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標題: 在人類細胞中利用核醣核酸開關調控-1 核醣體轉 譯軌道移轉的效率
Regulation of -1 Programmed Ribosomal Frameshifting by Synthetic Riboswitch in Human Cells
作者: 林雅惠
Ya-Hui Lin
關鍵字: 核醣核酸開關;-1 核醣體轉譯軌道移轉;riboswitch;-1 programmed ribosomal frameshifting
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核醣體在轉譯進行時必須維持正確的讀取框架,使細胞轉譯出正確的蛋白,維持細胞正常功能。而當核醣體在mRNAs上進行轉譯時,特定的核醣核酸訊息會影響核醣體讀取框架發生改變。這些特定的核醣核酸訊息為滑動序列 (slippery sequence,通常為X XXY YYZ的序列) 及下游刺激子假結結構 (stimulator),而二者間的適當距離,稱為spacer。當核醣體轉譯至滑動序列時,由於下游的假結結構未能被即時解開,使得核醣體往5'端後退一個核苷酸,造成核醣體讀取框架從0 frame改變至 -1 frame,當假結結構被解開,核醣體會依-1 frame讀取框架再繼續進行轉譯,因此產生具有 -1 frame的融合蛋白,此現象稱為 -1 核醣體框架轉移 (-1 programmed ribosomal frameshifting,簡稱為 -1 PRF)。另外,在滑動序列的上游發現在轉譯中再摺疊形成的髮夾結構,能減弱刺激子所促進的 -1 PRF 效率,而被稱為「減弱子 (attenuator)」,其與刺激子假結結構有相拮抗的作用。
在細菌中所發現核醣核酸開關 (Riboswitch),是由結構性的核醣核酸專一地結合細胞代謝物造成構形改變,來調控特定基因表現的方式,其包含可專一結合細胞代謝物的核醣核酸適體 (aptamer) 及調控平台 (expression plateform),常見於細菌中轉錄終結髮夾結構形成與否的調控,或是核醣體結合位 (RBS) 能被核醣體結合之狀態的調控。由於,原核生物和真核生物間轉譯及轉錄系統的差異性,使得目前在哺乳動物中尚未有適當的基因表現平台可以被核醣核酸開關來調控,因此造成核醣核酸開關難以被應用於哺乳動物細胞中。然而,細菌中的SAH假結結構,被發現能於結合SAH後具有-1 PRF刺激子的能力,刺激-1 PRF的發生,產生具有相同轉譯起始位,卻有不同功能的二種蛋白產物。因此,-1 PRF可以發展成一個能應用在哺乳動物細胞中,並在轉譯的層次上以核醣核酸開關調控基因表現的平台。
所以,我們發展以 theophylline誘導減弱子髮夾結構形成及破壞的迴路來調控-1 PRF 效率,並同時結合SAH假結結構,發現這二個能結合小分子的結構可以協同地調控-1 PRF 效率。為降低SAH造成的調控背景訊號,我們更發展以非細胞代謝物的小分子theophylline來誘導刺激子假結結構的形成,從構形的檢測及-1 PRF功能性分析的結果中,証實theophylline能誘導假結結構的形成,並具有刺激子的能力,促進 -1 PRF效率。結合所設計的減弱子髮夾結構及刺激子假結結構,在有無theophylline的誘導下,能使-1 PRF效率有較佳的相對倍數,對於調控基因的表現,可以有較佳的效果。此一以-1 PRF為基礎的基因表現調控平台,能在轉譯的層次以小分子來誘導蛋白的產生,相較於傳統轉錄層次的調控,或許能更有效率地調控基因的表現。

-1 programmed ribosomal frameshifting (-1 PRF) is a mechanism to regulate gene expression at the level of protein synthesis. -1 PRF occurs upon ribosomes decoding mRNA on a hepta-nucleotides slippery sequence (XXXYYYZ) followed by an optimally spaced stimulator pseudoknot. These signals on mRNA cause a fraction of ribosomes to shift into the -1 reading-frame. A co-translational refolding hairpin found in upstream of slippery sequence attenuates the stimulator pseudoknot-mediated -1 PRF efficiency, and was named 'attenuator'.
Riboswitch, structured RNA capable of binding cellular metabolites to control downstream gene expression by ligand-dependent RNA conformational change, has been characterized in the regulation of prokaryotic transcription termination and translation initiation. Because of the differences between prokaryotes and eukaryotes in transcription termination and translation initiation, it is difficult to apply riboswitch regulatory platform in eukaryotes. However, a SAH pseudoknot riboswitch found in prokaryotes has been demonstrated to stimulate -1 PRF after SAH binding. It provides an opportunity to apply riboswitch into eukaryote cells by -1 PRF gene expression platform. Furthermore, due to the similarity between co-translational attenuation hairpin and the co-transcriptional termination hairpin in prokaryotic rho-independent transcription termination, I rationalized that -1 PRF attenuation event might also be regulated by a synthetic riboswitch.
Here, I engineered a theophylline aptamer within the attenuator hairpin and demonstrated that the theophylline-responsive attenuators regulate -1 PRF by modulating the attenuator hairpin formation or disruption. The combination of the upstream theophylline-responsive attenuator and downstream SAH pseudoknot stimulator has synergistic effect on -1 PRF regulation. However, SAH pseudoknot stimulator causes the regulation leakages as SAH is a universal metabolite in cells. To overcome this problem, I engineered a non-cellular metabolite- theophylline aptamer into a stimulator pseudoknot to regulate -1 PRF by theophylline-dependent stimulator pseudoknot conformational rearrangements. Coupling of both theophylline-responsive upstream attenuator hairpin and downstream stimulator pseudoknot leads to better dynamic range on -1 PRF regulation. This synthetic riboswitch based -1 PRF gene expression extends the riboswitch application to a new gene expression platform and increases the gene regulation repertoire in mammalian cells.
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