転写共役修復開始反応の分子機能解析（遺伝性光線過敏症の分子基盤）

我们首先建立了一个细胞系文库，其中每个TC-NER因子使用CRISPR/Cas9技术敲除USP 7或UVSSA（例如UVSSA或Usp 7）;为了深入了解UVSSA依赖的RNA聚合物泛素化，我们建立了一种基于SILAC的定量蛋白质组学方法，用于有效分离和富集泛素化肽，结合高分辨率质谱，我们成功地鉴定了停滞RNA聚合物上的UV损伤特异性和UVSSA依赖性泛素化位点的数量;为了随后测试polIlo泛素化与TC-NER的生物学相关性，产生了“非泛素化”突变细胞系，并且几种产生的突变细胞系在UV照射时显示降低的TC-NER活性和RNA polIlo泛素化缺陷。为了详细了解各种TC-NER蛋白在RNA polIII泛素化中的作用，我们纯化了大多数TC-NER因子，包括RNA polII、UVSSA、CSA/B、Cul 4CSA E3连接酶和Usp 7。通过使用这些纯化的蛋白质，我们开始再现RNA polIio泛素化，通过在反应中加入RNA polII和Cul 4CSA E3连接酶观察到RNA polIio修饰的污点，这是先前报道的。但经过多次尝试，到目前为止，我们无法在试管中重现UVSSA特异性RNA polII泛素化，这意味着该反应需要更多的因子才能完成。我们正在建立一种蛋白质组学方法来鉴定可能参与这一过程的潜在因素，我们相信以下工作将为RNA聚合泛素化的分子基础提供进一步的信息。

We firstly generated a cell line library, in which each TC-NER factors (e.g. UVSSA or Usp7) is knocked out using CRISPR/Cas9 technique; To get insights into UVSSA-dependent RNA polIIo ubiquitination, we setup a SILAC-based quantitative proteomics method for efficiently isolation and enrichment of ubiquitinated peptides, in combination with high-resolution mass-spectrum and we successfully identified numbers of UV-damage specific and UVSSA-dependent ubiquitination sites on stalled RNA polIIo; To subsequently test the biological relevance of polIIo ubiquitination to TC-NER, the “non-ubiquitinable” mutant cell lines were generated and several generated mutant cell lines displayed reduced TC-NER activity and defects in RNA polIIo ubiquitination upon UV irradiation. To give a detail view of various TC-NER proteins in RNA polIIo ubiquitination, we have purified most of TC-NER factors including RNA polII, UVSSA, CSA/B, Cul4CSA E3 ligase and Usp7. By using these purified proteins, we set out to reproduce the RNA polIIo ubiquitination, a smear of RNA polIIo modification were observed by addition of RNA polII and Cul4CSA E3 ligase in reaction, which was previously reported. But after several attempts, so far we could not reproduce the UVSSA-specific RNA polII ubiquitination in test tube, implying that this reaction requires further factor (s) to complete. We are now setting up an proteomic method to identify potential factors that may involved in this process, and we believe the following work will provide further information for molecular basis of RNA polIIo ubiquitination.