Abstract
本論文的目的是研究酵母菌傳訊RNA剪接因子(Splicing factor)PRP19之生化性質。PRP19蛋白質在大腸桿菌中被大量製造後作為抗原，用以在兔子體內產生抗體，我們利用親和性管柱層析純化出抗－PRP19蛋白質抗體，由免疫沉澱的結果顯示PRP19在剪接反應(Splicing reaction)中會結合到剪接體(Spliceosome)上，但它並不屬於小核糖核蛋白體(snRNPs)上的蛋白質。我們將抗－PRP19蛋白質抗體加入細胞抽取液中會抑制其剪接反應的功能，因此証明PRP19蛋白質對剪接反應非常重要。藉由除去某些剪接因子或降低剪接反應中ATP的濃度，我們可以阻斷剪接體組合過程中的任一步驟，再由免疫沉澱的實驗我們發現在剪接體組合的過程中，PRP19參與在複合體A2-1至A1形成的步驟中。因為在此步驟中U4 snRNP 會離開剪接體，因此我們認為PRP19在剪接反應過程中可能扮演一個重要角色。prp19 突變種細胞抽取液經過熱處理後會失去剪接功能，在大腸桿菌中製造的PRP19蛋白質無法互補(complement)此細胞抽取液所失去的剪接功能，所以我們利用酵母菌來製造PRP19 蛋白質，結果顯示增加PRP19蛋白質的量，並不能同時增加其互補活性。被大量製造出來的PRP19 蛋白質也未具有互補活性。因此我們推測PRP19 與至少一個剪接分子形成一個蛋白複合體(protein complex)，我們已在一酵母菌株中將其PRP19基因的3'端接上一段序列，使其產生的PRP19 蛋白質之Ｃ端帶有一個會被單株抗體12CA5認識的抗原決定基。我們另將此單株抗體接到管柱內用以分離PRP19蛋白複合體。我們發現在此複合體內大約包含7個蛋白質 (polypeptides)，經由Far-Western 方法我們找到在此蛋白複合體中會與PRP19直接接觸的6個蛋白質。The yeast PRP19 protein was expressed in E.coli and used forraising antibodies. By immunoprecipitation studies, PRP19 isshown to be associated with the spliceosome during thesplicing reaction, but is not tightly associated with any ofthe snRNAs essential for the splicing reaction. PRP19 isdemonstrated to be essential for the in vitro splicingreaction by immunodepletion and immunoinhibition studies.Formation of splicing complexes was blocked at different stepsby depletion or inactivation of a specific splicing factor,or by limiting the concentration of ATP.Immunoprecipitation of specific splicing complexes revealedthat PRP19 is associated with the spliceosome duringtransition from complex A2-1 to A1, in which U4 becomesloosely associated with the spliceosome. We have found thatPRP19 does not function by itself, and the stoichiometricalassociation of the PRP19 protein with other components may berequired for its function. To study the componentsassociated with RPR19, we constructed a strain in which apeptide recognized by the monoclonal antibody 12CA5 islinked to the carboxyl terminus of PRP19. By use of theimmunoaffinity chromatographic procedure, we have isolated thePRP19 associated complex from the epitope-tagged strain.Approximately 7 polypeptides were identified to be in thePRP19 associated complex. Far Western analysis reveals 6polypeptides including PRP19 interact with PRP19 directly.