Functional Roles And Mechanisms Of Snornas In Pre-rrna P

Snornas在Pre-rna P中的功能作用和机制

• 批准号: 6673780
• 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/6673780
• 关键词: RNA binding protein; RNA biosynthesis; Saccharomyces cerevisiae; Xenopus oocyte; genetic manipulation; genetic regulatory element; intermolecular interaction; laboratory mouse; laboratory rat; molecular chaperones; nucleic acid hybridization; nucleic acid sequence; nucleic acid structure; nucleolus; point mutation; posttranscriptional RNA processing; protein structure function; ribosomal RNA; small nuclear RNA; small nuclear ribonucleoproteins; transcription factor; yeast two hybrid system

Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three separate structural RNAs that are transcribed as a single precursor molecule. This precursor must be correctly modified, folded, processed and assembled with proteins to yield the two mature ribosomal subunits that comprise 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 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 growth and 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 differentiate between two predicted structural models for an intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Identification of the structural conformation of the precursor is essential for understanding how the structure affects ribosome biogenesis. 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 assay the effects 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, 3). Over the past year we have identified essential alterations in structural conformation which are both required for efficient processing and are involved in specific recognition of the precursors (4). The data obtained in these yeast studies are being 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 prime 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 (5). We are continuing to characterize the X29 protein. In addition we have identified a heteroheptameric complex of proteins which bind U8 snoRNA in an evolutionarily conserved octamer sequence in U8 snoRNA (6). NMR on our purified complex of proteins has identified each member of the heteroheptamer. These proteins are evolutionarily conserved and present from Archaea to human (6). Thus, U8 snoRNA function probably requires binding of the LSm complex in all organisms. We are further characterizing this protein complex and the binding site on U8 RNA to learn why binding of this protein complex is essential for in vivo function of the U8 RNP. A third focus of the lab has been an examination of snoRNA localization in oocytes and the kinetics of snoRNA-mediated pre-rRNA processing in Xenopus oocytes (7). We previously examined the kinetics of pre-rRNA processing in oocytes treated with transcriptional inhibitors to separate transcription-dependent events from those involved in processing and the data were consistent with a scenario where these snoRNAs must be present co-transcriptionally to facilitate processing. These results supports our theory that the snoRNAs act, in part, as molecular chaperones to facilitate pre-rRNA folding and we are continuing to pursue direct evidence of our working model for U8 function in vivo (1). Characterization of U8 snoRNA genes in Xenopus identified naturally occurring U8 sequence variants that are functional in vivo (8). 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 the protein complex that binds this sequence (6) has begun to provide insight into conserved functional mechanisms and provide additional information about the unique role of U8 snoRNP in 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 processing. 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. Identification of conserved and unique cis- and trans-acting components involved in ribosome biogenesis will provide additional components to monitor in diseases involving the complex process of ribosome biogenesis.

核糖体生物发生是存在于所有活细胞中的重要且复杂的多步骤途径。前体 rRNA (pre-rRNA) 编码三个独立的结构 RNA，它们转录为单个前体分子。该前体必须经过正确的修饰、折叠、加工并与蛋白质组装，才能产生构成功能性核糖体的两个成熟核糖体亚基。该途径的严重突变是致命的。轻微扰动的特征是疾病，包括先天性角化不良和一些自身免疫性疾病。 我实验室的研究重点是识别和表征对前 rRNA 加工过程至关重要的顺式作用元件和反式作用因子，因此对细胞生长和存活至关重要。在过去的一年里，我的实验室在三个方面取得了进展。首先，我们利用酿酒酵母中可用的遗传学来区分后续处理步骤所需的 pre-rRNA 中分子内相互作用的两种预测结构模型。前体结构构象的鉴定对于理解该结构如何影响核糖体生物发生至关重要。其次，我们使用生化方法来鉴定非洲爪蟾 U8 小核仁核糖核蛋白颗粒 (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。第三，我们正在研究 pre-rRNA 加工的动力学，以便在机制水平上更多地了解 snoRNP，特别是 U8，在 pre-rRNA 加工中所起的作用。 实验室的一个重点是对我们之前描述的 U8 snoRNA 可能促进非洲爪蟾卵母细胞中前 rRNA 加工的机制模型进行更详细的检查 (1)。该模型预测 pre-rRNA 中特定分子内相互作用的形成对于 pre-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。酵母中遗传和生化操作的可行性使得分析该区域的点突变对处理前 rRNA 的能力的影响成为可能。我们在酵母中进行的早期实验明确证明，这种分子内相互作用的形成对于前 rRNA 加工至关重要 (2, 3)。在过去的一年里，我们已经确定了结构构象的重要改变，这些改变既是有效加工所必需的，也涉及前体的特异性识别 (4)。这些酵母研究中获得的数据正应用于非洲爪蟾的平行实验，非洲爪蟾是迄今为止唯一用于检查脊椎动物 rRNA 加工的现有模型系统。 第二个重点是我们对脊椎动物前 rRNA 加工所必需的反式作用因子的表征的延续。我之前证明了 U8 snoRNP 对于非洲爪蟾卵母细胞中前 rRNA 加工至关重要。在没有 U8 RNA 的情况下，前 rRNA 加工会受到抑制，并且不会积累成熟的 rRNA (1)。 U8 RNA 的诱变表明 U8 RNA 5 引物末端的序列是必要的，但不足以指导 pre-rRNA 加工；据推测，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与 U8 RNA 特异性结合的蛋白质。我们最近报道了从爪蟾卵巢提取物中鉴定出的 29 kDa 蛋白质，该蛋白质特异性结合 U8 RNA (5)。我们正在继续表征 X29 蛋白。此外，我们还鉴定了一种异七聚体蛋白质复合物，它以 U8 snoRNA 中进化保守的八聚体序列结合 U8 snoRNA (6)。我们纯化的蛋白质复合物的核磁共振鉴定了异七聚体的每个成员。这些蛋白质在进化上是保守的，并且存在于从古细菌到人类的整个过程中 (6)。因此，U8 snoRNA 功能可能需要所有生物体中 LSm 复合物的结合。我们正在进一步表征该蛋白质复合物和 U8 RNA 上的结合位点，以了解为什么该蛋白质复合物的结合对于 U8 RNP 的体内功能至关重要。 该实验室的第三个重点是检查 snoRNA 在卵母细胞中的定位以及爪蟾卵母细胞中 snoRNA 介导的前 rRNA 加工的动力学 (7)。我们之前检查了用转录抑制剂处理的卵母细胞中前 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，并且数据与这些 snoRNA 必须共转录存在以促进加工的情况一致。这些结果支持了我们的理论，即 snoRNA 在一定程度上充当分子伴侣，促进 rRNA 前体折叠，并且我们正在继续寻找 U8 体内功能工作模型的直接证据 (1)。对非洲爪蟾中 U8 snoRNA 基因的表征鉴定出天然存在的、在体内具有功能的 U8 序列变体 (8)。这种自然变异使我们能够鉴定出 U8 snoRNA 中保守的八聚体序列，该序列存在于迄今为止已知的所有脊椎动物 U8 snoRNA 中，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复合物的鉴定 (6) 已开始深入了解保守的功能机制，并提供有关 U8 snoRNP 在前 rRNA 加工中独特作用的更多信息。 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 pre-rRNA 加工的基本机制。通过了解核糖体生物合成中涉及的正常过程和组成部分，我们可以开始解决先天性角化不良、硬皮病和其他细胞增殖疾病等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且独特的顺式和反式作用成分将为监测涉及核糖体生物发生复杂过程的疾病提供额外的成分。