| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2016: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2015: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2014: ¥700,000 (Direct Cost: ¥700,000)
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| Outline of Annual Research Achievements |
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.
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