Browsing by Author "Catanese, Daniel James, Jr"

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    Biophysical and biochemical characterization of the disconnected interacting protein 1, DIP1, from Drosophila melanogaster
    (2005) Catanese, Daniel James, Jr; Matthews, Kathleen S.
    Transcription regulation and RNA metabolism, two essential cellular processes, are linked by Disconnected Interacting Protein 1 (DIP1) in Drosophila melanogaster. This linkage occurs through DIP1 interactions with double-stranded RNA (dsRNA) and its association with other proteins, such as Disconnected (zinc-finger transcription factor), Ultrabithorax (Ubx) (Hox transcription factor), Suppressor of variegation 3--9 (chromatin methyltransferase), and Adat (tRNA deaminase). This thesis explores the molecular mechanisms of DIP1 function by elucidating its interactions with dsRNA and two protein partners, Adat and Ubx. Proteins with dsRNA-binding domains bind dsRNA with high affinity and no sequence specificity in vitro. Nonetheless, these proteins are involved in many important facets of cellular metabolism in vivo that encompass splicing, deamination, and RNA interference. Analytical ultracentrifugation suggests that DIP1 is a homodimer in solution, consistent with data for other proteins with dsRNA-binding domains. Gel retardation demonstrates that DIP1 prefers dsRNA over dsRNA. Additionally, DIP1 binds the Adenovirus VA1 RNA with the highest affinity we have been able to identify for this class of proteins. DIP1 also binds to a stem-loop structure similar to those involved in long-range enhancer effects of the Bithorax complex as well as in the generation of two microRNAs that potentially regulate Ubx. Adat·DIP1 interaction was established in yeast two-hybrid assays, and the Adat interaction with DIP1 was confirmed with GST pulldown experiments. DIP1 binds with high affinity to the pre-tRNAala, a potential in vivo target of Adat. We hypothesize that Adat is recruited by the DIP1·RNA complex in order to properly deaminate the pre-tRNA ala. DIP1 also binds Ubx, a Hox transcription factor necessary for haltere formation during embryogenesis. All Hox proteins rely on protein-protein interactions to define their cellular function, although few such interactions have been identified. The Ubx·DIP1 interaction was demonstrated using phage display, immunoprecipitation, GST pulldown assays, and gel supershifts in complex with DNA. Yeast one-hybrid assays establish DIP1 can block Ubx activation, and a functional consequence of this interaction may be to differentiate Hox function in vivo. Elucidating these molecular mechanisms via DIP1 interactions potentially provide insight into cancer regulation and a physical bridge that links transcription and RNA metabolism.