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）编码三个单独的RNA，其被转录为单个前体分子，其必须被正确地修饰、折叠、加工并与蛋白质组装以产生构成功能性核糖体的两个成熟核糖体亚基。在我的实验室的研究重点一直是确定和表征顺式作用元件和反式作用因子的前rRNA加工的加工事件的关键，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，以检查预测的前rRNA分子内相互作用所需的后续处理步骤。第二，我们正在使用生物化学方法来确定蛋白质，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pre-rRNA加工的动力学，以在机械水平上了解更多关于snoRNP，特别是U8，在pre-rRNA加工中发挥的作用。 实验室的一个重点是更详细地检查我们先前描述的U8 snoRNA可能促进非洲爪蟾卵母细胞中前rRNA加工的机制模型（1）。该模型预测，前体rRNA中特异性分子内相互作用的形成对于前体rRNA加工至关重要。由于该模型涉及RNA加工的许多不同方面以及非洲爪蟾卵母细胞系统的复杂性，因此使用酵母系统直接测试该模型的这一方面。在酵母中进行遗传和生物化学操作的可行性使得可以直接测试该区域中的点突变对处理pre-rRNA的能力的影响。我们在酵母中的早期实验明确表明，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表明，其他顺式作用元件在加工过程中起着重要作用;这些元件似乎起着结构元件的作用，而序列在识别这些结构方面几乎没有作用（3）。在这些酵母研究中获得的数据稍后将应用于非洲爪蟾的平行实验，非洲爪蟾是迄今为止唯一存在的研究脊椎动物rRNA加工的模型系统。 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。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，并且没有成熟的rRNA积累（1）。U8 RNA的诱变表明，U8 RNA 5端的序列是必需的，但不足以指导pre-rRNA加工;推测U8 RNP蛋白影响U8 RNA的稳定性和加工效率（1）。’为了更好地了解U8 RNP在体内的功能，我们一直在体外鉴定特异性结合U8 RNA的蛋白质。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（4）。该蛋白质以高亲和力结合U8 RNA，并且可以与U8 snoRNA交联。体外竞争结合试验表明，这种蛋白质是U8 RNP特有的，不是其他snoRNP中常见或共有的蛋白质（4）。我们正在继续表征X29蛋白并鉴定其他U8 RNA结合蛋白，以更好地了解U8 RNP如何促进加工。 实验室的第三个重点是检查卵母细胞中snoRNA的定位和非洲爪蟾卵母细胞中pre-rRNA加工的动力学（5）。在用转录抑制剂处理的卵母细胞中检查前rRNA加工的动力学，以将转录依赖性事件与参与加工的事件分开。当在snoRNA耗尽的卵母细胞中进行时，拯救功能测定证明snoRNA介导先前积累的rRNA前体的加工失败。‘’这一结果与这些snoRNA必须共转录存在以促进加工的情况一致（5），并支持我们的理论，即snoRNA部分地作为分子伴侣促进前rRNA折叠。非洲爪蟾中U8 snoRNA基因的表征鉴定了在体内具有功能的天然存在的U8序列变体（6）。这种自然变异使我们能够鉴定出迄今为止已知的所有脊椎动物U8 snoRNA中存在的U8 snoRNA中的保守八聚体序列，包括非洲爪蟾，小鼠，大鼠和人类。八聚体的表征和结合该序列的蛋白质的鉴定可以深入了解保守的功能机制，并提供关于U8 snoRNP在前rRNA加工中的独特作用的额外信息。 通过使用两个模型系统并利用它们的差异，我们希望更好地了解前rRNA加工的基本机制，并鉴定参与前rRNA成熟的保守且独特的顺式和反式作用组分。鉴定共同的成分以及物种特异性元件将有助于我们理解在核糖体生物发生的普遍和复杂过程中起作用的基本机制。

Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three separate RNAs that are transcribed as a single precursor molecule that must be correctly modified, folded, processed and assembled with proteins to yield the two mature ribosomal subunits that comprise the functional ribosome. The focus of the research in my lab has been to identify and characterize cis-acting elements and trans-acting factors 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 three fronts. First, we are using the genetics available in yeast, S.cerevisiae, to examine a predicted intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Second, we are using biochemical methods to identify 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 of pre-rRNA processing to learn more, at a mechanistic level, about the roles that snoRNPs, particularly U8, play in pre-rRNA processing. One focus in the lab involves a more detailed examination of our previously described a model for the mechanism by which U8 snoRNA may facilitate pre-rRNA processing in the Xenopus oocyte (1). This model predicted that formation of a specific intramolecular interaction in pre-rRNA should be critical for pre-rRNA processing. Because of the many different aspects of RNA processing addressed by this model and complexity of the Xenopus oocyte system, the yeast system was used to directly test this one aspect of the model. The feasibility of genetic and biochemical manipulations in yeast made it possible to directly test the effect of point mutations in this region upon the ability to process pre-rRNA. Our early 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 that play important roles in processing; these appear to function as structural elements and sequence plays little role in recognition of these structures (3). The data obtained in these 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. A second focus is a continuation of 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 accumulates (1). Mutageneis of U8 RNA indicated that sequences at the 5’ end of U8 RNA were necessary, but not sufficient to direct pre-rRNA processing; presumably 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. We recently reported our identification of a 29 kDa protein from Xenopus ovary extracts which specifically binds U8 RNA (4). This protein binds U8 RNA with high affinity and can be crosslinked to U8 snoRNA. In vitro competition binding assays indicated this protein is unique to the U8 RNP and is not a common or shared protein present in other snoRNPs (4). We are continuing to characterize the X29 protein and identify other U8 RNA binding proteins to gain a better mechanistic understanding of how the U8 RNP facilitates processing. A third focus of the lab has been an examination of snoRNA localization in oocytes and the kinetics of pre-rRNA processing in Xenopus oocytes (5). The kinetics of pre-rRNA processing were examined in oocytes treated with transcriptional inhibitors to separate transcription-dependent events from those involved in processing. When performed in snoRNA-depleted oocytes, the ‘rescue-of-function’ assay demonstrated a failure of snoRNAs to mediated processing of previously accumulated rRNA precursors. This result is consistent with a scenario where these snoRNAs must be present co-transcriptionally to facilitate processing (5) and supports our theory that the snoRNAs act, in part, as molecular chaperones to facilitate pre-rRNA folding. Characterization of U8 snoRNA genes in Xenopus identified naturally occurring U8 sequence variants that are functional in vivo (6). This natural variation allowed us to identify a conserved octamer sequence in U8 snoRNA present in all vertebrate U8 snoRNAs known to date, including Xenopus, mouse, rat and human. Characterization of the octamer and identification of proteins that bind this sequence may give insight into conserved functional mechanisms and provide additional information about the unique role of U8 snoRNP in pre-rRNA processing. In using two model systems and taking advantage of their differences we hope to better understand the basic mechanisms of pre-rRNA processing and to identify conserved and unique cis- and trans-acting components involved in pre-rRNA maturation. Identification of common components as well as species specific elements will help us understand the basic mechanisms at play in the universal and complex process of ribosome biogenesis.