2010-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686035摘要：人類 hPuf-A/KIAA0020蛋白在2001年被發現是一個新的次要組織相容性抗原(minor histocompatibility antigen)，但它的生理功能至今仍是不清楚。hPuf-A/KIAA0020在結構上具有6個類似Pumilio-RNA結合區域，胺基酸序列比對發現hPuf-A應該是斑馬魚puf-A同源基因,相似度可達63%。斑馬魚puf-A基因在胚胎時期與眼睛和原始生殖細胞的發育有關；hPuf-A在酵母菌的同源基因Puf6p則會抑制菌株接合型轉換相關基因ASH1的轉譯，Puf6p與ASH1的結合能力會受到CK2磷酸化的調控，實驗也證明酵母菌Puf6p位在細胞核質內。 為了探討人類hPuf-A的生理功能，我們先製備抗hPuf-A的單株抗體，免疫螢光染色發現 hPuf-A主要位於細胞核仁內，只有極少量存在細胞核質；由於hPuf-A是一個可與RNA結合的蛋白質，因此利用 Actinomycin D 和 DRB (分別是RNA pol I和 pol II的抑制劑)處理細胞後，hPuf-A在核仁的分佈位置會轉移至核質，顯示hPuf-A的位置會受到RNA的存在與否而呈現動態的轉變；有趣的是當我們利用siRNA降解hPuf-A蛋白的表現，細胞週期雖無明顯變化，但缺少hPuf-A蛋白表現的細胞在處理camptothecin 和UV 後卻有大量死亡的情形；利用親和性層析與質譜分析發現HA-hPuf-A與DNA傷害相關蛋白PARP-1有相互作用情形，免疫沉澱分析也發現HA-hPuf-A與PARP-1在同一個複合物中。 根據上述尚未發表的實驗結果判斷hPuf-A可能在RNA轉譯或DNA傷害反應扮演了直接或間接的角色，因此我們提出三年計畫，希望能進一步詮釋 hPuf-A的生理功能： 第一、 hPuf-A可以和哪一種RNA結合 第二、 為何hPuf-A siRNA轉殖後的細胞會對camptothecin和UV的處理變成非常敏感 第三、 hPuf-A與PARP-1在同一個免疫沉澱後的複合物有何生理上的意義 <br> Abstract: Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001, but its function remained largely unknown. Structurally, hPuf-A/KIAA0020 contains six Pumilio-homology RNA binding domains and is an ortholog of zebrafish puf-A gene with 63% of identity. Zebrafish puf-A has been shown to participate in eye and primordial germ-cell (PGC) development. Another potential homolog of hPuf-A is yeast Puf6p, which represses ASH1 translation for mating-type switching and is required for ASH1 localization. Yeast Puf6p could be phosphorylated by CK2 and mainly localized in the nucleoplasm. To study the function of human hPuf-A, a monoclonal anti-hPuf-A antibody was generated. hPuf-A predominantly localized in the nucleoli and exhibited a minor punctate pattern in the nucleoplam. The localization of hPuf-A was redistributed from the nucleolus to the nucleoplasm after treatments of Actinomycin D and DRB (inhibitors for RNA pol I and RNA pol II, respectively), indicating that the localization of hPuf-A was affected by RNA transcription. Intriguingly, knockdown of hPuf-A sensitized HeLa cells to camptothecin and UV treatment compared with control siRNA, suggesting that hPuf-A-depleted cells were vulnerable to genotoxic stress. Using affinity purification, we found that HA-hPuf-A could pull down PARP-1 by Mass Spec. Reciprocal immunoprecipitation showed that HA-hPuf-A and PARP-1 were in the same immunocomplex. According to the above preliminary results, hPuf-A may play, directly or indirectly, important roles in RNA transcription and DNA damage response. At the initial stage to study hPuf-A, we would like to launch a three-year project for delineating the function of hPuf-A. Firstly, to identify which RNA is the binding target of hPuf-A and how this RNA is regulated by hPuf-A. Secondly, to address why hPuf-silenced cells were sensitive to the treatments of camptothecin and UV light. Finally, to investigate what is the biological significance regarding the interaction of hPuf-A and PARP-1.hPuf-APUFnucleolusPARP-1hPuf-APUFnucleolusPARP-1