ATXN8OS RNA 上不依賴Cap 的內部核醣體進入位置活性研究
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Date
2012-07-??
Authors
蕭欣杰 陳怡辰 林玄原 蘇銘燦 李桂楨
Hsin-Chieh Shiau, I-Cheng Chen, Hsuan-Yuan Lin, Ming-Tsan Su, Guey-Jen Lee-Chen
Journal Title
Journal ISSN
Volume Title
Publisher
國立臺灣師範大學生命科學學系
Department of Life Science, NTNU
Department of Life Science, NTNU
Abstract
第八型脊髓小腦運動失調症與ATXN8OS CUG重複擴增RNA及ATXN8多麩醯胺擴增的蛋白相關。雖然在CTG方向被轉錄、選擇性裁接及加上polyA尾，ATXN8OS RNA上僅存在小的開放解讀架構。為探究ATXN8OS RNA上一包含102胺基酸之ORF1及一包含41胺基酸與多白胺酸之ORF2是否被轉譯，我們選殖了人的ATXN8OS cDNA 5’片段，置入水母及螢火蟲雙冷光酶基因間。短暫轉染人類胚胎腎細胞及冷光報告測驗顯示，ATXN8OS RNA上帶有兩個不依賴Cap的內部核醣體進入位置。進一步表現在同一解讀架構上的ORF1-EGFP基因亦證實ORF1的轉譯。結合GFP抗體的西方轉漬分析顯示，上游的non-AUG密碼可能作為ORF1-EGFP蛋白的轉譯起始密碼。ATXN8OS ORF1的生理意義及其在SCA8致病機轉的角色仍有待釐清。
Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. Although being transcribed, alternatively spliced and polyadenylated in the CTG orientation, only small open reading frames (ORFs) were noted in ATXN8OS RNA. To investigate if a 102 amino acids containing-ORF (ORF1) and a 41 amino acids plus a polyleucine tract containing-ORF (ORF2) in the ATXN8OS RNA could be translated, we cloned 5’ region of the human ATXN8OS cDNA into dicistronic Renilla and firefly luciferase reporter. Transient transfection in human embryonic kidney (HEK)-293 cells and reporter assay revealed the presence of bipartite cap independent internal ribosome entry segments (IRESs) in the ATXN8OS RNA. The translation of ORF1 was further demonstrated by expression of chimeric construct with an in-frame ORF1-EGFP gene. Western blot with GFP antibody suggested that the expressed ORF1-EGFP protein may be initiated from an upstream non-AUG initiator. The biological meaning of the ATXN8OS ORF1 protein and its role in the pathogenesis of SCA8 remains to be determined.
Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. Although being transcribed, alternatively spliced and polyadenylated in the CTG orientation, only small open reading frames (ORFs) were noted in ATXN8OS RNA. To investigate if a 102 amino acids containing-ORF (ORF1) and a 41 amino acids plus a polyleucine tract containing-ORF (ORF2) in the ATXN8OS RNA could be translated, we cloned 5’ region of the human ATXN8OS cDNA into dicistronic Renilla and firefly luciferase reporter. Transient transfection in human embryonic kidney (HEK)-293 cells and reporter assay revealed the presence of bipartite cap independent internal ribosome entry segments (IRESs) in the ATXN8OS RNA. The translation of ORF1 was further demonstrated by expression of chimeric construct with an in-frame ORF1-EGFP gene. Western blot with GFP antibody suggested that the expressed ORF1-EGFP protein may be initiated from an upstream non-AUG initiator. The biological meaning of the ATXN8OS ORF1 protein and its role in the pathogenesis of SCA8 remains to be determined.