Title page for ETD etd-04122011-111053


Type of Document Dissertation
Author He, Fang
Author's Email Address hefang1234@hotmail.com
URN etd-04122011-111053
Title Functional Analysis of Nopp140 and P5CDH1 in Drosophila Melanogaster
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
DiMario, Patrick J. Committee Chair
Bartlett, Sue G. Committee Member
Donze, David Committee Member
Hart, Craig M. Committee Member
Roy, Alma F. Dean's Representative
Keywords
  • proline
  • mitochondria
  • Nopp140
  • cell stress
  • P5CDH1
  • nucleolus
  • ribosome biosynthesis
  • hyperprolinemia
  • snoRNP
Date of Defense 2011-03-30
Availability restricted
Abstract
Nopp140 is a nucleolar protein with several purported roles in ribosome biogenesis. To further characterize Nopp140 in Drosophila, we used FLP-FRT recombination to delete the Nopp140 gene. Genomic PCR, RT-PCR, and immuno-fluorescence microscopy confirmed the loss of Nopp140 and its products. Compared to embryos bearing the transposons used to generate the deletion, Nopp140-/- embryos displayed similar hatching rates, but resulting larvae died after 8 days, still in the second instar stage. Nucleoli were apparent in Nopp140-/- cells with no observable morphological defects, but the rRNA methyl-transferase, fibrillarin, redistributed partially to the nucleoplasm. BrU-labeling indicated that rDNA transcription is reduced overall in Nopp140-/- larvae, while Northern analysis showed that pre-RNA cleavage was un-affected. Surprisingly, Northern analyses of Nopp140-/- larvae showed an unusually large pre-rRNA bearing the R2 retro-transposon sequence and the processed R2 transcript. The observation suggests a fundamental shift in rDNA chromatin structure and expression upon loss of Nopp140. Transmission electron microscopy of Nopp140-/- cells showed excess nuclear virus-like copia particles, reduced cytoplasmic ribosomes, autophagosome-like structures, and many electron dense cytoplasmic granules that are likely to be stress bodies and proccessing (P) bodies.

Because the downstream piggyBac element used to delete Nopp140 resides in CG7145, phenotypes caused by this insertion were compared to those caused by Nopp140 deletion. Delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) is a nuclear-encoded mitochondrial enzyme that catalyzes the second step in proline degradation. Mutations in human P5CDh cause type II hyperprolinemia, a complex syndrome displaying increased serum proline and mental disabilities. Conceptual gene CG7145 in Drosophila melanogaster encodes the orthologous DmP5CDh1. The mutant allele, CG7145f04633, contains a piggyBac transposon that truncates the enzyme by 83 residues. Heterozygous (CG7145f04633/TM3) individuals developed normally, while homozygous (CG7145f04633/CG7145f04633) individuals displayed proline levels twice that of normal, swollen mitochondria, and ultimately larval and pupal lethality. These mutants showed normal ribosomal enrichment in cytoplasm, and normal rRNA gene transcription, processing, and pre-rRNA modifications. We conclude that the Nopp140 plays a critical role in ribosomal biosynthesis, and that the phenotypes in Nopp140-/- can be distinguished from the phenotypes caused solely by the CG7145 mutation.

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