Mechanisms of Response to DNA Damage Nuclear Factors

描述(申请人提供)：紫外线照射后，细胞的信使核糖核酸水平下降，反映了DNA修复、转录和RNA处理因子的协调作用。Poly(A)尾巴在调节信使核糖核酸的周转中起着重要作用，是基因表达调控的基础。本研究的长期目标是更好地了解RNA加工调控DNA损伤条件的基本机制及其生理意义。具体的假设是，多聚腺苷化因子CstF-50在协调这一核反应中发挥着重要作用。我们基于这些观察结果提出了这一假说：(1)DNA损伤后，由于BRCA1/BARD1/CstF抑制复合体的形成和蛋白酶体介导的多聚腺苷化激活物RNA聚合酶II(RNAP II)的降解，多聚腺苷化受到抑制；(2)DNA损伤诱导的BARD1磷酸化对于紫外线诱导的多聚腺苷化和RNAP II降解是至关重要的；(3)CstF在转录偶联修复反应中发挥作用；(4)CstF-50可以与聚(A)特异性核糖核酸酶(Parn)相互作用，调节其死基化活性。 基于这些观察和初步数据，具体目的是：(1)表征RNA加工因子CstF在TCR中的功能。将分析CstF在TCR反应的不同事件中的作用。将使用缺乏TCR的Cockayne综合征(CS)细胞。CstF和其他因素在紫外线诱导BRCA1/BARD1泛素化RNAP II和修复过程中的直接作用也将使用已经证明的生化分析来确定。(2)确定CstF-50与PARN相互作用的生物学意义。我们将确定这些相互作用的因素对多聚腺苷化复合体的形成、多腺化反应以及DNA损伤后细胞mRNA水平恢复的影响。将使用经过验证的生化分析和缺乏这些蛋白质的细胞。(3)发展目标：提高研究工作的生产力，这将使提交新的论文以供发表和后续向国家卫生研究院提出R01拨款申请成为可能。 这项拟议的工作具有创新性，因为它反映了肿瘤抑制因子，如BRCA1/BARD1，和无处不在的基因表达机制的功能-机制重叠，并表明RNA处理因子在复杂的DNA损伤反应中发挥核心作用。

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.