Year of Award

2021

Document Type

Thesis

Degree Type

Master of Science (MS)

Degree Name

Pharmaceutical Sciences

Department or School/College

Biomedical and Pharmaceutical Sciences

Committee Chair

Ekaterina Voronina

Commitee Members

Erica Woodahl, Kasper Hansen, J. Stephen Lodmell

Keywords

CSN-5, COP9 signalosome, FBF, PUM, stem cells, cancer

Subject Categories

Cancer Biology | Cell Biology | Developmental Biology

Abstract

RNA-binding proteins FBF-1 and FBF-2 (FBFs) are required for stem cell maintenance in C. elegans, although the mechanisms by which FBFs protein levels are regulated remain unknown. Using a yeast two-hybrid screen, we identified an interaction between both FBFs and CSN-5, a component of the COP9 (constitutive photomorphogenesis 9) signalosome. This highly conserved COP9 complex can affect protein stability through a range of mechanisms including deneddylation, deubiquitination, and phosphorylation (Wolf et al., 2003). Mapping protein-protein interactions between FBFs and CSN-5 suggested that the MPN (Mpr1/Pad1 N-terminal) metalloprotease domain of CSN-5 interacts with the RNA-binding domain of FBFs at physiologically relevant (micromolar) concentrations and this interaction is not RNA-dependent. Furthermore, these conserved domains of the human homologs PUM1 and CSN5 interact as well, thus identifying a protein complex conserved in evolution. We discovered that CSN-5 promotes the accumulation of FBF-1 and FBF-2 proteins in C. elegans stem and progenitor cells. Phenotypic analysis results were consistent with csn-5 contributing to FBF function since csn-5 germlines are masculinized (produce only sperm similar to fbf-1/2 loss of function) and show reduced numbers of stem cells. Similar phenotypes were observed in worms mutant for another COP9 holoenzyme component, csn-6. Curiously, phenotypes of csn-2 mutant were clearly distinct, where oocytes were still forming and stem cell numbers were not as affected. Additionally, FBFs protein levels were all reduced in csn(lf) mutants, but reduction of FBF-1 was only statistically significant in the csn(lf) mutant. This suggests that csn-5’s effect on FBFs might be independent of the COP9 holoenzyme. Interestingly, qPCR analysis of fbf-2 transcript revealed a significant reduction in csn(lf) mutants, indicating that we cannot exclude potential transcriptional effects on fbf and reduction of FBFs protein levels may partly be a result of a reduction in their transcript levels. Investigating CSN-5 contribution to FBF protein activity and stem cell maintenance will have implications for human stem cell biology and improve our understanding of diseases such as cancer.

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