Ribosomal RNA Processing: Removal of the Gap in Eukaryotic 26S-28S rRNA

通常，在前体45S核糖体RNA(RRNA)的加工过程中最大的成熟衍生物是26S-28S rRNA，它是大的核糖体亚单位的一个组成部分。人们对从真核rRNA中去除转录间隔区所需的结构元素、相互作用的分子和机械知之甚少。在一些真核生物中，Z6S-Z8S rRNA进一步裂解成α和β两个部分，通过氢键结合在一起。关于这一现象的信息也很匮乏，这就是所谓的缺口加工。该项目的长期目标是确定GAP处理过程中涉及的识别信号和机制，并确定其在核糖体功能中的作用。该建议的具体目的是：1)通过体外结合实验确定核糖体蛋白L25与真菌共亲和线虫GAP区的相互作用：2)以链特异核酸酶为探针并进行引物延伸分析，确定GAP区的rRNA结构；3)通过四膜虫的亚细胞分离来定位GAP加工的位置；以及4)通过杂交和S1核酸酶或Northern印迹分析来确定切除的rRNA的命运。这些研究将为更好地理解GAP加工和真核生物中成熟rRNA的表达提供依据。

In general the largest mature derivative in the processing of precursor 45S ribosomal RNA (rRNA) is 26S-28S rRNA, a component of the large ribosomal subunit. Little is known about the structural elements, interacting molecules, and machinery needed to remove transcribed spacers from eukaryotic rRNA. In some eukaryotic Z6S-Z8S rRNA is further fragmented into alpha and beta halves, held together by hydrogen bonding. There is also a dearth of information about the phenomenon, which is called gap processing. The long term goals of the project are to determine the recognition signals and mechanisms involved in gap processing and to ascertain its role in ribosome function. The specific objectives of this proposal are to: 1) determine the interaction of ribosomal protein L25 with the gap region in the fungus fly Sciara coprophila by in vitro binding experiments; 2) determine the structure of rRNA in the gap region using strand-specific nucleases as probes and primer extension analysis; 3) localize the site of gap processing by subcellular fractionation of Tetrahymena; and 4) determine the fate of the excised rRNA by hybridization and S1 nuclease or Northern blot analyses. These studies should provide a better understanding of gap processing and the expression of mature rRNA in eukaroyotes.