Cloning and analysis of centrosome-associated RNA

描述（由申请人提供）：动物细胞中的中心体由一对中心粒和相对不明确的中心粒周围基质组成。尽管经过了100多年的研究，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有限。中心体每个细胞周期只复制一次，以确保正常双极纺锤体的发育。中心粒的复制是生殖性的，半保守性的，独立于细胞核之外。这些和其他观察结果使研究人员提出中心体含有它们自己的核酸。事实上，关于DNA和RNA在中心体中的存在和可能的功能已经有了许多报道。在最近的评论中，该领域的共识是中心体不含有DNA，但可能含有RNA。我们实验室最近的工作为解决这个几十年的问题提供了新的视角和机会。我们分离并克隆了一个独特的rna文库，从纯化的海蛤，Spisula solidissima中心体。初步数据表明，如果不完全局限于中心体，这个池也明显丰富。我们的长期目标是确定这些中心体相关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。考虑到这一研究路线的争议历史和这种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，我们提交了R21申请，重点关注一个特定目标，以测试crna代表中心体不可或缺的独特分子家族的假设。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，这项研究将在细胞和发育生物学领域重新开启一条探究线，从细胞分裂的控制到真核生物的进化。

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.