探討酵母菌核醣核酸蛋白Rbp1p對絲狀生長中絮凝因子FLO11表現之調控

Abstract

酵母菌核醣核酸結合蛋白(Rbp1p)最初被定義為一負向調控的生長因子。在其結構上包含三個核醣核酸識別基序(RRM)和兩個富含麩氨酸區域，並且在碳端具有一個富含天門冬氨酸、甲硫胺酸和脯胺酸的區域。我們實驗室已發現當過度表現RRM突變株時，會誘導酵母菌產生過度附著(hyper-adhesion)的生長現象，此為一種絲狀生長(filamentous growth)現象。而絮凝因子(Flo11p)已被知道是調控此過度附著現象的關鍵因子。最近我們發現當S1278b基因背景下的酵母菌之RBP1基因被剃除會促使FLO11信使RNA(mRNA)增加，導致洋菜膠附著(agar-adhesion)、入侵(invasion)及細胞伸長(cell elongation)的現象產生。在本篇研究中，我們想要探討Rbp1p是如何去調控FLO11的表現。首先，我們先驗證在rbp1突變株中FLO11的信使RNA及蛋白質確實都有增加。然後，利用北方點墨法證實RBP1基因剔除並不會顯著延長FLO11信使RNA的半衰期，反而會促進FLO11基因的轉錄。我們藉由剃除一些涉及洋菜膠附著表現的候選基因之方式去尋找哪些基因可能參與Rbp1p介導的絲狀生長表現。除了Flo8p及Ste12p之外，我們發現rbp1 mss11突變株會失去rbp1誘導的過度附著生長及FLO11高表現的現象。此外，我們也證實剃除RBP1可以抑制ICR1的表現，其為一種非編碼RNA(ncRNA)並已知其表現會抑制FLO11的轉錄。在本篇研究中，我們也想探討過度表達Rbp1p-rrm3的突變株如何誘發FLO11的表現。西方點墨法及北方點墨法的分析都顯示過度表達Rbp1p-rrm3會促使FLO11的轉錄。此外，在BY4741基因背景下分別剔除SNF1及BCY1基因都會減弱Rbp1p-rrm3所引發的過度附著生長及FLO11高表現的現象。然而，還有許多Rbp1p及Rbp1p-rrm3調控FLO11轉錄的詳細分子機制仍需要再進一步研究。

The RNA-binding protein, Rbp1p was first identified as a negative growth regulator in Saccharomyces cerevisiae. Structure of Rbp1p contains three RNA-recognition motifs (RRMs), two glutamine-rich regions, and asparagine-methionine-proline-rich (NMP-rich) region in the C-terminus. In our lab, we had found that over-expressing Rbp1p-RRM mutants into yeast induced a hyper-adhesion growth phenotype, a filamentous growth phenotype. Flo11p has been known as the key factor in hyper-adhesion phenotype. Recently, we found that deletion of RBP1 enhanced FLO11 mRNA level resulting in agar-adhesion, invasion, and cell elongation in S1278b strain. In this study, we went to investigate how Rbp1p regulates the FLO11 expression. Firstly, we showed that FLO11 mRNA and Flo11p exactly increased in RBP1-deleted strain. Then, Northern blot analyses indicated that deletion of RBP1 did not significantly prolong the half-life of FLO11 mRNA, but enhanced the FLO11 transcription. To study which genes would involve in Rbp1p-mediated filamentous growth, we disrupted several candidate genes involving in adhesion phenotype. In addition to Flo8p and Ste12p, we further found that rbp1 mss11 double mutant lost the rbp1-induced hyper-adhesion growth phenotype and high FLO11 expression. Moreover, we also found that the deletion of RBP1 could suppress the level of ICR1, which is the ncRNA that represses FLO11 transcription. Here, we also asked how over-expression of Rbp1p-rrm3 mutant induces FLO11 expression. Western blot and Northern blot analysis showed that over-expressing the Rbp1p-rrm3 mutant activated the FLO11 transcription. Furthermore, the deletion of SNF1 or BCY1 decreased the Rbp1p-rrm3-induced hyper-adhesion and high FLO11 expression in BY4741. Thus, the more detail molecular mechanism of FLO11 transcription regulated by Rbp1p or Rbp1p-rrm3 is still need to be further investigated.