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標題: RNA解旋酶A核酸結合能力對其轉錄活化功能影響之探討
Study of the effect of nucleic-acid binding property of RNA helicase A on its transcriptional activation activity
作者: 張崇德
Chang, Chung-Te
關鍵字: RNA helicase A;RNA解旋酶A;RHA;CREB-binding protein;CBP;SUMO-1;轉錄共活化因子;dsDNA結合能力
出版社: 生物化學研究所
RNA解旋酶A (RNA helicase A; RHA)屬於DExH家族的一員,具有解開雙股螺旋之DNA及RNA的能力。近來的研究指出,RHA在CREB所引導的轉錄過程中,扮演連結CREB-binding protein (CBP)與RNA聚合酶II的轉錄共活化因子角色;但RHA的helicase活性及其與dsDNA結合能力對轉錄活化的重要性,卻始終沒有深入的研究。為了進一步探討此一課題,我們在RHA 1-257片段(包含dsRBM1 & 2)上的PEST-like motif做點突變,經由in vitro及in vivo的實驗證明,該突變可使RHA無法與dsDNA 結合,卻無損其與CBP的交互作用。我們接著利用transient transfection assays來測試此突變蛋白對CREB所引導之轉錄活性的影響;實驗結果顯示,當RHA無法與dsDNA結合時,CRE下游基因的轉錄活性隨之下降,這表示RHA並不僅只擔任橋樑的角色,與dsDNA的結合與否亦會對本身轉錄共活化因子的功能造成影響。
經由序列比對,我們發現在RHA dsRBM1 C端的PEST-like motif上,具有兩個SUMO-1的可能價接位點(a.a. K76及K120)。並經由實驗證明了RHA確實可為SUMO-1所標定;且若於細胞中大量表達SUMO-1蛋白,則會使RHA的活性受到明顯的抑制。此結果暗示了RHA可能會受到SUMO-1蛋白標定的調控,而造成對其轉錄功能的影響。

RNA helicase A (RHA) is a member of the DExH family of RNA helicase and is capable of unwinding duplex RNA and DNA. Recent studies have shown that RHA is also a component of holo-RNA polymerase II (Pol. II) complexes and recruits Pol. II to the CREB-binding protein (CBP). To further understand the role of RHA on gene expression, we have focused on the 257-amino-acid dsRNA binding motifs (dsRBMs) of RHA that interact with dsRNA/dsDNA and CBP. Using site-directed mutagenesis, we created mutants of RHA that contain point mutation in the PEST-like motif of RHA. In vitro and in vivo biochemical analysis show that these mutants lacking dsDNA binding activity can still interact with CBP. In transient transfection assays, these mutants of RHA reduced the activity of CREB-mediated transcription. We thus propose that RHA may not only serve as a bridging factor, but also contribute its dsDNA binding ability to its transcription co-activator activity.
In addition, we also identified two potential modification sites (a.a. K76 & K120) for small ubiquitin-like modifier 1 (SUMO-1) by sequence analysis of RHA. Experimental data further show that RHA can be sumoylated, and over-expression of SUMO-1 can reduced the transcription activation activity of RHA. This observation suggests sumoylation may regulate the transcriptional co-activation activity of RHA.
Appears in Collections:生物化學研究所

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