Retrotransposon expression within ribosomal gene loci

核糖体基因座内的逆转录转座子表达

• 批准号: 6917266
• 负责人: Thomas H. Eickbush
• 金额: $ 41.87万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917266
• 关键词: DNA binding protein; Drosophilidae; RNA binding protein; RNA directed DNA polymerase; developmental genetics; enzyme activity; eukaryote; gene expression; genetic transcription; genetic translation; intermolecular interaction; northern blottings; nucleic acid repetitive sequence; posttranscriptional RNA processing; ribosomal RNA; site directed mutagenesis; transposon /insertion element

DESCRIPTION (provided by applicant): A significant fraction of all eukaryotic genomes is composed of sequences derived from the reverse transcription of RNA. The integration of these elements results in spontaneous insertional mutations and their combined accumulation has played a significant role is shaping the size, structure and function of eukaryotic genomes. For example, nearly 40% of the human genome is composed of these reverse transcripts. The machinery largely responsible for these insertions is that encoded by the non-long terminal repeat (non-LTR) retrotransposable elements. One of the best characterized non-LTR elements, and the only element in which detailed in vitro studies are being conducted, is the R2 element. The single protein encoded by R2 cleaves its chromosomal target site and polymerizes the reverse transcript directly onto this site. One specific aim of this proposal is to continue the characterization of the R2 retrotransposition mechanism with emphasis on the means by which the R2 protein specifically binds its own RNA template and positions this template for reverse transcription. R2 elements specifically insert into the 28S rRNA genes of their host. In Drosophila, these insertions are frequently co-transcribed with the 28S rRNA but then rapidly degraded. Because ribosome structure and assembly are similar in all eukaryotes, an expression system in yeast will be developed to study the assembly and processing of R2/28S rRNA subunits. The second aim is to determine whether such subunits can serve as biologically relevant templates for R2 integration. A critical issue in the study of any transposable element is regulation. Strains of Drosophila have been identified in which R2 activity has been documented. The third aim of the proposal is to compare R2 transcription, RNA processing and translation in these active strains to that of inactive strains with special emphasis on determining when in germ line development R2 retrotransposition events occur. The last aim is to begin a genetic analysis of R2 activity by determining whether factors involved in its control map to the rDNA locus itself. The rDNA locus, and the nucleolus that assembles about it, plays a key role in many aspects of cellular metabolism. R2 elements are so directly integrated into the structure and regulation of the rDNA locus that their study should provide important insights and useful tools to study the rRNA genes.

描述（由申请人提供）：所有真核基因组的很大一部分是由源自 RNA 逆转录的序列组成。这些元件的整合导致自发插入突变，它们的组合积累在塑造真核基因组的大小、结构和功能方面发挥了重要作用。例如，近 40% 的人类基因组是由这些逆转录本组成的。主要负责这些插入的机制是由非长末端重复（非LTR）反转录转座元件编码的机制。 R2 元件是特征最明确的非 LTR 元件之一，也是唯一正在进行详细体外研究的元件。 R2 编码的单一蛋白质切割其染色体靶位点并将逆转录物直接聚合到该位点上。该提案的一个具体目标是继续表征 R2 逆转录转座机制，重点关注 R2 蛋白特异性结合其自身 RNA 模板并定位该模板进行逆转录的方式。 R2 元件特异性插入宿主的 28S rRNA 基因中。在果蝇中，这些插入经常与 28S rRNA 共转录，但随后迅速降解。由于所有真核生物的核糖体结构和组装都相似，因此将开发酵母表达系统来研究 R2/28S rRNA 亚基的组装和加工。第二个目标是确定此类亚基是否可以作为 R2 整合的生物学相关模板。任何转座因子研究中的一个关键问题是调控。已鉴定出具有 R2 活性的果蝇菌株。该提案的第三个目标是将这些活性菌株与非活性菌株中的 R2 转录、RNA 加工和翻译进行比较，特别强调确定在种系发育中 R2 逆转录转座事件何时发生。最后一个目标是通过确定参与其控制的因素是否映射到 rDNA 基因座本身来开始对 R2 活性进行遗传分析。 rDNA 基因座以及围绕其组装的核仁在细胞代谢的许多方面发挥着关键作用。 R2 元件如此直接地整合到 rDNA 位点的结构和调控中，因此他们的研究应该为研究 rRNA 基因提供重要的见解和有用的工具。