My laboratory is interested in pursuing questions regarding the structure and function of the nucleolus, the eukaryotic organelle that assembles ribosomal subunits and performs several recently discovered functions in small nuclear RNA processing. Our current efforts focus on nucleoli in Drosophila which affords a vast array of molecular and genetic tools to dissect nucleolar function. We are particularly interested in nucleolar-specific proteins, mostly Nopp140, but also fibrillarin, nucleostemin, and Nop56.
The Nopp140 gene in Drosophila (see http://flybase.bio.indiana.edu/ for a complete description) encodes two potential isoforms by alternative splicing. The first isoform, Nopp140-True, is a close sequence homologue to mammalian Nopp140. The second, Nopp140-RGG, is identical to the first throughout most of its length, except that the highly conserved carboxy terminus normally found in Nopp140 proteins is replaced by a RGG-rich domain much like that found in vertebrate nucleolin and fibrillarin proteins. We have recently transformed D. melanogaster to express GFP-tagged versions of the two isoforms and to express RNAi in a GAL4-mediated tissue-specific manner to knock down endogenous Nopp140 expression. Current and future research efforts include:
• determining the cell type expression patterns for the two Nopp140 isoforms during embryonic and larval development, and then later in adults;
• using GFP-tagged Nopp140 isoforms as cytological markers for the study of nucleologenesis in early Drosophila embryos;
• using GFP-tagged Nopp140 isoforms as cytological markers in genetic backgrounds that disrupt normal nucleolar structure and function (e.g. the many bobbed and Minute mutations),
• using RNAi to knock down the respective transcripts encoding Nopp140 and other nucleolar proteins;
• establishing the relationship between Drosophila’s Minute syndrome observed by RNAi knock down of Nopp140 and the human Treacher Collins syndrome (craniofacial birth defects) caused by mutations in treacle, a nucleolar protein very similar to Nopp140.
McCain, J., Danzy, L., Hamdi, A., Dellafosse, O., and DiMario, P. (In press). Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis. Cell. Tiss. Res.
DiMario, P.J. (2004). Cell and molecular biology of nucleolar assembly and disassembly. Int. Rev. Cytol. 239:99-178.
Pellar, G.J. and DiMario, P.J. (2003). Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain. Chromosoma 111:461-469.
Gilchrist, J.S.C., Abrenica, B., DiMario, P.J., Czubryt, M.P., and Pierce, G.N. (2002). Nucleolin is a calcium-binding protein. J. Cell. Biochem. 85:268-278.
Waggener, J.M. and DiMario, P.J. (2002). Two splice variants of Nopp140 in Drosophila melanogaster. Mol. Biol. Cell 13:362-381.
Zhu, Y., Lu, D., and DiMario, P. (1999). Nucleolin, defective for MPF phosphorylation, localizes normally during mitosis and nucleologenesis. Histochem. Cell Biol. 111:477-487.
Shah, S.B., Terry, C.D., Wells, D.A., and DiMario, P.J. (1996). Structural changes in oocyte nucleoli of Xenopus laevis during oogenesis and meiotic maturation. Chromosoma 105:111-121.
Heine, M.A., Rankin, M.L., and DiMario, P.J. (1993). The Gly/Arg rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization. Mol. Biol. Cell. 4:1189-1204.
DiMario, P.J. and Gall, J.G. (1990). Nucleolin from the multiple nucleoli of amphibian oocyte nuclei. Chromosoma 99:87-94.
DiMario, P.J. and Mahowald, A.P. (1987). Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within developing oocytes. J. Cell Biol. 105:199-206.