探討酵母菌核糖核酸蛋白Rbp1p核糖核酸識別基序突變珠對絲狀生長之調控

Abstract

酵母菌核糖核酸結合蛋白Rbp1p最初被發現為會負向調控生長的因子。其結構包含三個核糖核酸識別基序(RRM)和兩個富含麩氨酸區域，在其碳端具有一個富含天門東氨酸、甲硫胺酸和脯氨酸的區域。實驗室之前已經發現在∑1278b基因背景下，剔除RBP1基因後會導致酵母菌對於洋菜膠的過度貼附與侵入性生長。最近實驗中發現，核糖核酸識別基序突變珠的貼附與侵入性生長能力皆會提高，尤其是在第三核糖核酸識別基序突變珠中。此現象不只在∑1278b基因背景下被發現，也可以在BY4741基因背景下發現，而BY4741因為FLO8基因變異，原本是不具有貼附與侵入性生長能力的。 在前人的研究中，發現Rbp1p的碳端有八個假定磷酸化位點，而其中的一個假定磷酸化位點－第637號位點，此位點上蘇氨酸之磷酸化會受到環境中葡萄糖的有無而改變，因此我們製造了可以專一辨認第637號位點上蘇氨酸之磷酸化會的抗體。我們也發現，相較於一般菌株，在第三核糖核酸識別基序突變珠中，第637號位點上蘇氨酸之磷酸化會升高。但當我們改變第637號位點上蘇氨酸之磷酸化後，第三核糖核酸識別基序突變珠的貼附與侵入性生長能力並沒有顯著影響，說明了單一位點的磷酸化並不足以影響其貼附與侵入性生長能力。但我們發現這八個假定磷酸化位點可以共同調控第三核糖核酸識別基序突變珠的貼附與侵入性生長能力；此外，我們也發現剔除SNF1和BCY1基因後，第三核糖核酸識別基序突變珠的貼附與侵入性生長能力皆會減弱。 在先前的微陣列分析中，發現FLO基因家族的信使核糖核酸表現量在第三核糖核酸識別基序突變珠中會提高。信使核糖核酸表現量提高的FLO基因包含FLO1、FLO9、FLO10和FLO11，這些FLO基因會調控酵母菌的菌絲生長，包含絮凝作用、貼附能力和侵入性生長。我們發現這些FLO基因之信使核糖核酸表現量的提高和菌絲生長之間有正相關，不過FLO基因的轉錄並不能完全反映在表現型上，所以FLO基因的轉譯還需更進一步的研究。

Rbp1p, as a RNA binding protein, was first identified as a negative growth regulator in Saccharomyces cerevisiae. Protein composition of Rbp1p contains three RNA recognition motifs (RRMs), two glutamine-rich regions, and one asparagine-methionine-proline-rich (NMP) region in the C terminus. Our previous studies have shown that deletion of RBP1 resulting in a hyper ager-invasive growth in ∑1278b strain. Recently, we had found that over-expressing Rbp1p-RRM mutants, especially Rbp1p-rrm3, into yeast induced a hyper-invasion growth phenotype. This hyper-invasion growth phenotype had not only been found in ∑1278b strain but also in BY4741 strain, which had no invasive ability because of its flo8-mutation. We had previously predicted eight putative phosphorylation sites of Rbp1p, which mainly are located at C-terminus. According to the mass-spectrometry-based results, phosphorylation at threonine 637 (T637) would change in response to glucose deprivation. We generated an antibody specifically recognizing T637 phosphorylation, and observed a high phosphorylation level at T637 when over-expressing Rbp1p-rrm3 into yeast. However, the invasion phenotype of Rbp1p was unchanged regardless of T637 phosphorylation state. It indicated that T637 phosphorylation is not sufficient to regulate the Rbp1p-dependant invasive ability. Here we showed that eight putative phosphorylation sites of Rbp1p partially participated in regulating the Rbp1p-dependant invasive ability. Furthermore, the deletion of SNF1 and BCY1 decreased the Rbp1p-rrm3-induced hyper-invasion. According to previous microarray results, we found that the mRNA levels of FLO genes family increased when over-expressing Rbp1p-rrm3 as compared to Rbp1p. The increased FLO genes were FLO1, FLO9, FLO10 and FLO11. These FLO genes were involved in filamentous growth in Saccharomyces cerevisiae, such as flocculation, adhesion and invasion. Here we showed that most increasing mRNA levels of these FLO genes were consistent to the filamentous growth relative phenotypes; however, transcription was not sufficient to reflect these phenotypes. The translation of these FLO genes are needed to be further investigated.