Area of Interest
My laboratory studies the molecular and cellular aspects of eukaryotic cell nuclei. Specifically, we are studying the nucleolus, the nuclear compartment responsible for the assembly of ribosomal subunits. We use the fruit fly, Drosophila melanogaster, to model nucleolar stress (disruption of ribosome biogenesis leading to apoptosis or autophagy depending upon cell type) and the ribosomopathies (human diseases caused by disruption in ribosome biogenesis or function). Our experimental approach uses transgenic flies, various forms of microscopy (phase contrast, confocal, transmission electron microscopy), and molecular techniques such as RNA interference and CRISPR to deplete nucleolar ribosome assembly factors, with qRT-PCR and proteomics to document depletion of these gene products. We are particularly interested in the Nucleolar and Cajal body Phosphoprotein of 140 kDa (Nopp140), Nucleostemin 1 and 2 (NS1 and NS2), Fibrillarin, and several other nucleolar proteins.
Loss of nucleolar ribosome assembly factors leads to nucleolar stress and phenotypes in Drosophila that are reminiscent of the human ribosomopathies. We have shown that the Drosophila Nopp140 gene encodes two protein isoforms. Partial loss of both isoforms in larval imaginal wing discs by RNAi expression leads to apoptosis and thus deformed wings in the adult. This phenotype is reminiscent of the loss of the related protein, Treacle, in human embryonic neural crest cells which leads to craniofacial deformities of the Treacher Collins syndrome, a ribosomopathy. However, we showed this apoptosis caused by nucleolar stress in Drosophila was p53-independent, but JNK-dependent. At the sub-cellular level, we showed that complete loss of the Nopp140 isoforms by gene deletion or CRISPR disruption redistributes Fibrillarin to the nucleoplasm, and this likely explains the observed reduction in 2’-O-methylation on the rRNA. With the complete loss of Nopp140, we also showed a substantial loss of cytoplasmic ribosomes, but the simultaneous accumulation of unusual electron dense granules that we suspect are related to Processing (P) bodies. Finally, we have shown that loss of NS1 or 2 blocks release of the large ribosomal subunit from nucleoli; with NS2 depletion, we showed enlarged granular regions within nucleoli.
We are currently investigating the effects of nucleolar stress on the developing nervous system in Drosophila embryos and larvae, and we are beginning a new effort to assess nucleolar activity as a function of age in Drosophila adults. In a third effort we are employing rDNA promoter fusions ectopically inserted at defined loci on the polytene chromosomes to study how RNA Pol I transcription machinery interacts with pre-ribosomal RNA processing components. Finally, we are also interested in the reactivation of normally silent rDNA genes that harbor R1 and R2 retro-transposons.
Selected publications include (* denotes undergraduates):
Wang, Y. and DiMario, P.J. (2017). Loss of Drosophila nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress. Chromosoma 126:375-388. Link
He, F., James, A., Raje, H., *Ghaffari, H., and DiMario, P.J. (2015). Deletion of Drosophila Nopp140 induces subcellular ribosomopathies. Chromosoma 124:191-208. Link
James, A., Raje, H., Wang, Y., and DiMario, P. (2014). Nucleolar stress with and without p53. Nucleus 5(2):402-426. Link
DiMario, P.J., James, A., and Raje, H., (2013). rDNA and Nucleologenesis in Drosophila. In Proteins of the Nucleolus, eds: O’Day and A. Catalano. Publisher: Springer. pp. 39-78. Link
James, A. *Cindass, R., *Mayer, D., *Terhoeve, S., *Mumphrey, C., and DiMario, P.J. (2013). Nucleolar stress in Drosophila melanogaster. Nucleus 4(2):1-11. Link
He, F. and DiMario, P.J. (2011). Structure and Function of Nopp140 and Treacle. In The Nucleolus, ed: M.O.J. Olson. Publisher: Springer. pp. 253-278. Link
He, F. and DiMario, P.J. (2011). Drosophila mitochondrial delta-1-pyrroline-5-carboxylate dehydrogenase is required for normal proline breakdown and mitochondrial integrity. Mitochondrion 11:397-404. Link
Rosby, R., Cui, Z., *Rogers, E., deLivron, M.A., Robinson, V.L., and DiMario, P.J. (2009). Knockdown of the Drosophila GTPase Nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance. Mol. Biol. Cell 20:4424-4434. Link
Cui, Z. and DiMario, P.J. (2007). RNAi knockdown of Nopp140 in Drosophila induces Minute-like phenotypes in Drosophila. Mol. Biol. Cell 18:2179-2191. Link
*McCain, J.E., *Danzy, L., *Hamdi, A.A., and *Dellefosse, O., DiMario, P.J. (2006). Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis. Cell Tissue Res. 323:105-115. Link