斑馬魚胚胎發育中 prmt8 基因之表現及功能分析

蛋白質精胺酸甲基化（arginine methylation）為蛋白質轉譯後修飾的一種，是藉由 protein arginine methyltransferases（PRMT）進行催化。此作用參與細胞中許多的功能，包含訊號傳遞（signal transduction）、轉錄後的調節（transcriptional regulation）、蛋白質間的交互作用（protein-protein interaction）、DNA修復（DNA repair）、RNA 的剪接（RNA splicing）等。PRMT8為第一型的甲基轉移酶，與哺乳動物中最主要的精胺酸甲基轉移酶 PRMT1 有高達80％以上的相同度。PRMT8被報導其表現侷限於腦部，且N端可能因具有荳蔻酸化而表現於細胞膜。但在最近研究指出，PRMT8表現於細胞核，其N端有不同的轉譯起始點。除此之外，尚未有其他針對PRMT8功能上分析的報導。斑馬魚與人類之PRMT8具有高度的保留性，因此我們以斑馬魚為模式動物，探討PRMT8在胚胎發育時期的表現及功能。RT-PCR及全胚體原位雜交實驗得知，zfprmt8 mRNA 特異性表現於成魚腦部，且其從胚胎合子時期持續表現至早期幼體時期。從全胚體原位雜交結果觀察，zfprmt8 mRNA 於 24 hpf（hours post fertilization）廣泛表現於魚體之頭部及體節。有趣的是，zfprmt8 mRNA 於 72 hpf開始表現主要在體節，到了96 hpf，頭部的zfprmt8 mRNA訊號明顯上升，而體節的訊號則減弱。接著利用反股寡核酸antisense morpholino oligonuceotide 抑制 zfprmt8 基因的功能，在胚胎早期造成高致死率及不同程度的發育異常，包含體軸彎曲且變短，卵黃形狀異常及心包膜水腫。利用兩種不同可能的轉譯起始點位置之反股寡核酸來進行 knockdown zfprmt8，在同樣的劑量下造成不同的嚴重程度，且觀察到 morphants中的精胺酸甲基化反應下降。為了確認 morphants型態之專一性，我們成功的以 zfprmt8 cRNA 進行型態上的回復。在局部的標示基因分析下，可以看到 morphants有外包作用、早期細胞匯聚及延展、體軸發育上的異常，利用acridine orange對morphants 染色分析，觀察到大量的細胞凋亡。在本研究中，我們確認了zfprmt8 基因的表現，且對PRMT8 在胚胎發育上的功能角色進行分析。

關鍵字

蛋白質精胺酸甲基轉移酶斑馬魚

並列摘要

Protein arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification involved in signal transduction, transcriptional regulation, protein-protein interaction, DNA repair and RNA splicing. PRMT8 is the type I methyltransferase which is about 80% identical to PRMT1, the major protein arginine methyltransferase in mammals. PRMT8 was reported to be broadly distributed in brain and localizes at the plasma membrane via N- terminal myristoylation. However, a recent study reported nuclear localization of PRMT8 and suggested a different N-terminal translational start site. We use zebrafish (Danio rerio) as a model system to analyze the unsettled issues of PRMT8 as it is highly conserved from zebrafish to human. RT-PCR and whole-mount in situ hybridization（WISH）experiments show that zfprmt8 mRNA is specifically expressed in adult zebrafish brain and during development from zygote period to the early larva stage. Whole-mount in situ hybridization reveals that zfprmt8 mRNA is expressed in most parts of the head and somites at 24 hours post fertilization (hpf). Interestingly, zfprmt8 mRNA is expressed solely in somites at 72 hpf and then migrates to brain at 96 hpf. Knockdown of the prmt8 gene in zebrafish embryos by antisense morpholino oligonuceotides (AMOs) leads to higher lethality and different degrees of phenotype defects including shortened and curved body axes, abnormal yolk shape and heart edema. AMOs against different putative translational start sites appear to result in phenotypes of different severity at the same dosage. We rescue the morphants by zfprmt8 cRNA and confirmed the specificities of phenotype as well as reduced arginine methylation in the morphants. Detailed marker analysis revealed defection in epiboly, convergence and extension of the body plan and somite development. Our study contributes to the understanding of specific zfprmt8 expression and analysis of Prmt8 function in embryonic development.

並列關鍵字

PRMT8 ； zebrafish ； protein arginine methyltransferases

參考文獻

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斑馬魚胚胎發育中 prmt8 基因之表現及功能分析

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