Aberrant centrosome function underlies pathologies with profound significance for human health, including growth deficiency syndromes and cancer. Centrosomes function as microtubule organizing centers, build the mitotic spindle, form the basal body to template cilia in ciliated cells, and serve as platforms for signaling cascades including cell cycle signaling. The centrosome consists of a pair of centrioles surrounded by a protein matrix termed pericentriolar material (PCM). The composition and quantity of PCM determines the microtubule nucleating activity of the centrosome and changes rapidly in cycling cells. The mechanisms responsible for rapid changes to centrosome composition and structure are incompletely understood. Intriguingly, a screen for localized mRNA in Drosophila early embryos identified multiple mRNA transcripts enriched at spindle poles. Mutations to several of these genes disrupt centrosome function and/or the mitotic spindle, suggesting that mRNA enrichment and local translation at the centrosome may be an unexplored mechanism to modulate centrosome composition. This proposal aims to fill this gap in knowledge by leveraging the genetically tractable Drosophila melanogaster early embryo, an ideal system to visualize hundreds of active MTOC centrosomes. My central hypothesis is that specific RNAs are actively localized to the centrosome by RNA binding proteins to regulate its structure and function. I will test this hypothesis with two complementary aims.
In Specific Aim 1, I will define the role of centrosome mRNAs in centrosome composition and function using single molecule FISH, spinning disk confocal microscopy, super-resolution microscopy, quantitative image analysis, biochemical isolation of centrosomes, and 3?UTR swapping experiments in well-characterized Drosophila centrosome mutants and controls.
In Specific Aim 2, I will identify mechanisms of centrosome mRNA localization by testing the hypothesis that Orb, a Drosophila cytoplasmic element binding protein ortholog and RNA binding protein, targets mRNAs to centrosomes. I will test this hypothesis using single molecule FISH, RNA immunoprecipitation, and chimeric reporters with mutated Orb consensus binding sequences.
In Aim 2, I will also take an unbiased approach to identify other RNA binding proteins that contribute to centrosome mRNA localization via an RNA interference screen, which will form the basis for my research program as an independent investigator. This proposal exploits the Drosophila early embryo system to provide insight into the rapid changes in centrosome composition during the cell cycle, a biological process with implications for cancer pathogenesis and growth deficiency syndromes.
Centrosomes are small organelles that build the mitotic spindle in dividing cells and undergo dramatic changes to their shape and composition when they alter their activity, such as during cell division. This project studies the localization of RNA to centrosomes, which is an unexplored mechanism to regulate centrosome function. The results of this proposal will have implications for centrosome-associated diseases, such as microcephaly and cancer.
