GGGGCC重複序列與其轉譯產物Poly-GR的特異性交互作用研究
Abstract
位於人類第九號染色體72號開放閱讀框架 (C9ORF72) 之 GGGGCC 六核苷酸重複序列擴增是肌萎縮性脊髓側索硬化症 (Amyotrophic lateral sclerosis, ALS) 及額顳葉失智症 (Frontotemporal dementia, FTD) 的共同致病基因之一，其詳細之致病機制尚不明確。GGGGCC重複序列經研究發現會折疊成二級結構，使RNA結合蛋白 (RNA binding proteins) 於轉錄過程與其RNA重複序列聚集成RNA foci；在轉譯過程則會誘發重複區關聯非AUG轉譯 (repeat associated non-AUG translation)，進而產生毒性聚二肽 (dipeptide protein repeats)。本篇研究即以轉譯產物毒性聚二肽為理論基礎，其中富含精氨酸 (R) 之蛋白擁有細胞穿透性質，Poly-GR可穿透細胞核且具細胞毒性，進而可干擾及阻礙位於細胞核內DNA與RNA之相關生理機制，因此本篇研究致力於觀測 GGGGCC重複序列與其轉譯產物Poly-GR兩者間之特異性交互作用。本篇研究以單分子螢光共定位追蹤 (GR)25 與 (GGGGCC)3 的動態變化，實驗結果顯示 (GR)25 與 (GGGGCC)3 之反應具有立即性且會隨時間恢復。螢光標記之 (GR)25 與 (GGGGCC)3 的特異性結合於反應初期較為頻繁，而後逐漸緩和，並且未有長時間滯留的情形。因此推測 (GGGGCC)3 對 (GR)25 具催化性質，而 (GR)25 於反應達平衡後則推測將聚集於系統中他處亦或是被消耗。於實驗即時影像中觀察到 (GR)25 螢光亮斑逐漸形成，而後聚集的現象，然而僅於反應初期偵測到至多三顆 (GR)25 結合於 (GGGGCC)3 的訊號。因此推測於反應初期，(GR)25 透過與 (GGGGCC)3 的特異性結合，(GGGGCC)3 可協助 (GR)25 成核 (nucleation)，隨後已成核至特定大小之 (GR)25 則會與 (GGGGCC)3 分離，轉移至系統中他處繼續聚集成低聚體 (oligomer)，即為影像中所觀察到之螢光亮斑聚集。
GGGGCC hexanucleotide repeat expansion in the first intron of C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The precise C9-ALS/FTD pathogenic mechanisms remain unclear. The main hypothesized mechanisms are C9ORF72 haploinsufficiency and toxicity induced by the formation of RNA foci and dipeptide protein repeats (DPRs). DPRs can be synthesized from both sense and anti-sense strands of the hexanucleotide repeat sequences via repeat-associated non-AUG (RAN) translation, forming five distinct DPRs: Poly-GA, Poly-GP, Poly-GR, Poly-PR, and Poly-PA. In particular, Poly-GR, the arginine(R)-rich DPR can enter the nucleus and reduce cell viability. Moreover, once penetrate the plasma membrane, Poly-GR can disturb intracellular functions. The concept of Poly-GR-mediated toxicity and its colocalization with the nucleus thus motivates this study to observe the specific interactions between GGGGCC repeats and its translational product Poly-GR.Here, we present our single-molecule fluorescence microscopy study on the interaction dynamics between (GGGGCC)3 and (GR)25. Adding (GR)25 induced the structural change of (GGGGCC)3 repeat hairpins instantly, followed by a slow recovery. The transient binding events of (GR)25 on (GGGGCC)3 also decrease gradually. Thus, we speculate (GGGGCC)3 hairpins act as a catalyst in this interaction, whereas (GR)25 could be consumed or aggregated elsewhere. Despite some bright fluorescent spots brightened over time, we only captured a maximum of three fluorescent-labeled (GR)25 bound on (GGGGCC)3 during the early time. Based on our findings, we presume (GGGGCC)3 will mediate the formation of (GR)25 nucleation seed. Subsequently, the (GR)25 seed will dissociate from (GGGGCC)3 and form (GR)25 oligomer elsewhere.
GGGGCC hexanucleotide repeat expansion in the first intron of C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The precise C9-ALS/FTD pathogenic mechanisms remain unclear. The main hypothesized mechanisms are C9ORF72 haploinsufficiency and toxicity induced by the formation of RNA foci and dipeptide protein repeats (DPRs). DPRs can be synthesized from both sense and anti-sense strands of the hexanucleotide repeat sequences via repeat-associated non-AUG (RAN) translation, forming five distinct DPRs: Poly-GA, Poly-GP, Poly-GR, Poly-PR, and Poly-PA. In particular, Poly-GR, the arginine(R)-rich DPR can enter the nucleus and reduce cell viability. Moreover, once penetrate the plasma membrane, Poly-GR can disturb intracellular functions. The concept of Poly-GR-mediated toxicity and its colocalization with the nucleus thus motivates this study to observe the specific interactions between GGGGCC repeats and its translational product Poly-GR.Here, we present our single-molecule fluorescence microscopy study on the interaction dynamics between (GGGGCC)3 and (GR)25. Adding (GR)25 induced the structural change of (GGGGCC)3 repeat hairpins instantly, followed by a slow recovery. The transient binding events of (GR)25 on (GGGGCC)3 also decrease gradually. Thus, we speculate (GGGGCC)3 hairpins act as a catalyst in this interaction, whereas (GR)25 could be consumed or aggregated elsewhere. Despite some bright fluorescent spots brightened over time, we only captured a maximum of three fluorescent-labeled (GR)25 bound on (GGGGCC)3 during the early time. Based on our findings, we presume (GGGGCC)3 will mediate the formation of (GR)25 nucleation seed. Subsequently, the (GR)25 seed will dissociate from (GGGGCC)3 and form (GR)25 oligomer elsewhere.
Description
Keywords
肌萎縮性脊髓側索硬化症, 額顳葉失智症, GGGGCC, amyotrophic lateral sclerosis, frontotemporal dementia, C9ORF72, dipeptide protein repeats, Poly-GR