Mining Localized mRNAs in Late-Stage Drosophila Oocytes

Huadi Zhang, Vassar College ’11 and Profs. Kathleen Susman and Nancy Pokrywka

Localization of mRNAs to discrete regions of oocyte during oogenesis of the fruit fly Drosophila melanogaster is an important manifestation of cell polarity. It results in a mature egg with a distinct anterior-posterior axis and thus enables the embryo to follow well-defined developmental programs. The cytoskeleton, which establishes the intracellular scaffold, is known to play important roles in mRNA localization. In this study, genome-wide behavior of the Drosophila mRNA population in late-stage oocytes was assayed. A biochemical fractionation approach was employed to isolate cytoskeletal elements in the oocyte. Major components of the cell were disrupted by a non-ionic detergent buffer while the cytoskeleton was expected to remain insoluble. Thus, by low-speed centrifugation, we were able to precipitate a fraction of the cell lysate that is expected to be highly enriched for the cytoskeleton and associated elements. Real-time PCR (qPCR) of transcripts known to localize via the cytoskeletal machinery or remain unlocalized in the cytoplasm were used to validate the fractionation. Relative abundance of mRNAs in the soluble and insoluble fractions of the lysate was then determined by microarray analyses, allowing us to identify mRNAs that are enriched in the detergent-insoluble fraction of the cell. By further cross-referencing various databases and running cluster analyses, we hope to shed light on gene expression during late-stage oogenesis of Drosophila, explore the functional relation and biological significance among mRNAs that have similar associations with the cytoskeleton, and uncover novel mRNAs that are localized via the cytoskeletal machinery.