Retrotransposon expression within ribosomal gene loci

描述（由申请人提供）：所有真核生物基因组的很大一部分由来自RNA逆转录的序列组成。这些元素的整合导致了自发的插入突变，它们的联合积累在塑造真核生物基因组的大小、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。这些插入的主要机制是由非长末端重复序列（non-long terminal repeat，非ltr）反转录转座元件编码的。R2元素是表征最好的非ltr元素之一，也是目前正在进行详细体外研究的唯一元素。由R2编码的单一蛋白切割其染色体靶位点，并将反向转录物直接聚合到该位点上。本提案的一个具体目的是继续表征R2逆转录转位机制，重点是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法。R2元件特异性地插入宿主的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。由于核糖体的结构和组装在所有真核生物中都是相似的，因此我们将在酵母中建立一个表达系统来研究R2/28S rRNA亚基的组装和加工。第二个目标是确定这些亚基是否可以作为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。已经确定的果蝇菌株中有记录的R2活性。本研究的第三个目的是比较这些活性菌株与非活性菌株的R2转录、RNA加工和翻译，特别强调确定在种系发育中何时发生R2反转录转位事件。最后一个目标是通过确定其控制中涉及的因素是否映射到rDNA位点本身来开始R2活性的遗传分析。rDNA位点和围绕它聚集的核仁在细胞代谢的许多方面起着关键作用。R2元件是如此直接地整合到rDNA位点的结构和调控中，它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。

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.