梨形鞭毛蟲中nonsense-mediated mRNA decay (NMD)的重要因子upf1之鑑定

Abstract

我們實驗室之前利用具有無意義突變(nonsense mutation)的螢火蟲發光基因(luciferase gene)的系統，發現了nonsense-mediated mRNA decay (NMD)的效應存在於梨形鞭毛蟲中。為了要確定梨形鞭毛蟲自身的基因也有NMD效應，我將梨形鞭毛蟲的囊壁蛋白(cwp3)基因中加入一個無意義突變，在北方墨點法中偵測到具有無意義突變的cwp3 mRNA量降低了約70%，因此NMD的效應也存在於梨形鞭毛蟲自身的基因中。為了要更深入探討NMD在梨形鞭毛蟲中的效應，我利用在酵母菌NMD途徑中最conserved的upf1基因來尋找梨形鞭毛蟲基因組資料庫中是否也有相似的基因，blast的結果找到一個相似upf1的基因，在序列分析上，梨形鞭毛蟲的Upf1也和酵母菌及人類的Upf1一樣擁有NTPase, 核酸解螺旋酵素(helicase)以及具RNA結合力的區域。將upf1基因接入可以在梨形鞭毛蟲中穩定表現的質體內，利用免疫螢光染色確認了Upf1蛋白質分佈在梨形鞭毛蟲的細胞質中，其分佈區域和酵母菌、人類以及線蟲的Upf1相同。 為了了解梨形鞭毛蟲的upf1的功能，我將upf1的表現質體與含有無意義突變的螢火蟲發光基因的表現質體同時轉染到梨形鞭毛蟲中，在北方墨點法偵測到在同時有upf1的表現質體的梨形鞭毛蟲中，螢火蟲發光基因的mRNA量跟對照組比較起來少了約50%而其螢光測定值也少了約90％。此外，加入RNA合成抑制劑Actinomycin D 25分鐘後抽取pPW1與pPW1m的RNA，再利用北方墨點法分析，結果顯示有nonsense mutation的pPW1m luc mRNA不穩定，相較於不加Ac.D約少了八成。酵母菌的upf1基因除了對含有無意義點突變的基因表現有影響外，對許多正常基因的表現也有影響，因此我將upf1大量的表現在梨形鞭毛蟲中，觀察一些正常基因的表現，結果發現囊壁蛋白cwp1, cwp2和cwp3 的mRNA量都有下降約85%，除了囊體蛋白以外，也有其他的基因受到upf1大量表現而改變其mRNA量，如：cyclophilin、thioredoxin peroxidase、translation elongation factor、p21以及arginine deiminase等，但是ran基因的mRNA卻不受影響。為了要釐清其囊壁蛋白cwp1, cwp2和cwp3 mRNA下降的原因是否是因為RNA 穩定性的下降所致，我加入RNA合成抑制劑Actinomycin D於大量表現upf1的梨形鞭毛蟲中，在不同時間後抽取RNA並利用北方墨點法來偵測cwp1的mRNA的量，發現到cwp1 mRNA在不同的時間點都維持在較少的量，所以大量表現upf1會造成cwp1的mRNA的穩定性下降。 近年來發現到Upf1被SMG1磷酸化後可以跟Upf2、Upf3形成NMD core complex。我利用線蟲的smg1在梨形鞭毛蟲基因組資料庫中也找到一個類似的基因稱為smg1-like，序列分析上梨形鞭毛蟲smg1除了跟線蟲smg1一樣擁有HEAT、PI3PI4 kinase以及FATC domain以外，它還有一個Rapamycin binding domain，將smg1基因接入可以在梨形鞭毛蟲中穩定表現的質體內，利用免疫螢光染色確認了Smg1蛋白質分佈在梨形鞭毛蟲的細胞質中，其分佈區域和Upf1相同，相同的分佈位置顯示出Upf1以及Smg1-like protein之間有作用的可能性。

Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). To understand whether NMD is present in the primitive protozoan Giardia lamblia, we constructed a reporter vector, pPW1m, which contains a nonsense mutation in the luciferase (luc) coding region and stably transfected it into Giardia. Northern analysis revealed that the luc mRNA decreased in the pPW1m cell line compared to that in the control cell line harboring pPW1 vector which has a normal luc gene. This indicated that the NMD effect might be present in Giardia. To further analyze the NMD effect in G. lamblia, a blast search against Giardia genome database with the sequence of the most conserved NMD factor, Upf1, from Saccharomyces cerevisiae were performed. A putative upf1 gene in the Giardia genome database was identified. The putative Giardia Upf1 is longer than the Saccharomyces Upf1. Like the S. cerevisiae Upf1, Giardia Upf1 also has a zinc finger domain at N-terminus and the DEAD box at C-terminus. Immunofluorescence assay revealed that the Giardia Upf1, like Upf1 in S. cerevisiae. is expressed in the cytoplasm. The luc mRNA decreased by ~50％ in the co-transfectants pPW1m+pUPF1neo compared to that in the control cell line pPW1m+pRANneo suggesting that the Giardia Upf1 enhances decay of the nonsense-containing transcripts. It has also been observed that S. cerevisiae Upf1 not only functions in the NMD pathway but also functions in regulating some wild-type mRNAs. In agreement with above notion, overexpression of the upf1 gene in Giardia trophozoites resulted in a significant decrease of a number of wild-type gene transcripts, including cwp1 (cyst wall protein 1), cwp2, cwp3, cyclophilin, thioredoxin peroxidase, translation elongation factor, p21 and arginine deiminase. I also found that overexpression of Upf1 decreased cwp1-3 mRNA stability. Moreover, deletion analysis indicated that the regions responsible for the Upf1 dependent RNA instability of cwp1 mRNA are located in the 5’ and 3’terminal coding region. Recently, a novel phosphatidylinositol 3-kinase-related protein kinase called Smg1 has been found in Caenorhabditis elegans and human. Smg-1 phosphorylates Upf1 at specific serine residues in SQ motifs at C-terminus inC. elegans and human. A blast search against Giardia genome database was also performed, resulting in identification of a putative smg1 gene in the Giardia genome database. The immunofluorescence assay reveals that the Giardia Smg1-like protein, like Giardia Upf1, is also localized in the cytoplasm, indicating that they may interact with each other.