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

核糖体的生物发生是一种存在于所有活细胞中的基本而复杂的多步骤途径。前体rRNA(前-rRNA)编码三个不同的RNA，但转录为单一的前体分子，必须正确地与蛋白质修饰、折叠、加工和组装，才能产生两个亚基，共同形成一个成熟的核糖体。我的研究重点一直是检查和鉴定顺式作用元件和反式作用因子，这些元件和反式作用因子对前rRNA加工的处理事件至关重要，从而对细胞生存至关重要。在过去的一年里，我的实验室在两个方面取得了进展。首先，我们正在使用酵母中可用的遗传学来检查所提出的分子内相互作用，这是加工前rRNA所必需的。其次，我们正在使用生化方法来鉴定包括U8小核仁核糖核蛋白颗粒(U8 SnoRNP)的蛋白质，U8 snoRNP是积累新形成的大的核糖体亚单位所必需的反式作用因子。以前我描述了一个模型，描述了U8 snoRNA似乎促进非洲爪哇卵母细胞处理前rRNA的机制(1)。该模型预测了Pre-rRNA中的特定分子内相互作用。这种茎的形成应该是前rRNA加工的关键。由于非洲爪哇卵母细胞的复杂性，以及这个模型处理RNA的许多不同方面，我使用酵母系统直接测试了这个模型的一个较小的方面。在酵母中可能进行的遗传和生化操作使直接测试和分析非洲爪哇提出的模型的特定方面成为可能。在酵母中的实验明确地证明，这种分子内相互作用的形成是前rRNA加工的关键(2)。在过去的一年里，在酵母中进行的更多实验已经涉及到其他顺式作用元件，这些元件可能作为一级序列或二级结构，也在加工过程中发挥重要作用。我们已经开始对rRNA的这一区域进行更详细的突变，以确定那些影响酵母中前rRNA处理效率或准确性的因素。酵母研究中产生的数据稍后将应用于非洲爪哇的平行实验，这是迄今为止唯一存在的检查脊椎动物rRNA加工的模型系统。我们正在继续表征脊椎动物加工rRNA前所必需的反式作用因子。我先前证明了U8 snoRNP在非洲爪哇卵母细胞中处理前rRNA是必不可少的。在没有U8 RNA的情况下，前rRNA的加工被抑制，不会积累成熟的rRNA(1)。对U8 RNA的突变表明，U8 RNA是必要的，但不足以指导前rRNA的加工；存在影响U8 RNP稳定性和加工效率的蛋白质(1)。为了更好地了解U8蛋白是如何促进U8 RNA的稳定性以及它们如何影响U8 RNP在体内的功能，我们一直在体外寻找与U8 RNA特异结合的蛋白质。在过去的一年里，我们已经在非洲爪哇卵巢提取液中鉴定并部分鉴定了一个29 kDa的蛋白，它与U8 RNA结合。该蛋白与U8 RNA结合具有很高的特异性，并能与U8(3)发生交联反应。体外竞争结合分析表明，该蛋白是U8所特有的，并不代表所有snoRNPs上存在的共同蛋白。初步的蛋白质分析表明，这个新的蛋白质代表了U8 RNP的第一个被鉴定的成分，U8 RNP是处理5.8S和28S rRNA所需的唯一脊椎动物snoRNP。未来的工作将继续表征x29蛋白，并确定其他假定的U8 RNA结合蛋白。通过利用酵母和非洲爪哇这两个不同的模型系统，我们希望更好地了解Pre-rRNA加工的基本机制，并识别所涉及的顺式和反式作用成分。鉴定共同的成分以及物种特有的元素将有助于我们理解在前rRNA成熟的普遍过程中发挥作用的基本机制1)Peculis，B.A.(1997)U8小核仁RNA的5端序列对5.8S和28S rRNA的成熟至关重要。Mol Cell Biol17：3702-3713.2)Peculis，B.A.和Greer，C.L.(1998年)在酵母中加工前rRNA需要ITS2-近端茎的结构。RNA4：1610-1622.3)Tomasevic，N.和Peculis，B.A.一种U8特异结合蛋白的鉴定。已提交。

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.