The RNA-associated splicing-factor p54nrb regulates survival of human cells following UV irradiation
Corduff, Aoife
Corduff, Aoife
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2013-04
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Thesis
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Abstract
UV radiation causes several forms of DNA damage; the most prominent of these are cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-photoproducts (6-4 PPs). Nucleotide excision repair (NER) is a repair process that repairs these DNA lesions. Defects in the NER pathway lead to cancer-related diseases such as xeroderma pigmentosum (XP) that is caused by a defect in the XPA to XPG genes that are involved in the NER pathway. To investigate protein recruitment to chromatin after DNA damage, SV40 minichromosomes containing pyrimidine-rich DNA were established to create a tool with high sensitivity to UV for in vivo studies. Using this tool these findings suggest that p54nrb, which exhibits multi-functional characteristics in a variety of cellular processes, including DNA and RNA binding, nuclear RNA processing and regulation of transcription, is a novel protein putatively involved in UV DNA damage response. The objective of this study was to gain a better insight into the mechanism(s) by which p54nrb is involved in the DNA damage response following UV irradiation, primarily using human cells as a model system. Immunofluorescence microscopy using human cells showed that p54nrb, a 54-kDa nuclear RNA-binding protein, is recruited to nucleoli after UV irradiation. This recruitment is evident in a number of cell lines and depends on transcription since inhibition of transcription abolishes the DNA damage-dependent recruitment of p54nrb. Fusion constructs of green fluorescent protein (GFP) with N- and C-terminal domains of p54nrb deleted have been constructed to determine that the coiled-coil domain (C-terminal) of p54nrb is required for its localisation to nucleoli. By performing local UV irradiation ¿micropore¿ experiments, whereby specific areas of the cell are irradiated with UV, it was determined that p54nrb does not localise to sites of DNA damage and nucleoli specifically do not need to be irradiated for the nucleolar localisation of p54nrb. DNA damage signalling (PIKkinases) drug inhibitors reduce p54nrb nucleolar localisation following UV irradiation that suggests that p54nrb could be a downstream target of these factors. Notably, the use of ATM- and ATR-specific inhibitors revealed that both DNA damage response kinases cooperate in the regulation of p54nrb recruitment to the nucleolus after UV treatment. Depletion of p54nrb demonstrates higher cell viability following UV irradiation. Model consistent with data is that sequestering of p54nrb to the nucleolus could be a cellular mechanism to allow an optimal response of cells to repair DNA damage.
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Attribution-NonCommercial-NoDerivs 3.0 Ireland