酵母菌核醣核酸蛋白Rbp1p絲胺酸-脯胺酸及核醣核酸辨識區域突變株之功能探討

Abstract

真核細胞中，基因的調控需要核醣核酸結合蛋白與核醣核酸形成複合體，稱為核醣核蛋白。核醣核酸結合蛋白(Rbp1p)轉譯成一個六百七十二個胺基酸組成，約八十kD的蛋白質；包含三個核醣核酸識別基序 (RRM)及兩個富含麩氨酸區域。核醣核酸結合蛋白(Rbp1p)在發現時，被定義為一個負調控生長的因子。在我們實驗室中，已經發現在細胞內大量表現核醣核酸結合蛋白(Rbp1p) 能夠抑制酵母菌生長、促進粒腺體外模孔蛋白(porin)訊息核醣核酸降解、並抑制蛋白質的轉譯。核醣核酸結合蛋白(Rbp1p)在細胞中會座落到細胞質中特殊的聚集顆粒，稱為P-bodies。本實驗中，我們發現在第四百二十八個胺基酸由絲胺酸變成脯胺酸的突變株，會部份或完全喪失上述已知三項核醣核酸結合蛋白(Rbp1p)的功能。但這樣的核醣核酸結合蛋白(Rbp1p)突變株依然能夠座落到P-bodies。我們也發現：核醣核酸結合蛋白(Rbp1p)的絲胺酸-脯胺酸突變株及三個核醣核酸識別基序(RRM) 突變株均能夠引發絮凝現象(flocculation) 及洋菜入侵生長現象(invasive growth)這兩種附著現象。絮凝基因(Flo1p)和(Flo11p)是細胞膜上能夠進行附著的糖化蛋白。絮凝基因(Flo1p)剔除細胞株表現核醣核酸結合蛋白絲胺酸-脯胺酸突變株時不能夠引發絮凝現象；絮凝基因(Flo11p)剔除細胞株表現核醣核酸結合蛋白絲胺酸-脯胺酸突變株時則會降低洋菜入侵生長現象。絮凝基因(Flo1p)和(Flo11p)共同的轉錄因子(Mss11p)對於絲胺酸-脯胺酸突變株引發的附著現象也是必需的，推測核醣核酸結合蛋白(Rbp1p)的絲胺酸-脯胺酸突變株透過調控轉錄因子MSS11引發絮凝現象及洋菜入侵生長現象。與核醣核酸結合蛋白(Rbp1p)有交互作用的蛋白質中，Dhh1p、Xrn1p和Kre6p是可能的目標蛋白，藉由與絲胺酸-脯胺酸突變株及三個核醣核酸識別基序(RRM) 突變株的交互作用，調控附著現象。

RNA-binding proteins forming dynamic messenger ribonucleoproteins with the transcript are required for the regulation of eukaryotic gene expression. The RNA-binding protein RBP1 encodes a 672-amino acid, ~80-kD protein containing three RNA recognition motifs (RRM) and two glutamine-rich stretches. It is first identified as a negative growth regulator. Our lab has demonstrated overexpression of Rbp1p shows a slow-growth phenotype, decreases the porin mRNA level by enhancing degradation, and suppresses the translation. Rbp1p localized to cytoplasm foci, named P-bodies. Here we showed that serine-428-Proline mutation loses the function of Rbp1p in slow-growth, translation repression and decreasing the stability of POR1 mRNA, but it still can localize to P-bodies. Rbp1p-S428P and Rbp1p RRM motif mutants induce flocculation and invasive growth. Flocculins, FLO1 and FLO11, are membrane surface glycoproteins for adhesion. FLO1 deletion suppresses flocculation; however FLO11 deletion decreases the invasive-growth level. It is also demonstrated that MSS11, transcription factor of FLO1 and FLO11 is required for both adhesion phenotypes, suggesting RNA-binding proteins mutation might regulate flocculation and invasive growth through MSS11. Rbp1p interacting proteins, DHH1, XRN1 and KRE6 might be possible candidates interacting with Rbp1p-S428P and Rbp1p RRM motif mutants to regulate adhesion phenotypes.