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）

我们首先生成了一个细胞系库，其中每个TC-NER因子（例如uvsa或Usp7）使用CRISPR/Cas9技术敲除；为了深入了解uvsa依赖的脊髓灰质炎RNA泛素化，我们建立了一种基于silac的定量蛋白质组学方法，用于有效地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RNA脊髓灰质炎上的紫外线损伤特异性和uvsa依赖的泛素化位点；随后，为了测试脊髓灰质炎泛素化与TC-NER的生物学相关性，生成了“非泛素化”突变细胞系，并且生成的一些突变细胞系在紫外线照射下显示TC-NER活性降低和RNA脊髓灰质炎泛素化缺陷。为了更详细地了解各种TC-NER蛋白在RNA脊髓灰质炎泛素化中的作用，我们纯化了大部分TC-NER因子，包括RNA polII、uvsa、CSA/B、Cul4CSA E3连接酶和Usp7。利用这些纯化的蛋白，我们开始复制RNA polII泛素化，通过加入RNA polII和Cul4CSA E3连接酶反应，观察到RNA polII修饰的涂片，这是之前报道的。但经过多次尝试，目前我们还无法在试管中重现uvsa特异性RNA polII泛素化，这表明该反应需要进一步的因子才能完成。我们现在正在建立一种蛋白质组学方法来确定可能参与这一过程的潜在因素，我们相信接下来的工作将为RNA脊髓灰质炎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.