FUNCTIONAL ROLES AND MECHANISMS OF SNO-RNAS IN PRE-RRNA PROCESSING

SNO-RNA 在 RRNA 前处理中的功能作用和机制

• 批准号: 6289811
• 负责人: BRENDA A PECULIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289811
• 关键词: RNA; RNA biosynthesis; Xenopus oocyte; intermolecular interaction; molecular chaperones; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; nucleolus; ribosomal RNA; yeast two hybrid system

Ribosome biogenesis is an essential but complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three distinct RNAs, but is transcribed as a single precursor molecule that must be correctly modified, folded, processed and assembled with proteins in order to yield the two subunits that together form a mature ribosome. The focus of my research has been to examine and identify cis-acting elements and trans-acting factors which are critical for the processing events of pre-rRNA processing, and thus essential for cell survival. Over the past year my lab has made progress on two fronts. First, we are using the genetics available in the yeast, S.cerevisiae to examine a proposed intramolecular interaction necessary for pre-rRNA processing. Second, we are using biochemical methods to identify proteins which comprise the U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits.Previously I described a model describing the mechanisms by which U8 snoRNA appeared to facilitate pre-rRNA processing in the Xenopus oocyte (1). This model predicted a specific intramolecular interaction in pre-rRNA. Formation of this stem should be critical for pre-rRNA processing. Because of the complexity of the Xenopus oocyte and the many different aspects of RNA processing addressed by this model, I used the yeast system to directly test a smaller aspect of this model. The genetic and biochemical manipulations which are possible in yeast made it possible to directly test and analyze specific aspects of the model proposed in Xenopus. The experiments in yeast unequivocally demonstrated that formation of this intramolecular interaction is critical for pre-rRNA processing (2). Over the past year additional experiments in yeast have implicated other cis-acting elements, which may act either as primary sequences or secondary structures, that also play an important roles in processing. We have begun to undertake more detailed mutagenesis of this region of the rRNA to identify those elements that affect the efficiency or the accuracy of pre-rRNA processing in yeast. The data generated in the yeast studies will later be applied to parallel experiments in Xenopus, which to date is the only existing model system for examining rRNA processing in vertebrates.We are continuing our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. I previously demonstrated that U8 snoRNP is essential for pre-rRNA processing in Xenopus oocytes. In the absence of U8 RNA, pre-rRNA processing is inhibited and no mature rRNA will accumulate (1). Mutageneis of U8 RNA indicated that U8 RNA was necessary, but not sufficient to direct pre-rRNA processing; proteins were present that affected the stability of the U8 RNP and the efficiency of processing (1). To better understand how the U8 proteins contribute to stability of U8 RNA and how they affect in vivo function of U8 RNP, we have been identifying proteins which specifically bind U8 RNA in vitro. In the past year we have identified and partially characterized a 29 kDa protein in Xenopus ovary extracts which binds U8 RNA. This protein binds U8 RNA with high specificity and can be crosslinked to U8 (3). In vitro competition binding assays indicated this protein is unique to U8 and does not represent a common shared protein present on all snoRNPs. Preliminary protein analysis indicates this novel protein represents the first identified constituent of the U8 RNP, the only vertebrate snoRNP required for processing of 5.8S and 28S rRNA. Future work will continue to characterize the X29 protein and identify other putative U8 RNA binding proteins. By taking advantage of the two different model systems, yeast and Xenopus, we hope to better understand the basic mechanisms of pre-rRNA processing and to identify and the cis- and trans-acting components involved. Identification of common components as well as species specific elements will help us understand the basic mechanisms at play in the universal process of pre-rRNA maturation.1) Peculis, B. A. (1997) The sequence of the 5 end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation. Mol Cell Biol 17:3702-3713.2) Peculis, B.A. and Greer, C.L. (1998) The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast. RNA 4:1610-1622.3) Tomasevic, N. and Peculis, B.A. Identification of a U8-specific binding protein. Submitted.

