Cloning and analysis of centrosome-associated RNA

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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.