posted on 2020-02-03, 10:48authored byRyan D. Mordue
Cytoplasmic control of gene expression allows for a rapid reprogramming of specific cellular processes in response to stresses, such as exposure to agents that damage nucleic acids. These responses dictate cell fate in the absence of transcriptional regulation, by regulating the expression of proteins that permit a cell to promote cell survival pathways, or alternatively trigger cell death in situations where homeostasis cannot be restored. In general, cytoplasmic control of gene expression is achieved by the action of RNA-binding proteins, therefore, techniques that allow the analysis of the RNA-bound proteome, such as RNA-interactome capture (RIC), permit the identification of novel RNA-binding proteins (RBPs) that have a key regulatory role in these responses. Work within the Willis laboratory has previously identified Activating signal cointegrator 1 (ASC-1) complex subunit 3 (ASCC3) as a cytoplasmic protein that has increased affinity for RNA in response to UVB irradiation. ASCC3 is a member of the ASC-1 complex, which also includes ASCC2, TRIP4, and ASCC1.
In this thesis, the ASC-1 complex members were investigated using immunoaffinity purification and size exclusion chromatography-based approaches. The data show that ASCC3 has increased binding to RNA in a range of cell lines following exposure to UVB and 4NQO, and that the response of ASCC3 and the ASC-1 complex to nucleic acid damage is conserved. FLAG-tagged versions of ASCC3 were generated, and when used as a bait in immunoaffinity purification reactions, the deubiquitinase OTUD4 was identified as a novel interactor of ASCC3. Further studies showed that OTUD4 was part of the cytoplasmic ASC-1 complex, although this interaction may occur with only a small proportion of total cytoplasmic OTUD4. While OTUD4 did not influence control of global protein synthesis rates or cell proliferation, it demonstrated increased binding to polyribosomes following treatment of cells with 4NQO, suggesting that it may function in the ribosome quality control pathway.