Cloning and analysis of centrosome-associated RNA

中心体相关RNA的克隆和分析

• 批准号: 7140361
• 负责人: MARK Collin ALLIEGRO
• 金额: $ 17.15万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140361
• 关键词: RNA; animal genetic material tag; biochemical evolution; cell component structure /function; centrosome; chemical structure function; genetic screening; genetic transcription; genome; immunocytochemistry; molecular biology information system; molecular cloning; nucleic acid sequence; protein localization

DESCRIPTION (provided by applicant): The centrosome in animal cells is composed of a pair of centrioles and a relatively ill-defined pericentriolar matrix. Despite over 100 years of research, our understanding of centrosome composition, organization, and mechanisms of action is poor compared with other organelles. Centrosomes replicate only once per cell cycle to ensure development of a normal bipolar spindle. Replication of the centrioles is generative, semiconservative, and independent of the nucleus. These and other observations have led investigators to propose that centrosomes contain their own nucleic acids. Indeed, a number of reports have been made regarding both the presence and possible functions of DNA and RNA in centrosomes. The consensus in the field as expressed in the most recent reviews is that centrosomes do not contain DNA, but may contain RNA. Recent work in our laboratory provides a new perspective and opportunity to resolve this decades-old question. We have isolated and cloned a unique library of RNAs from purified centrosomes of the surf clam, Spisula solidissima. Preliminary data suggest this pool is markedly enriched in, if not fully restricted to centrosomes. Our long term goals are to determine if these centrosome-associated RNAs (cRNAs) are critical for centrosome function and possibly representative of an organellar genome. Given the controversial history of this line of inquiry and the potential impact of this high risk research, clear proof of principle must first be established. We therefore submit this R21 application with a focus on one Specific Aim, to test the hypothesis that cRNAs represent a unique family of molecules integral to the centrosome. We will perform detailed structural analyses to determine if cRNAs encode proteins, identify functional domains, and discover homologues in nucleic acid and protein databases. We will determine if they are closely associated with the centrosome in vitro and in situ. If our hypothesis is correct, this study will reopen a line of inquiry with impact in areas of cell and developmental biology ranging from control of cell division to evolution of eukaryotes.

描述(申请人提供)：动物细胞中的中心体由一对中心粒和一个相对模糊的中心粒周围基质组成。尽管经过了100多年的研究，但与其他细胞器相比，我们对中心体的组成、组织和作用机制的了解还很差。中心体在每个细胞周期内只复制一次，以确保正常的两极纺锤体的发育。中心粒的复制是生殖性的、半保守性的和独立于核的。这些和其他观察结果导致研究人员提出中心体含有自己的核酸。事实上，已经有许多关于中心体中DNA和RNA的存在和可能的功能的报道。在最近的综述中，该领域的共识是中心体不包含DNA，但可能包含RNA。我们实验室最近的工作为解决这个几十年来的问题提供了一个新的视角和机会。我们已经从纯化的文蛤Spisula solidissima的中心体中分离和克隆了一个独特的RNA文库。初步数据表明，如果不是完全局限于中心体，这个池明显丰富。我们的长期目标是确定这些中心体相关的RNA(CRNAs)是否对中心体功能至关重要，并可能代表细胞器基因组。考虑到这一调查路线有争议的历史和这项高风险研究的潜在影响，必须首先建立明确的原则证据。因此，我们提交这个R21申请的重点是一个特定的目标，以检验CRNAs代表中心体所必需的一个独特的分子家族的假设。我们将进行详细的结构分析，以确定CRNAs是否编码蛋白质，识别功能结构域，并在核酸和蛋白质数据库中发现同源物。我们将在体外和原位确定它们是否与中心体密切相关。如果我们的假设是正确的，这项研究将重新开启一条在细胞和发育生物学领域产生影响的研究路线，范围从细胞分裂的控制到真核生物的进化。