Genetic Analysis of U14 snoRNP Core Protein Function

U14 snoRNP核心蛋白功能的遗传分析

• 批准号: 9727945
• 负责人: E Stuart Maxwell
• 金额: $ 28.9万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-15 至 2002-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9727945&HistoricalAwards=false
• 关键词: Genetic; Analysis; U14; snoRNP; Core

9727945 MAXWELL Eukaryotic ribosome biogenesis occurs in the nucleolus where rRNA is synthesized as a large precursor transcript that undergoes processing and subsequent assembly into new ribosomal subunits. The eukaryotic nucleolus contains a large population of small nucleolar RNAs (snoRNAs) which are critical for ribosome biogenesis. Investigations have demonstrated that the snoRNAs are important for pre-rRNA folding, rRNA processing, and rRNA base modification. U14 is one such eukaryotic snoRNA species, essential for pre-rRNA processing and 18S rRNA accumulation in both vertebrates and yeast. Previous investigations have defined the RNA sequences and structures within the U14 snoRNA:pre-mRNA precursor required for U14 excision from its pre-mRNA intron. These essential processing elements include nucleotide boxes C and D, consensus sequences important not only for processing but also for snoRNA transport to the nucleolus and pre-rRNA methylation. A "box C/D core motif" has been defined within this U14 snoRNA element and shown to serve as a binding site for nucleolar proteins that are required for snoRNA synthesis. The widespread occurrence of the box C/D core motif indicates that this snoRNA element as well as its associated nucleolar proteins are critical for the biogenesis and function of the box C/D snoRNA family. While the essential snoRNA elements are well defined, the nucleolar proteins that bind this core motif remain uncharacterized. Therefore, this project will isolate and define these critical nucleolar proteins, characterize their genes, and then genetically examine in yeast the role individual proteins play in snoRNA processing, nucleolar transport, and rRNA methylation. Ribosomes are essential components of all living cells, necessary for the synthesis of proteins crucial for cell structure, growth, and function. Eukaryotic ribosome biogenesis occurs in the nucleolus where numerous RNAs and proteins participate in a coordinated series of events necessary for ribos ome assembly. U14 snoRNA is an essential snoRNA species required for pre-rRNA processing and the accumulation of 18S rRNA. Strikingly, U14 was the first of what is now a rapidly-growing class of snoRNA species that are encoded within interruptions in the protein coding regions of genes (introns) and synthesized via an intron-processing pathway. Analysis of mouse U14 snoRNA biosynthesis has demonstrated that an RNA structural motif is essential for processing. Additional experiments have shown that nuclear proteins bind this core motif and are also crucial for snoRNA synthesis and function. These experiments will define these important but as yet uncharacterized proteins. These results will provide new and fundamental insight into the role these proteins play in snoRNA biosynthesis as well as snoRNP function during ribosome biogenesis.

9727945 MAXWELL 真核核糖体生物发生发生在核仁中，rRNA 被合成为大的前体转录物，经过加工并随后组装成新的核糖体亚基。 真核生物的核仁含有大量的小核仁 RNA (snoRNA)，它们对于核糖体的生物发生至关重要。 研究表明，snoRNA 对于 rRNA 前体折叠、rRNA 加工和 rRNA 碱基修饰非常重要。 U14 就是这样一种真核 snoRNA 物种，对于脊椎动物和酵母中前 rRNA 加工和 18S rRNA 积累至关重要。 先前的研究已经确定了 U14 snoRNA 内的 RNA 序列和结构：U14 从其前 mRNA 内含子中切除所需的前体 mRNA。 这些必需的加工元件包括核苷酸盒 C 和 D，它们是共有序列，不仅对于加工很重要，而且对于 snoRNA 转运到核仁和前 rRNA 甲基化也很重要。 U14 snoRNA 元件内定义了“C/D 盒核心基序”，并显示其可作为 snoRNA 合成所需核仁蛋白的结合位点。 Box C/D 核心基序的广泛出现表明该 snoRNA 元件及其相关核仁蛋白对于 Box C/D snoRNA 家族的生物发生和功能至关重要。 虽然基本的 snoRNA 元件已明确定义，但结合该核心基序的核仁蛋白仍然未知。 因此，该项目将分离和定义这些关键的核仁蛋白，表征它们的基因，然后在酵母中对单个蛋白在 snoRNA 加工、核仁转运和 rRNA 甲基化中所起的作用进行遗传学检查。 核糖体是所有活细胞的重要组成部分，是合成对细胞结构、生长和功能至关重要的蛋白质所必需的。 真核核糖体生物发生发生在核仁中，其中大量 RNA 和蛋白质参与核糖体组装所需的一系列协调事件。 U14 snoRNA 是前 rRNA 加工和 18S rRNA 积累所需的重要 snoRNA 种类。 引人注目的是，U14 是目前快速增长的一类 snoRNA 物种中的第一个物种，这些物种在基因（内含子）蛋白质编码区的中断内进行编码，并通过内含子加工途径合成。 对小鼠 U14 snoRNA 生物合成的分析表明，RNA 结构基序对于加工至关重要。 其他实验表明核蛋白结合这个核心基序，对于 snoRNA 的合成和功能也至关重要。 这些实验将定义这些重要但尚未表征的蛋白质。 这些结果将为了解这些蛋白质在 snoRNA 生物合成以及核糖体生物合成过程中 snoRNP 功能中的作用提供新的基本见解。