酵母菌核醣體結合蛋白Rbp1p及其結合蛋白的功能性探討

Abstract

正確控制訊息核醣核酸的降解，對於適當表達細胞基因來說是關鍵的。酵母菌中一個包含三個核醣核酸辨識基序(RRM)及兩個富含麩氨酸區域的核醣核酸結合蛋白Rbp1p，被鑑定為負向生長調控因子。我們已經證實Rbp1p可以促進粒線體外膜孔蛋白porin的訊息核醣核酸降解。在這個研究中，我們發現Rbp1p可以直接與DEAD-box核醣核酸解旋酶Dhh1p結合，並且鑑定出Dhh1p的功能性區域，可專一性的用來參與Rbp1p所媒介的porin核醣核酸降解。酵母菌雙雜交實驗以及體外拉下分析皆可證實Rbp1p與Dhh1p碳端非保留區的八十一個胺基酸進行交互作用。Dhh1p-dC81具有完全補足DHH1剔除菌株的生長缺陷以及EDC1核醣核酸的量。此結果指出Dhh1p-dC81在體內仍具有Dhh1p的其他的生物功能。然後，剔除碳端八十一個胺基酸的Dhh1p確實失去降解porin核醣核酸及調控Rbp1p媒介的porin核醣核酸降解的功能。我們也發現Dhh1p結合至porin核醣核酸是依賴Rbp1p存在的。表現辨識基序突變的Rbp1p或是剔除碳端天冬醯胺、甲硫胺酸和脯胺酸區域的Rbp1p，都不能補足在剔除RBP1菌株中，Dhh1p結合至porin核醣核酸的能力。這些結果顯示與Rbp1p交互作用對於Dhh1p結合至porin核醣核酸是必須的，並且暗示Rbp1p吸引Dhh1p解旋酶將porin核醣核酸進行降解。而且，當哺乳動物的Dhh1p相似物DDX6的碳端聚合了Dhh1p的非保留區胺基酸序列後，DDX6變具有與Rbp1p交互作用以及可以進行Rbp1p媒介的porin降解的能力。因此，我們提出Dhh1p的碳端非保留區扮演確定Dhh1p可以專一性與核醣核酸因子結合，進而促進不同核醣核酸降解的角色。

The control of mRNA degradation is critical for proper gene expression. In Saccharomyces cerevisiae, an RNA-binding protein Rbp1p was first identified as a negative growth regulator, which contains three copies of RNA recognition motifs (RRMs) and two glutamine-rich stretches. We have demonstrated that Rbp1p decreased the level of mitochondrial porin mRNA by enhancing its degradation. Here, we show that Rbp1p directly interacts with the DExD/H-box RNA helicase Dhh1p and identify the Dhh1p-specific functional region required for Rbp1p-mediated porin mRNA decay. Yeast two-hybrid and in vitro pull-down assays showed that Rbp1p interacts with the C-terminal nonconserved 81 residues of Dhh1p. Dhh1p-dC81 fully complements the growth defect and EDC1 mRNA steady level of dhh1Δ mutant, indicating that Dhh1p-dC81 still contains other biological function of Dhh1p in vivo. However, deleted C-terminal 81 residues in Dhh1p lost its function to restore the porin mRNA degradation in dhh1Δ mutant and in modulating Rbp1p- mediated porin mRNA decay. Dhh1p binds to porin mRNA is Rbp1p dependent. Expression of Rbp1p-rrm1, a mutant lost its ability to bind porin mRNA, in rbp1Δ mutant does not restore Dhh1p to bind porin mRNA. In addition, expression of Rbp1-dNMP, a mutant does not interact with Dhh1p, does not rescue the binding of porin mRNA to Dhh1p in rbp1Δ mutant. These data indicate that Rbp1p is required for binding of porin mRNA to Dhh1p in vivo and suggest that Rbp1p recruits the Dhh1p helicase to promote porin mRNA decay. Moreover, mammalian DDX6 becomes capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1Δ strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the nonconserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.