BIOCHEMISTRY OF SMALL NUCLEOLAR RNAS IN YEAST

真核生物大核糖体RNA产生的途径是很好的 定义，但对具体反应知之甚少。 本 应用程序提出，以描述特定的小核武器的作用， 在酿酒酵母中rRNA加工所需的RNA（snRNA）。 snRNA将在两种情况下进行研究，包括： 正在进行的关于一种被称为U14的物种的研究，以及，B）评估 与初级加工相关的尚未表征的snRNAs 复杂. U14在遗传上是保守的，是生产所必需的。 18S rRNA的缺失导致35S rRNA的不正确加工 转录和快速周转的异常中间体含有18S 核糖核酸 体内交联数据显示，U14直接与 前体rRNA，通过一个必要的互补序列。 几 U14的其他基本元素已经被定义， 结构图可用。 项目期间的目标包括：1）定义U14的相互作用 2）建立U14功能的无细胞测定，3） 表征U14 snRNP的蛋白质，4）描述U14 snRNP的合成 U14本身，以及5）表征90S rRNA加工的新snRNA 复杂. 通过序列突变检查U14和18S RNA的相互作用 体内交联和加工功能所需的。 努力 建立U14功能的无细胞测定法将集中于前rRNA 切割反应5'近端18S RNA（A1位点），使用天然和在 体外产生的rRNA底物和耗尽U14的提取物。 蛋白 的U14 snRNP将通过表征抑制基因来鉴定， U14功能缺陷。 将鉴定U14上的蛋白结合位点 生物化学和snRNP形成和rRNA蛋白质的作用 将从遗传学和免疫学的角度对加工过程进行分析。 DNA信号 参与U14转录、加工和调节的基因 通过诱变分析。 最后，与这些基因相关的snRNAs 处理复杂将被编目和新物种的作用 通过体内消耗分析评估。 拟议研究的结果将为我们提供有关 snRNA在真核细胞中的作用，并建立通用方法 用于在更复杂的生物体中表征这些重要的RNA，包括 人类

The path by which eucaryotic large ribosomal RNAs are produced is well defined, but little is known about the specific reactions. The present application proposes to characterize the role of specific small nuclear RNAs (snRNAs) required for rRNA processing in Saccharomyces cerevisiae. The snRNAs will be investigated in two contexts, involving: a) extension of ongoing studies of a species known as U14 and, b) assessing the roles of yet-uncharacterized snRNAs associated with the primary processing complex. U14 is phylogenetically conserved and required for production of 18S rRNA; its loss results in incorrect processing of the 35S rRNA transcript and rapid turnover of abnormal intermediates containing 18S RNA. In vivo cross-linking data show that U14 interacts directly with precursor rRNA, through an essential complementary sequence. Several other essential elements of U14 have been defined and a secondary structure map is available. Goals for the project period include: 1) defining the interaction of U14 with pre-rRNA, 2) establishing a cell-free assay of U14 function, 3) characterizing proteins of the U14 snRNP, 4) describing the synthesis of U14 itself, and 5) characterizing novel snRNAs of the 90S rRNA processing complex. Interaction of U14 and 18S RNAs will be examined by mutation of sequences required for in vivo cross-linking and processing function. Efforts to establish a cell-free assay of U14 function will focus on the pre-rRNA cleavage reaction 5' proximal to 18S RNA (A1 site), using natural and in vitro produced rRNA substrates and extracts depleted of U14. Proteins of the U14 snRNP will be identified by characterizing genes that suppress defects in U14 function. Protein binding sites on U14 will be identified biochemically and the role of the proteins in snRNP formation and rRNA processing will be analyzed genetically and immunologically. DNA signals involved in U14 transcription, processing and regulation will be defined by mutagenic analysis. Finally, the snRNAs associated with the processing complex will be catalogued and the roles of new species assessed by in vivo depletion analysis. Results from the proposed studies will yield important insights into the role of the snRNAs in eucaryotic cells and establish general approaches for characterizing these vital RNAs in more complex organisms, including humans.