yeast) element retrotransposition in the budding yeast Saccharomyces cerevisiae. Ty elements comprise five related families of long terminal repeat retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes, TYA and TYB, which correspond to the gag and pol genes of retroviruses, respectively. The retrotransposon is transcribed into a nearly genome-length RNA, which is the template for reverse transcription by the self-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 sub-VLP preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins probably is required to transit the nuclear membrane, and mediate integration at preferred chromosomal locations. We are particularly interested in the biology of Ty1 elements because these elements are the most abundant, competent for transposition, and their RNA transcripts accumulate to an exceptionally high level. Despite the abundance of Ty1 RNA, however, mature Ty1 proteins and VLPs are present at low levels, and Ty1 transposition events are also very rare. Although Ty1 elements preferentially integrate upstream of genes transcribed by RNA polymerase III, Ty1 insertions can mutate essentially any yeast gene, form large complex multimeric insertions of 100 kb or more, and can also initiate chromosomal deletions, inversions and translocations by homologous recombination with other Ty1 elements in the genome. Information gained from studying Ty elements has been successfully applied to several other areas of biomedical research. For example, understanding how Ty elements transpose in yeast has led to a greater understanding of how retroelements in other organisms including humans function, because many of these elements are related. Over 40% of the human genome is comprised of retroelement sequences, such as LINE and SINE, intracisternal A-type particle, and endogenous retroviral elements. Most importantly, genome rearrangements and insertional events involving these elements have been implicated in human disease and cancer. Further computational analyses of mammalian genome sequences coupled with functional genomic analyses of cancerous cells will likely reveal new roles for retroelements that can be modeled in yeast using Ty elements or their mammalian counterparts. In addition, many aspects of the retrotransposon replication cycle are similar to those of retroviruses, including HIV. Therefore, steps in the process of retrotransposition can be compared and contrasted with similar processes in retroviruses to learn more about both classes of elements. Over the past year, we have made progress in the following areas. We, in collaboration with an international consortium of yeast researchers headed by Dr. Mark Johnston (Washington University), have developed a near complete set (95% of all ORFs) of single gene deletions to systematically survey gene function. These mutations are currently being screened for their affects on Ty1 retrotransposition. In our continuing effort to identify cellular genes that modulate Ty1 retrotransposition, we have surveyed all members of the RAD2 family of nucleases, which are required for genome stability. We have shown that only Rad27/Fen1, a highly conserved structure-specific nuclease important for lagging strand DNA replication, inhibits Ty1 mobility by altering the fate of unincorporated cDNA. We have also discovered a novel form of post-transcriptional cosuppression that controls Ty1 element copy number. A decrease in the synthesis of Ty1 cDNA is strongly correlated with post-transciptional cosuppression. Unlike the conserved RNAi pathways that cosuppress transposable elements in a variety of eukaryotes, the level of Ty1 RNA increases with the addition of elements to the genome, yet genomic Ty1 retrotransposition decreases. In addition, Saccharomyces cerevisiae lacks the conserved RNAi pathway, therefore, identifying the factors responsible for Ty1 cosuppression may provide new insight into how eukaryotic genomes evolve and are stably maintained.

我们的研究涉及TY(转座子酵母)元件在芽殖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反转录转座子家族组成，这些反转录转座子通过RNA中间体转座。Ty基因组包含两个基因TYA和TYB，分别对应于逆转录病毒的Gag和Pol1基因。反转录转座子被转录成接近基因组长度的RNA，这是自我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-VLP聚集在细胞质中，但可能需要一个亚VLP预整合复合体，该复合体包含Ty cDNA、元件编码的整合酶和其他蛋白质，以通过核膜，并介导在首选染色体位置的整合。 我们对Ty1元素的生物学特别感兴趣，因为这些元素是最丰富的，能够转座，并且它们的RNA转录产物积累到异常高的水平。然而，尽管Ty1 RNA丰富，但成熟的Ty1蛋白和VLP存在于低水平，Ty1转座事件也非常罕见。虽然Ty1元件优先整合在RNA聚合酶III转录的基因的上游，但Ty1插入可以突变任何酵母基因，形成100kb或更多的大型复杂多聚体插入，也可以通过与基因组中其他Ty1元件的同源重组来启动染色体缺失、倒位和易位。 从研究Ty元素中获得的信息已经成功地应用于生物医学研究的其他几个领域。例如，了解酵母中TY元素的转座方式有助于更好地理解包括人类在内的其他生物中的逆转录元素是如何发挥作用的，因为这些元素中的许多都是相关的。人类基因组的40%以上由反转录元件序列组成，如直线和正弦、脑池内的A型颗粒和内源性逆转录病毒元件。最重要的是，涉及这些元素的基因组重排和插入事件与人类疾病和癌症有关。对哺乳动物基因组序列的进一步计算分析与对癌细胞的功能基因组分析相结合，可能会揭示逆转录元件的新作用，这些逆转录元件可以在酵母中使用Ty元件或其哺乳动物对应元件进行建模。此外，逆转录转座子复制周期的许多方面与逆转录病毒相似，包括艾滋病毒。因此，逆转录转座过程中的步骤可以与逆转录病毒中的类似过程进行比较和对比，以了解更多关于这两类元件的信息。 一年来，我们在以下方面取得了进展。我们与以Mark Johnston(华盛顿大学)博士为首的国际酵母研究人员联盟合作，开发了一套近乎完整的单基因缺失(占所有ORF的95%)，以系统地研究基因功能。这些突变目前正在筛选它们对Ty1逆转录转座的影响。在我们继续努力确定调节Ty1逆转录转座的细胞基因的过程中，我们调查了基因组稳定所需的RAD2核酸酶家族的所有成员。我们已经证明，只有Rad27/Fen1，一个高度保守的结构特异性核酸酶，对于滞后的链DNA复制很重要，通过改变未掺入的cDNA的命运来抑制Ty1的移动。我们还发现了一种控制Ty1元件拷贝数的转录后共抑制的新形式。Ty1基因合成的减少与转运后的共抑制密切相关。与真核生物中共抑制转座元件的保守RNAi途径不同，Ty1 RNA的水平随着基因组中元素的增加而增加，而基因组中的Ty1逆转录转座减少。此外，酿酒酵母缺乏保守的RNAi途径，因此，识别导致Ty1共抑制的因素可能为真核基因组的进化和稳定维持提供新的见解。