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

核糖体的生物发生是一个重要而复杂的多步骤过程，存在于所有的活细胞中。前体rRNA （pre-rRNA）编码三种不同的rna，但转录为一个前体分子，必须正确修饰、折叠、加工和与蛋白质组装，才能产生两个亚基，共同形成成熟的核糖体。我的研究重点是检查和鉴定对前rrna加工事件至关重要的顺式作用元件和反式作用因子，因此对细胞存活至关重要。在过去的一年里，我的实验室在两个方面取得了进展。首先，我们利用酵母的遗传学来研究预rrna加工所必需的分子内相互作用。其次，我们使用生化方法鉴定包含U8小核核核糖核蛋白颗粒（U8 snoRNP）的蛋白质，这是新形成的大核糖体亚基积累所需的重要反式作用因子。之前我描述了一个模型，该模型描述了U8 snoRNA似乎促进爪蟾卵母细胞中pre-rRNA加工的机制(1)。该模型预测了pre-rRNA中特定的分子内相互作用。该茎的形成对于pre-rRNA的加工至关重要。由于非洲爪蟾卵母细胞的复杂性以及该模型处理RNA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。在酵母中可能进行的遗传和生化操作使得可以直接测试和分析在非洲爪蟾中提出的模型的特定方面。酵母实验明确表明，这种分子内相互作用的形成对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，在加工过程中也起重要作用。我们已经开始对rRNA的这个区域进行更详细的诱变，以确定那些影响酵母中pre-rRNA加工效率或准确性的元素。酵母研究中产生的数据将随后应用于爪蟾的平行实验，爪蟾是迄今为止唯一用于检查脊椎动物rRNA加工的模型系统。我们正在继续研究脊椎动物中前rrna加工所必需的反式作用因子。我之前证明了U8 snoRNP对于爪蟾卵母细胞的pre-rRNA加工至关重要。在缺乏U8 RNA的情况下，pre-rRNA的加工受到抑制，不会积累成熟的rRNA(1)。U8 RNA的诱变表明U8 RNA是必需的，但不足以指导pre-rRNA的加工；存在影响U8 RNP稳定性和加工效率的蛋白(1)。为了更好地了解U8蛋白如何促进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。在过去的一年中，我们已经在非洲爪蟾卵巢提取物中鉴定并部分表征了一个结合U8 RNA的29 kDa蛋白。该蛋白以高特异性结合U8 RNA，并可与U8交联(3)。体外竞争结合实验表明，该蛋白是U8所特有的，并不代表所有snornp中存在的共同蛋白。初步的蛋白质分析表明，这种新蛋白代表了U8 RNP的第一个鉴定成分，U8 RNP是脊椎动物加工5.8S和28S rRNA所需的唯一snoRNP。未来的工作将继续表征X29蛋白并鉴定其他可能的U8 RNA结合蛋白。通过利用酵母和爪蟾这两种不同的模型系统，我们希望更好地了解pre-rRNA加工的基本机制，并识别其中的顺式和反式作用成分。识别共同成分以及物种特异性成分将有助于我们了解在普遍的pre-rRNA成熟过程中起作用的基本机制。1) Peculis， B. A. (1997) U8小核仁RNA 5端序列是5.8S和28S rRNA成熟的关键。Peculis, ba . and Greer， C.L.（1998）酵母中its2 -近端茎结构对pre-rRNA加工的影响。王晓明，王晓明，王晓明，等。一种u8特异性结合蛋白的鉴定。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.