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

SNO-RNA 在 RRNA 前处理中的功能作用和机制

• 批准号: 6105761
• 负责人: BRENDA A PECULIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6105761
• 关键词: RNA; RNA biosynthesis; Xenopus oocyte; intermolecular interaction; molecular chaperones; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; nucleolus; ribosomal RNA; yeast two hybrid system

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.

核糖体RNA（rRNA）是一种关键的催化剂， 核糖体的组成部分，一个复杂而巨大的 存在于所有活细胞中的核糖核蛋白颗粒。前rRNA是 转录为单个长的前体分子， 与蛋白质一起折叠、加工和组装， 核糖体在过去的一年里，我的实验室取得了重大进展。 在更好地了解一些顺式和反式作用的 参与前rRNA加工的组分。更具体地说， 我们已经确定了前rRNA中的顺式作用元件， 这对于处理产生成熟rRNA的事件至关重要， 酵母另外，我们已经开始分析 包含U8snoRNP，一种反式作用的小核仁 核糖核蛋白颗粒，我以前已经证明， 非洲爪蟾卵母细胞模型中前rRNA加工的关键 系统在不存在U8 RNA的情况下， 抑制，没有成熟的rRNA积累。去年我 发表了一篇论文，介绍了数据和模型，描述了 U8似乎促进前rRNA的机制 在非洲爪蟾卵母细胞中进行处理。该模型预测了一个特定的 分子内相互作用，其形成应该是关键的 用于前rRNA加工。由于复杂的 非洲爪蟾卵母细胞和模棱两可的结果的高可能性， 在这个系统中，我首先使用酵母系统来测试一个较小的方面， 这个模型直接，使用遗传学方法。中的实验 酵母已经非常富有成效，明确地证明， 这种分子内相互作用的影响对于加工至关重要。 酵母实验中的其他实验也暗示了 顺式作用元件（一级序列或二级序列 结构），在加工过程中也起着重要作用。这些 目前正在审查其他内容。达成的成果 在酵母中的工作是，结果预测了额外的实验， 现在需要在非洲爪蟾身上进行， 系统去年发表的论文也提供了证据， U8 RNP的U8 RNA成分是必需的，但 足以促进加工。与之相关的蛋白质，或 由U8 RNA招募的蛋白质也是加工所必需的， 它们似乎以一种特定的方式起作用。审查这些 蛋白质及其在加工过程中的作用，我的实验室一直在 使用凝胶位移测定和交联实验来鉴定和 通过标准柱富集U8特异性蛋白 色谱法为此，我们已经确定了假定的 我们正在研究的U8 RNA特异性结合蛋白 更详细地描述。通过利用这两个优势 不同的模型系统，酵母和爪蟾，我们希望更好地 了解pre-rRNA加工的机制， 以及所涉及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素将有助于 我们了解在宇宙进程中起作用的基本机制， rRNA前体成熟