Functional Roles And Mechanisms Of Snornas In Pre-rrna P

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）编码三种结构RNA，它们必须正确地被修饰、折叠、加工并与大约80种核糖体蛋白组装，并且在组装过程中可能受到数百种反式作用因子的作用。这个复杂的途径产生两个成熟的核糖体亚基，组成功能性核糖体。这一途径的严重突变是致命的。轻微的扰动的特点是疾病，包括先天性角化不良和一些自身免疫性疾病。 在我的实验室的研究的重点是通过识别和学习的顺式序列和反式因子在真核生物中的ITS 2内加工所需的体内功能作用，以了解更多关于核糖体组装过程。在过去的一年里，我的实验室在三个方面取得了进展。首先，我们正在使用酵母中的遗传学，酿酒酵母，以区分两个预测的结构模型的前rRNA的分子内相互作用所需的后续处理步骤。前体内初级切割位点的结构构象和序列识别的鉴定对于理解结构如何影响核糖体生物发生是必不可少的。其次，我们正在使用生物化学和生物物理方法来识别和表征的蛋白质，包括非洲爪蟾U8小核仁核糖核蛋白颗粒（U8 snoRNP），一个必要的反式作用因子所需的积累新形成的大核糖体亚基。第三，我们正在研究前rRNA加工中的动力学和snoRNA：pre-rRNA相互作用，并直接测试我们的工作模型在体内U8 RNA的作用（1）。 该实验室的一个重点是利用酵母酿酒酵母中可用的遗传学，使用我们的体内功能遗传分析来了解pre-rRNA中的细微序列和结构突变是否会影响pre-rRNA加工的效率或准确性。我们在酵母中的早期实验明确表明，形成一个特定的分子内相互作用是前rRNA加工的关键（2，3）。此后，我们鉴定了在成熟事件中起关键作用的ITS 2的相互转化结构构象（4）。我们现在正在产生的构建体，将解决在ITS 2内的主要切割位点的结构构象和序列识别的要求。关于切割位点识别的信息将与我们关于前体rRNA的该区域的替代结构的知识相结合，以允许我们开始对结构改变和反式因子（包括加工机器）的组装的时间进行排序，从而影响前体rRNA加工的效率。 第二个重点是继续我们的反式作用因子的特性在脊椎动物前rRNA加工必不可少的。U8 snoRNP是非洲爪蟾卵母细胞中前rRNA加工所必需的。在不存在U8 RNA的情况下，前rRNA加工受到抑制，并且没有成熟的rRNA积累（1）。U8 RNA的突变表明，U8 RNA 5 ′端的序列是必需的，但不足以指导pre-rRNA加工; U8 RNP蛋白影响U8 RNA的稳定性和加工效率（1）。为了更好地理解U8 RNP如何在体内发挥作用，我们一直在体外鉴定特异性结合U8 RNA的蛋白质（5，6）。我们最近报道了从非洲爪蟾卵巢提取物中鉴定出一种特异性结合U8 RNA的29 kDa蛋白质（5）。我们将继续在生化、生物物理和细胞水平上表征X29蛋白。我们最近证明了X29是在真核生物中鉴定的核去帽蛋白的第一个例子。我们已经开始使用结构方法（X射线晶体学）来研究这种蛋白质，以更好地了解这种蛋白质如何结合U8 RNA。我们正在研究这种蛋白质的体内定位，以更好地将其体内作用与其体外功能相关联。结构生物学、生物化学和细胞生物学的结合将使我们更好地了解这种蛋白质如何影响U8 snoRNA，从而影响核糖体的生物合成。 实验室的第三个重点是直接检查U8体内功能的工作模型。我们之前提出U8在体内作为RNA伴侣蛋白发挥作用，通过促进前rRNA中5.8S和28 S之间的相互作用，这种相互作用持续存在于成熟核糖体中（1）。我们已经产生的构建体，将用于在非洲爪蟾卵母细胞体内直接测定所提出的U8碱基配对的要求。这些实验是特别关键的，因为非洲爪蟾卵母细胞是唯一的脊椎动物模型系统的前rRNA加工。 使用我们的两个模型系统并利用它们的差异将使我们能够更好地理解前rRNA组装的基本机制和加工事件的时间。了解了核糖体生物合成中的正常过程和成分，我们就可以开始讨论在先天性角化不良、硬皮病和其他核糖体生物合成速率发生改变的细胞增殖疾病中哪些方面是异常的。鉴定参与核糖体生物发生的保守和独特的顺式和反式作用组分将提供额外的组分来监测涉及核糖体生物发生复杂过程中的缺陷的疾病。

Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three structural RNAs that must be correctly modified, folded, processed and assembled with approximately 80 ribosomal proteins and acted upon by probably several hundred trans action factors during the assembly process. This intricate pathway yields the two mature ribosomal subunits comprising the functional ribosome. Severe mutations in this pathway are lethal. Minor perturbations are characterized by diseases including dyskeratosis congenital and some autoimmune diseases. The focus of the research in my lab is to learn more about the ribosomal assembly process by identifying and learning the in vivo functional roles of the cis sequences and trans factors required for processing within ITS2 in eukaryotes. Over the past year my lab has made progress on three fronts. First, we are using the genetics available in yeast, S.cerevisiae, to differentiate between two predicted structural models for an intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Identification of the structural conformation and sequence recognition of primary cleavage sites within the precursor is essential for understanding how the structure affects ribosome biogenesis. Second, we are using biochemical and biophysical methods to identify and characterize proteins that comprise the Xenopus U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits. Third, we are examining the kinetics and snoRNA:pre-rRNA interactions in pre-rRNA processing and directly testing our working model for the role of U8 RNA in vivo (1). One focus of the lab takes advantage of the genetics available in yeast, S.cerevisiae, to use our in vivo functional genetic assay to learn