Functional Roles And Mechanisms Of snoRNAs In pre-rRNA P

pre-rRNA P 中 snoRNA 的功能作用和机制

• 批准号: 6507329
• 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/6507329
• 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 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.

核糖体的生物发生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）编码三个独立的rna，这些rna转录为一个前体分子，必须正确修饰、折叠、加工和与蛋白质组装，以产生两个成熟的核糖体亚基，构成功能性核糖体。我实验室的研究重点是识别和表征对pre-rRNA加工事件至关重要的顺式作用元件和反式作用因子，因此对细胞存活至关重要。在过去的一年里，我的实验室在三个方面取得了进展。首先，我们利用酵母S.cerevisiae的遗传学技术，来检验预测的pre-rRNA分子内相互作用，这是后续加工步骤所必需的。其次，我们正在使用生化方法鉴定包含爪蟾U8小核核核糖核蛋白颗粒（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，以在机制水平上了解更多关于snoRNPs，特别是U8在pre-rRNA加工中的作用。实验室的一个重点是对我们之前描述的U8 snoRNA可能促进爪蟾卵母细胞中pre-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个方面。酵母遗传和生化操作的可行性使得直接测试该区域点突变对预处理前rrna能力的影响成为可能。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，其他顺式作用元素在加工过程中起着重要作用；这些似乎是结构元素，序列在这些结构的识别中作用很小(3)。在这些酵母研究中获得的数据将随后应用于爪蟾的平行实验，这是迄今为止唯一存在的用于检查脊椎动物rRNA加工的模型系统。第二个重点是我们对脊椎动物中pre-rRNA加工所必需的反式作用因子的描述的延续。我之前证明了U8 snoRNP对于爪蟾卵母细胞的pre-rRNA加工至关重要。在缺乏U8 RNA的情况下，pre-rRNA加工受到抑制，没有成熟rRNA积累(1)。U8 RNA的诱变实验表明，U8 RNA 5&#8217；末端的序列是必需的，但不足以指导pre-rRNA的加工；推测U8 RNP蛋白影响了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。我们最近报道了从爪蟾卵巢提取物中鉴定出一个29 kDa的蛋白，该蛋白特异性结合U8 RNA(4)。该蛋白以高亲和力结合U8 RNA，并可与U8 snoRNA交联。体外竞争结合实验表明，该蛋白是U8 RNP所特有的，而不是其他snornp中常见或共享的蛋白(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对爪蟾卵母细胞中snoRNA定位和前rrna加工动力学的研究(5)。在用转录抑制剂处理的卵母细胞中，研究了pre-rRNA加工的动力学，以分离转录依赖事件和参与加工的事件。当在缺乏snorna的卵母细胞中进行时，功能拯救试验表明，snorna无法介导先前积累的rRNA前体的加工。这一结果与这些snoRNAs必须共同转录以促进加工的情况一致(5)，并支持我们的理论，即snoRNAs在一定程度上作为分子伴侣促进pre-rRNA折叠。对非洲爪蟾中U8 snoRNA基因的表征鉴定出在体内具有功能的自然发生的U8序列变异(6)。这种自然变异使我们能够在迄今已知的所有脊椎动物（包括爪蟾、小鼠、大鼠和人类）的U8 snoRNA中鉴定出一个保守的八聚体序列。八聚体的表征和结合该序列的蛋白质的鉴定可能有助于深入了解保守的功能机制，并提供有关U8 snoRNP在pre-rRNA加工中的独特作用的额外信息。通过使用两种模型系统并利用它们之间的差异，我们希望更好地了解pre-rRNA加工的基本机制，并确定参与pre-rRNA成熟的保守和独特的顺式和反式作用成分。鉴定核糖体的共同成分以及物种特异性成分将有助于我们了解核糖体生物发生的普遍和复杂过程中的基本机制。