磷酸化對於酵母菌核醣核酸結合蛋白Rbp1p之功能特性探討

Abstract

在細胞當中，核醣核酸結合蛋白對於訊息核醣核酸(mRNA)的修飾作用(processing)、轉錄(translation)和穩定性(stability)中扮演著重要的角色。目前已經知道核醣核酸結合蛋白 Rbp1p 具有抑制酵母菌生長和調控某些特定訊息核糖核酸(mRNA)穩定性的功能。然而，對於Rbp1p是如何調控生長與訊息核糖核酸穩定度的機制還不是非常清楚。之前實驗室利用質譜儀分析而找到了在Rbp1p的碳端有八個可能會被磷酸化的位置。為了進一步了解這些位置的磷酸化是否會影響到Rbp1p的蛋白質功能。我們先利用定點突變(site-directed mutagenesis)的技術建構出這八個磷酸化的位置被突變成丙胺酸(alanine)或天門冬胺酸(aspartate)來模擬完全去磷酸化(Rbp1-S8A)和完全磷酸化(Rbp1-S8D)的狀態，我們發現Rbp1-S8D喪失了抑制酵母菌生長的能力。此外利用酵母菌雙雜交實驗(Yeast-two-hybrid)發現在這八個位置被磷酸化的狀態之下，Rbp1p的自相互作用(self-interaction)的能力會上升而沒辦法被磷酸化的Rbp1p的自相互作用則會降低。實驗當中也發現這八個位置的磷酸化與否並不會影響Rbp1p與Pub1p和Hrp1p之間的交互作用。 Rbp1p在細胞中的位置會隨著細胞生長時期(growth stage)的不同而改變且Rbp1p在延滯期 (lag phase)被發現會座落在細胞核的位置。Rbp1-S8A大部分是分佈在細胞質當中且呈現細小顆粒狀，Rbp1-S8D則是一直是聚集在細胞核的位置。當面對葡萄糖被剝奪(glucose deprivation)的壓力之下，這些突變型的Rbp1p依舊能聚集到細胞質處理小體 (Processing bodies)。另外，在熱休克(heat shock)的處理之下，突變形的Rbp1p仍然能與Rbp1p的結合蛋白Psp1p有位置重合的現象(co-localization)。最後，我們也發現這些突變型的Rbp1p一樣可以回復由Rbp1p做為媒介來誘導過度的洋菜附著現象(hyper-agar adhesive growth)。 總而言之，我們發現了位於碳端的這八個胺基酸的磷酸化確實會影響Rbp1p在細胞中的位置和喪失抑制酵母菌生長的能力。

In cells, RNA binding proteins play an important role in regulating RNA processing, translation and stability. The RNA binding protein Rbp1p was known to repress cell growth and regulate the stability of a subset of mRNAs in yeast. However, the regulatory mechanism of Rbp1p function is still unclear. In this study, we indentified eight putative phosphorylation sites at C-terminus of Rbp1p by mass spectrometry. To characterize the biological importance of these phosphorylation modifications of Rbp1p, we first generated a non-phosphorylatable construct at eight serine residues in the C-terminal region of Rbp1p, Rbp1-S8A, and a mimicking phosphorylation at these residues, Rbp1-S8D. We observed that, unlike wild-type Rbp1p or Rbp1-S8A, Rbp1-S8D mutant lacked the ability to repress cell growth. Using yeast-two-hybrid analysis, we observed that Rbp1-S8D, but not Rbp1-S8A, increased self-interaction of Rbp1p. However, Rbp1-S8D did not affect its interaction with Hrp1p or Pub1p. We also found that Rbp1p exhibited dynamics cellular localization at different growth stage and was partially localized to the nucleus at the lag growth phase. Interestingly, we found that, at different growth stage, most of Rbp1-S8D was sequestered to localize in the nucleus and Rbp1-S8A exhibited diffuse and small cytoplasmic granules. Furthermore, Rbp1-S8A or Rbp1-S8D mutant did not affect their localization to P-bodies in response to glucose deprivation or heat-stress granules under heat shock. In addition, Rbp1-S8A and Rbp1-S8D still can restore Rbp1p mediated hyper-adhesive or invasive growth. Together, we demonstrate that subcellular localization and cellular function of Rbp1p is modulated by phosphorylation of serine residues in the C-terminal region.