whether subtle sequence and structural mutations in pre-rRNA affects the efficiency or accuracy of pre-rRNA processing. Our early experiments in yeast unequivocally demonstrated that formation of a particular intramolecular interaction is critical for pre-rRNA processing (2, 3). We have since identified interconverting structural conformations of ITS2 which play critical roles in the maturation event (4). We are now generating constructs which will address requirements for structural conformation and sequence recognition at the primary cleavage site within ITS2. The information about cleavage site recognition will be combined with our knowledge about alternative structures of this region of the pre-rRNA to allow us to begin to order the timing of the structural alterations and assembly of trans-factors (including the processing machinery) to affect the efficiency of pre-rRNA processing. A second focus is a continuation of our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. 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 accumulates (1). Mutagenesis of U8 RNA indicated that sequences at the 5 prime end of U8 RNA were necessary, but not sufficient to direct pre-rRNA processing; U8 RNP proteins affected the stability of the U8 RNA and the efficiency of processing (1). To better understand how the U8 RNP functions in vivo, we have been identifying proteins which specifically bind U8 RNA in vitro (5, 6). We recently reported our identification of a 29 kDa protein from Xenopus ovary extracts which specifically binds U8 RNA (5). We are continuing to characterize the X29 protein on biochemical, biophysical and cellular levels. We have recently demonstrated X29 is the first example of a nuclear decapping protein identified in eukaryotes. We have begun to look at this protein using structural methods (X-ray crystallography) to better understand how this protein binds U8 RNA. We are looking at the in vivo localization of this protein to better correlate the in vivo role with its identified in vitro function. Together the structural biology, biochemistry and cell biology will provide us with a better understanding of how this protein affects U8 snoRNA and thus ribosome biogenesis. A third focus of the lab has been a direct examination of the working model for U8 function in vivo. We previously proposed that U8 functioned in vivo as an RNA chaperone by facilitating an interaction between 5.8S and 28S in pre-rRNA, which persists in the mature ribosome (1). We have been generating constructs which will be used in vivo in Xenopus oocytes to directly assay the requirement for this proposed U8 base pairing. These experiments are particularly critical since the Xenopus oocyte is the only vertebrate model system for pre-rRNA processing. Using our two model systems and taking advantage of their differences will allow us to better understand the basic mechanisms of pre-rRNA assembly and the timing of the processing events. With an understanding of the normal processes and components involved in ribosome biogenesis we can begin to address what aspects are abnormal in diseases like dyskeratosis congenita, scleroderma and other cell-proliferation diseases where the rate of ribosome biogenesis has been altered. Identification of conserved and unique cis- and trans-acting components involved in ribosome biogenesis will provide additional components to monitor in diseases implicating defects in the complex process of ribosome biogenesis.