Retrotransposon expression within ribosomal gene loci

描述(申请人提供)：所有真核基因组的很大一部分是由来自RNA反转录的序列组成的。这些元素的整合导致了自发的插入突变，它们的结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%的人类基因组由这些逆转录本组成。主要负责这些插入的机制是由非长末端重复序列(非LTR)反转录转座元件编码的。R2元件是特征最好的非LTR元件之一，也是唯一正在进行详细体外研究的元件。由R2编码的单个蛋白质切割其染色体靶点，并将反向转录直接聚合到该位点上。这一建议的一个具体目的是继续对R2逆转录转座机制的表征，重点是R2蛋白质特异性地结合其自己的RNA模板并定位该模板进行反转录的方法。R2元件特异性地插入其宿主的28S rRNA基因。在果蝇中，这些插入片段经常与28S rRNA共转录，但随后迅速降解。由于核糖体的结构和组装在所有真核生物中都是相似的，因此将建立一个酵母表达系统来研究R2/28S rRNA亚基的组装和加工。第二个目的是确定这些亚基是否可以作为R2整合的生物相关模板。在研究任何转座元件时，一个关键问题是调控。已经鉴定出具有R2活性的果蝇品系。该建议的第三个目的是比较这些活性菌株和非活性菌株中的R2转录、RNA加工和翻译，特别强调确定在生殖系发育中R2逆转座子事件发生的时间。最后一个目的是通过确定R2的控制因素是否映射到rDNA基因座本身，来开始R2活性的遗传分析。RDNA基因座，以及围绕它组装的核仁，在细胞代谢的许多方面起着关键作用。R2元件直接整合到rDNA基因的结构和调控中，它们的研究将为rRNA基因的研究提供重要的见解和有用的工具。

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.