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

核糖体rna(Rrna)是一种重要的催化剂。 核糖体的组成部分，是一种复杂而巨大的 存在于所有活细胞中的核糖核蛋白颗粒。前rRNA是 转录成单一的长的前体分子，然后被修饰， 与蛋白质折叠、加工和组装，以产生成熟的 核糖体。在过去的一年里，我的实验室取得了重大进展 更好地理解一些顺式和反式作用的进展 参与前rRNA加工的成分。更确切地说， 我们已经在前rRNA中确定了一种顺式作用元件，即形成 其中对于处理在体内产生成熟rRNA的事件至关重要 酵母。此外，我们已经开始分析蛋白质 包括U8snoRNP，一种反式作用的小核仁 我之前演示过的核糖核蛋白颗粒是 非洲爪哇卵母细胞模型中对前rRNA处理的关键 系统。在没有U8 RNA的情况下，pre-rRNA的加工是 被抑制，不会积累成熟的rRNA。去年我 发表了一篇论文，介绍了数据和描述 U8促进Pre-rRNA的机制 在非洲爪哇卵母细胞中进行加工。这个模型预测了一个特定的 分子内相互作用，它的形成应该是至关重要的 用于核糖核酸前处理。因为这个问题很复杂 非洲爪哇卵母细胞与结果不明确的可能性很高 这个系统，我首先使用酵母系统测试了一个较小的方面 这个模型直接使用了遗传方法。在中国的实验 酵母菌一直非常高产，毫不含糊地证明了这种形成 这种分子内相互作用对加工至关重要。 酵母实验中的其他实验已经牵连到了其他 顺式作用元件(初级序列或次级序列 结构)，它们在加工过程中也起着重要作用。这些 目前正在审查其他要素。一个后果就是 在酵母中的工作是，结果预测了更多的实验 现在需要在非洲爪哇，脊椎动物模型中进行 系统。去年发表的这篇论文也提出了证据 U8 RNP的U8 RNA成分是必需的，但不是 足以方便加工。与之相关的蛋白质，或 被U8 RNA招募也是加工必不可少的，一些 它们似乎以物种特有的方式起作用。来研究这些 蛋白质及其在加工过程中的作用，我的实验室一直在 使用凝胶移位分析和交联实验来鉴定和 用标准柱富集U8特异性蛋白质 层析方法。为此，我们确定了推定的 我们正在研究的U8 RNA特异性结合蛋白 更详细地刻画。通过利用这两个优势 不同的模型系统，酵母和非洲爪哇，我们希望更好 了解Pre-rRNA加工的机制并识别 以及所涉及的顺式和反式作用成分。身份识别 共同的成分以及物种特有的元素将有所帮助 美国了解在普遍进程中发挥作用的基本机制 核糖核糖核酸成熟前。

Ribosomal RNA (rRNA) is a critical catalytic component of the ribosome, a complex and massive ribonucleoprotein particle present in all living cells. Pre-rRNA is transcribed as a single long precursor molecule that is modified, folded, processed and assembled with proteins to yield a mature ribosome. Over the past year my laboratory has made significant progress in better understanding some of the cis- and trans-acting components involved in pre-rRNA processing. More specifically, we have identified a cis-acting element in pre-rRNA, the formation of which is critical for processing events that yield mature rRNA in yeast. In addition, we have begun to analyze the proteins comprising the U8snoRNP, a trans-acting small nucleolar ribonucleoprotein particle which I have previously demonstrated is critical for pre-rRNA processing in the Xenopus oocyte model system. In the absence of U8 RNA pre-rRNA processing is inhibited and no mature rRNA will accumulate. Last year I published a paper presenting data and a model describing the mechanisms by which U8 appeared to facilitate pre-rRNA processing in the Xenopus oocyte. This model predicted a specific intramolecular interaction, the formation of which should be critical for pre-rRNA processing. Because of the complexity of the Xenopus oocyte and the high likelihood of ambiguous results in that system, I first used the yeast system to test a smaller aspect of this model directly, using genetic methods. The experiments in yeast have been very fruitful, proving unequivocally that formation of this intramolecular interaction is critical for processing. Additional experiments in yeast experiment have implicated other cis-acting elements (either primary sequence or secondary structure) which also play an important roles in processing. These additional elements are currently being examined. One outcome of the work in yeast is that the results predict additional experiments which now need to be performed in Xenopus, the vertebrate model system. The paper published last year also presented evidence that the U8 RNA component of the U8 RNP was necessary but not sufficient to facilitate processing. The proteins associated with, or recruited by U8 RNA are also essential for processing, some of which appear to act in a species specific manner. To examine these proteins and their role in processing more closely, my lab has been using gel shift assays and crosslinking experiments to identify and enrich for U8-specific proteins via standard column chromatography methods. To this end, we have identified putative U8 RNA-specific binding proteins that we are in the process of characterizing in more detail. By taking advantage of the two different model systems, yeast and Xenopus, we hope to better understand the mechanisms of pre-rRNA processing and 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.