Functional analysis of proteins that interact with Centrosomin. L.-R. Kao , T.L. Megraw , T.C. Kaufman. HHMI and Dept Biol, Indiana Univ, Bloomington, IN.

   Centrosomin (Cnn) is a core centrosomal protein that is localized to mitotic centrosomes throughout development. Mutations in cnn (null and C-terminal truncated alleles) are viable but female and male sterile. Without Cnn both mitotic and meiotic spindle organization as well as the assembly of the spermatid basal body are affected. Cnn contains three putative leucine zipper motifs and several coiled-coil domains, both of which may be involved in protein-protein interactions. In order to identify proteins that interact with Cnn, we have carried out yeast two-hybrid screens and several interesting candidates were identified. Two of these are related proteins, encoded by separate genes, that may be involved in regulating the phosphorylation state of Cnn. Several additional cDNAs (EST clones) were acquired from the BDGP and their sequences demonstrate that both genes have complicated 5' and 3' UTR sequences, likely resulting from alternative splicing. Polyclonal antibodies were raised against both proteins and immunofluorescence microscopy showed that both proteins are principally localized in the cytoplasm in ovaries, early embryos and S2 cells. To assay the function(s) of these proteins, RNA-mediated interference (RNAi) experiments were performed in embryos and S2 cells. RNAi caused multiple centrosomes to appear at the spindle poles, implicating a role for these proteins in centrosome replication. P-element mutant screens have also been carried out to isolate mutations in both genes. Several short deletion mutations of one gene were isolated, and all of these alleles are recessive lethal. Analysis of the mutant phenotype is in progress.