Identifier

etd-10082015-214831

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Nucleolar stress results when ribosome biogenesis is disrupted. An excellent example is the human Treacher Collins syndrome in which the loss of the nucleolar chaperone, Treacle, leads to p53-dependent apoptosis in embryonic neural crest cells, and ultimately to craniofacial birth defects. We show that depletion of the related nucleolar and Cajal body phospho-protein, Nopp140, in Drosophila melanogaster led to nucleolar stress and eventual lethality when multiple tissues were depleted of Nopp140 by RNAi. We used TEM, immuno-blot analysis, and metabolic protein labeling to show the loss of ribosomes. Targeted loss of Nopp140 only in larval wing discs caused apoptosis, which eventually led to defects in the adult wings. These defects were not rescued by a p53 gene deletion, as the craniofacial defects were in the murine model of TCS, thus suggesting that apoptosis caused by nucleolar stress in Drosophila is induced by a p53-independent mechanism. Loss of Nopp140 in larval polyploid midgut cells induced premature autophagy as marked by the accumulation of mCherry-ATG8a into autophagic vesicles. We also found elevated phenoloxidase A3 levels in whole larval lysates and within the hemolymph of Nopp140-depleted larvae, coincident with the appearance of melanotic tumors. The occurrence of apoptosis, autophagy, and phenoloxidase A3 release to the hemolymph upon nucleolar stress correlated well with the activation of JNK in Nopp140-depleted larvae. Nopp140 is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, RT-PCR, and immunofluorescence microscopy confirmed the loss of Nopp140, its mRNA, and protein products. Nopp140-/- larvae arrested in the second instar stage and died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D snoRNP methyl-transferase, fibrillarin, redistributed to the nucleoplasm in variable amounts; RT-PCRs showed that 2’-O-methylation of rRNA in Nopp140-/- cells was reduced. Ultra structural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron dense cytoplasmic granules. Furthermore, metabolic labeling showed a significant drop in protein translation. We believe the phenotypes described here define novel intracellular ribosomopathies.

Date

2015

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

DiMario, Patrick

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