Genetic Analysis of U14 snoRNP Core Protein Function

真核核糖体的生物发生发生在核核中，在核核中，rRNA作为一个大的前体转录物被合成，经过加工和随后组装成新的核糖体亚基。真核生物核仁含有大量的小核仁rna (snoRNAs)，它们对核糖体的生物发生至关重要。研究表明，snorna对pre-rRNA折叠、rRNA加工和rRNA碱基修饰很重要。U14就是这样一种真核snoRNA物种，在脊椎动物和酵母中对pre-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：从其前mrna内含子中切除U14所需的前mrna前体。这些基本的加工元件包括核苷酸盒C和D，它们不仅对加工，而且对snoRNA转运到核仁和前rrna甲基化都很重要。在这个U14 snoRNA元件中定义了一个“盒C/D核心基序”，并被证明是snoRNA合成所需的核核蛋白的结合位点。box C/D核心基序的广泛存在表明，该snoRNA元件及其相关核仁蛋白对box C/D snoRNA家族的生物发生和功能至关重要。虽然基本的snoRNA元件已被很好地定义，但结合该核心基序的核仁蛋白仍未被表征。因此，本项目将分离和定义这些关键的核仁蛋白，表征它们的基因，然后在酵母中遗传检测单个蛋白在snoRNA加工、核仁运输和rRNA甲基化中所起的作用。核糖体是所有活细胞的基本组成部分，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。真核生物的核糖体发生在核仁中，在核仁中有许多rna和蛋白质参与一系列协调的事件，这些事件是核糖体组装所必需的。U14 snoRNA是pre-rRNA加工和18S rRNA积累所必需的一种snoRNA。引人注目的是，U14是现在快速增长的一类snoRNA物种中的第一个，这些物种在基因的蛋白质编码区域（内含子）的中断中编码，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结构基序对加工至关重要。另外的实验表明，核蛋白结合这个核心基序，对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。这些结果将为这些蛋白在snoRNA生物合成中的作用以及在核糖体生物发生过程中的snoRNP功能提供新的和基本的见解。

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.