Mechanisms of Response to DNA Damage Nuclear Factors

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DESCRIPTION (provided by applicant): Following UV irradiation the cellular mRNA levels decrease, reflecting a coordinated interaction of DNA repair, transcription, and RNA processing factors. The poly(A) tail is important in the regulation of mRNA turnover and it is fundamental for the control of gene expression. The long-range goal of this research project is to better understand the basic mechanisms of RNA processing regulation upon DNA damage conditions and its physiological significance. The specific hypothesis is that the polyadenylation factor CstF-50 plays an important role in coordinating this nuclear response. We base that hypothesis on these observations: (1) Polyadenylation is inhibited after DNA damage as a result of the formation of the BRCA1/ BARD1/ CstF inhibitory complex and of the proteasome-mediated degradation of the polyadenylation activator RNA polymerase II (RNAP II); (2) DNA damage-induced BARD1 phosphorylation is critical for the UV-induced inhibition of polyadenylation and of RNAP II degradation; (3) CstF functions in the transcription-coupled repair response; (4) CstF-50 can interact with poly(A)-specific ribonuclease (PARN) and regulate its deadenylation activity. Based on these observations and preliminary data, the specific aims are to: (1) Characterize the function of the RNA processing factor CstF in TCR. The role of CstF in different events of the TCR response will be analyzed. Cockayne's syndrome (CS) cells, which are deficient in TCR, will be used. The direct role of CstF and other factors in UV-induced ubiquitination of RNAP II by BRCA1/ BARD1 and in the repair process will also be determined using already proven biochemical assays. (2) Determine the biological significance of the interaction of CstF-50 with PARN. We will determine the effect of these interacting factors on polyadenylation complex formation on the polyadenylation reaction and on the recovery of cellular mRNA levels after DNA damage. Proven biochemical assays and cells deficient in these proteins will be used. (3) Developmental goals: increase the productivity of the research work, which will allow the submission of new papers for publication and the follow-up for an R01 grant application to NIH. The proposed work is innovative because it reflects a functional-mechanistic overlapping of tumor suppressors, like BRCA1/BARD1, and the ubiquitous gene expression machinery, and it suggests a central role for an RNA processing factor in the intricate DNA damage response.