ASSEMBLY, LOCALIZATION AND FUNCTION OF THE U3 SNRNP

描述(改编自申请人的摘要)：核糖体生物发生是 对于真核细胞在细胞周期中的进展、生长在 大小，然后分割。这一应用重点是核糖体RNA的加工 (RRNA)，目标是识别和研究关键因素的功能 参与导致18S形成的前rRNA的切割 RRNA。使用酵母S.cerevisiae作为模型系统，拟议的工作将 关注U3小核仁核糖核蛋白(SnoRNP)和 它在这些分裂步骤中的亚基。建议的研究是重要的。 因为U3 snoRNP在真核生物中普遍存在，并且是 到目前为止所研究的所有生物体中新生rRNA转录本的处理。在……里面 酵母，三个依赖U3 snoRNP的切割步骤，导致释放 18S rRNA通过两种不同U3 snoRNA发生：前-rRNA碱基配对 互动。对其蛋白质组分的功能知之甚少 在这些RNA-RNA相互作用中的U3 snoRNP，或者如果蛋白质-蛋白质和 蛋白质-RNA的相互作用是功能所必需的。许多预测的U3 SnoRNP蛋白组分尚未鉴定。初步结果 从调查员的实验室里发现了几部小说 U3 snoRNP组件：MpplOp、Imp3p和Imp4p。他们的刻画有 提供了大量证据，表明 SnoRNP功能中的蛋白质-蛋白质、蛋白质-RNA和RNA-RNA相互作用。目标 2、3和4将测试有关这些交互的特定假设，并将 检查每一种相互作用在前RNA加工中的重要性。这将是 通过各种遗传方法完成，包括突变分析 包括蛋白质和RNA两种成分。在目标1中，调查员将继续她的 有希望的初步努力是纯化U3 snoRNP并鉴定剩余的 亲和捕获-质谱法测定未知蛋白质组分 光谱分析。在目标5中，他们提议利用一些 他们已经产生的MPP10、JMP3和IMP4的条件等位基因 设置一系列精选的基因筛查。这些屏幕旨在 确定在同一途径中对前rRNA加工起作用的新因素 作为U3 snoRNP或以某种方式调节它。总而言之，这项工作将 导致了对这些基础机制的全面理解 基本的U3 snoRNP介导的RNA处理事件。

DESCRIPTION (Adapted from the applicant's abstract): Ribosome biogenesis is essential for eukaryotic cells to progress through the cell cycle, grow in size, and divide. This application focuses on the processing of ribosomal RNA (rRNA), and the goal is to identify and study the function of critical factors involved in the cleavage of the pre-rRNAs that lead to the formation of the 18S rRNA. Using the yeast S. cerevisiae as a model system, the proposed work will focus on the function of the U3 small nucleolar ribonucleoprotein (snoRNP) and its subunits in these cleavage steps. The proposed research is important because the U3 snoRNP is ubiquitous in eukaryotes and is required for processing of the nascent rRNA transcript in all organisms studied so far. In yeast, the three U3 snoRNPdependent cleavage steps that lead to the release of the 1 8S rRNA occur via two different U3 snoRNA:pre-rRNA base-pairing interactions. Little is known about the function of the protein components of the U3 snoRNP in these RNA-RNA interactions or if protein-protein and protein-RNA interactions are essential for function. Many of the predicted U3 snoRNP protein components have not yet been identified. Preliminary results from the investigator's laboratory have led to the discovery of several novel U3 snoRNP components: MpplOp, Imp3p, and Imp4p. Their characterization has provided substantial evidence pointing to the critical nature of protein-protein, protein-RNA, and RNA-RNA interactions in snoRNP function. Aims 2, 3, and 4 will test specific hypotheses regarding these interactions and will examine the importance of each interaction in prerRNA processing. This will be accomplished by varied genetic approaches, including mutational analysis of both protein and RNA components. In Aim 1, the investigator will continue her promising initial efforts to purify the U3 snoRNP and to identify the remaining unknown protein components using affinity capture followed by mass spectrometry. In Aim 5, they propose to take advantage of a number of conditional alleles of MPP1O, JMP3, and IMP4 that they have already generated to set up a selected series of genetic screens. These screens are designed to identify new factors that function in the same pathway for pre-rRNA processing as the U3 snoRNP or regulate it in some manner. Taken together, this work will lead to a comprehensive understanding of the mechanisms underlying these essential U3 snoRNP-mediated RNA processing events.