BIOCHEMISTRY OF SMALL NUCLEOLAR RNAS IN YEAST

生产真核大核糖体rna的途径很好。 定义了，但对具体反应知之甚少。现在 应用建议表征特定小核的作用 酿酒酵母处理rRNA所需的RNA(SnRNAs)。 将在两个背景下调查SNRNAs，涉及：a)扩展 对一种被称为U14的物种正在进行的研究，以及，b)评估 与初级加工相关的尚未鉴定的SnRNA 很复杂。U14在系统发育上是保守的，是生产所必需的 18S rRNA；它的丢失导致对35S rRNA的错误处理 含有18S的异常中间体的转录本和快速周转 核糖核酸。体内交联数据表明，U14直接与 前体rRNA，通过一个重要的互补序列。几个 已经定义了U14的其他基本要素和次要要素 结构图可用。 项目期的目标包括：1)确定U14的互动 Pre-rRNA，2)建立U14功能的无细胞检测方法，3) U14-SnRNP的蛋白质特征，4)描述合成 U14本身，以及5)表征了90年代rRNA加工的新的SnRNA 很复杂。 U14和18S RNA的相互作用将通过序列突变来检测 体内所需的交联和加工功能。努力实现 建立无细胞检测U14功能的方法将重点放在前rRNA上 18S RNA(A1位)附近的5‘切割反应，使用Natural和In 体外产生的rRNA底物和提取物耗尽了U14。蛋白质 将通过鉴定抑制U14基因的基因来鉴定 U14功能缺陷。将确定U14上的蛋白质结合位点 生物化学及其在SnRNP和rRNA形成中的作用 处理过程将进行遗传和免疫学分析。DNA信号 将定义参与U14转录、加工和调控的因素 通过诱变分析。最后，与该基因相关的小分子RNA 加工综合体将被编目，新物种的作用 通过体内耗竭分析进行评估。 拟议研究的结果将对 小核糖核酸在真核细胞中的作用及建立一般方法 在更复杂的生物体中描述这些重要的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.