核糖体的生物合成是存在于所有活细胞中的一个重要而复杂的多步骤途径。前体rRNA（pre-rRNA）编码三种不同的RNA，但转录为单个前体分子，其必须被正确修饰、折叠、加工并与蛋白质组装，以产生共同形成成熟核糖体的两个亚基。我的研究重点一直是检查和确定顺式作用元件和反式作用因子，这是至关重要的前rRNA加工的加工事件，从而对细胞的生存至关重要。在过去的一年里，我的实验室在两个方面取得了进展。首先，我们正在使用酵母，酿酒酵母中可用的遗传学来检查一个拟议的前rRNA加工所必需的分子内相互作用。第二，我们正在使用生物化学方法来鉴定包含U8小核仁核糖核蛋白颗粒（U8 snoRNP）的蛋白质，U8 snoRNP是新形成的大核糖体亚基积累所需的一种基本反式作用因子。该模型预测了前rRNA中的特异性分子内相互作用。这个茎的形成对于前rRNA加工是至关重要的。由于非洲爪蟾卵母细胞的复杂性和该模型所涉及的RNA加工的许多不同方面，我使用酵母系统直接测试该模型的一个较小方面。酵母中可能的遗传和生化操作使得直接测试和分析非洲爪蟾中提出的模型的特定方面成为可能。酵母中的实验明确表明，这种分子内相互作用的形成对于pre-rRNA加工至关重要（2）。在过去的一年里，在酵母中进行的其他实验涉及其他顺式作用元件，这些元件可以作为一级序列或二级结构，也在加工中发挥重要作用。我们已经开始对rRNA的这一区域进行更详细的诱变，以确定影响酵母中前rRNA加工效率或准确性的那些元件。在酵母研究中产生的数据将被应用到非洲爪蟾的平行实验，这是迄今为止唯一存在的模型系统检查rRNA加工在脊椎动物中。我们正在继续我们的反式作用的前rRNA加工在脊椎动物中必不可少的因素的特性。我以前证明，U8 snoRNP是必不可少的前rRNA处理非洲爪蟾卵母细胞。在不存在U8 RNA的情况下，前rRNA加工受到抑制，不会积累成熟的rRNA（1）。U8 RNA的致突变性表明，U8 RNA是必需的，但不足以指导pre-rRNA加工;存在影响U8 RNP稳定性和加工效率的蛋白质（1）。为了更好地理解U8蛋白如何有助于U8 RNA的稳定性以及它们如何影响U8 RNP的体内功能，我们一直在体外鉴定特异性结合U8 RNA的蛋白质。在过去的一年中，我们已经确定和部分特征的29 kDa的蛋白质在非洲爪蟾卵巢提取物结合U8 RNA。该蛋白质以高特异性结合U8 RNA，并可与U8交联（3）。体外竞争结合试验表明，这种蛋白质是U8所特有的，并不代表所有snoRNP上存在的共有蛋白质。初步的蛋白质分析表明，这种新的蛋白质代表了U8 RNP的第一个确定的成分，U8 RNP是加工5.8S和28 S rRNA所需的唯一脊椎动物snoRNP。未来的工作将继续表征X29蛋白，并确定其他推定的U8 RNA结合蛋白。通过利用两个不同的模型系统，酵母和非洲爪蟾，我们希望更好地了解前rRNA加工的基本机制，并确定和所涉及的顺式和反式作用的组件。共同成分以及物种特异性元件的鉴定将帮助我们理解在前rRNA成熟的普遍过程中起作用的基本机制。1）Peculis，B. A.（1997）U8小核仁RNA的5端序列对于5.8S和28 S rRNA成熟至关重要。Mol Cell Biol 17：3702-3713.2）Peculis，B.A.和Greer，C.L.（1998）ITS 2近端茎的结构是酵母中前rRNA加工所需的。RNA 4：1610-1622.3）Tomasevic，N.和Peculis，B.A. U8特异性结合蛋白的鉴定。已提交。