探討 A-to-I RNA 編輯作用在海膽神經發育扮演的功能

Abstract

RNA編輯可以藉由微調RNA的序列來影響RNA二級結構、microRNA結合位置、RNA穩定度以及氨基酸序列來達到增加蛋白質多樣性的功能。大多數的RNA編輯是由adenosine deaminases acting on RNA (ADARs)來執行，ADARs能夠將雙股RNA上的adenosine藉由水解脫氨的作用修飾成inosine。在許多不同物種的研究上顯示ADAR經常作用於神經傳導物質受體以及離子通道的mRNA上。此外，ADAR適當的RNA編輯程度對於腦部正常功能扮演重要角色。然而現在我們仍然不了解ADAR如何影響神經系統的發育。海膽胚胎擁有相較簡單的神經系統並且可以容易的用於研究早期胚胎發育，因此我們使用海膽胚胎來研究ADAR在神經系統發育過程當中所扮演的角色。海膽胚胎的基因體包含兩個ADAR基因，adar1以及adar2，我們發現adar1專一的表現在神經細胞當中。adar1與一些神經標記物的雙重染色證實了adar1表現的細胞是會分泌乙醯膽鹼的神經細胞。抑制adar1蛋白質轉譯的胚胎，其形態以及軸突的分佈與正常胚胎沒有差異。為了要了解抑制adar1是否會影響神經活化的程度，我們利用鈣離子偵測物GCaMP6紀錄海膽胚胎活體中的神經活性。為了進一步尋找海膽胚胎當中可能的RNA編輯位點，我們利用次世代定序發現大約一萬一千個RNA編輯位點，其中96%都是adenosine修飾成inosine的RNA編輯並且落在大約100個基因上。我們已經利用一代定序確定其中一個基因choline acetyltransferase (chat)的mRNA會被ADAR1編輯。此外，我們已經成功的在Hela細胞當中過度表現海膽胚胎的ChAT蛋白，未來我們將利用體外ChAT分析來了解adenosine修飾成inosine的RNA編輯作用是否會影響到ChAT的酵素活性，這可能會導致海膽胚胎當中神經活性的改變。這個研究首次發現在神經系統發育的過程當中ADAR1可能會藉由微調ChAT的氨基酸序列，進而影響到分泌乙醯膽鹼的神經細胞活性。

RNA editing provides proteomic complexity by fine-tuning RNA sequences, which may result in a wide range of functional changes by affecting RNA secondary structures, microRNA binding sites, RNA stability and amino acid sequences. The majority of RNA editing is A-to-I RNA editing by adenosine deaminases acting on RNA (ADARs) that modify double-stranded RNA to alter adenosine into inosine by deamination. Numerous studies on different species have demonstrated that ADARs frequently act on mRNAs encoding neurotransmitter receptors and ion channels. Furthermore, proper RNA editing levels mediated by ADARs are important for normal brain functions. However, how ADARs affect nervous system development remains unknown. Sea urchin embryos have a relatively simple nervous system and are easily accessible for early developmental studies. Therefore, I set to investigate the roles of ADARs during nervous system development in sea urchin embryos. The sea urchin genome contains two adar genes, adar1 and adar2. I discovered that adar1 is specifically expressed in neuronal cells during embryogenesis. Double staining of adar1 and several neuronal markers confirmed that adar1-positive cells are cholinergic neurons. Adar1-knockdown embryos displayed no difference in morphology and axonal organizations. In order to examine whether knockdown of adar1 affects neuronal activity, I utilized the calcium indicator GCaMP6 to record neuronal activity in live sea urchin embryos. To further identify possible RNA editing sites in sea urchin embryos, I performed next generation sequencing and uncovered 11,000 RNA editing sites, and 96% of which were A-to-I RNA editing sites located in ~100 gene products. One of such gene products, which encodes choline acetyltransferase (ChAT), was confirmed to be a direct target of ADAR1 by Sanger sequencing. Furthermore, I have successfully expressed the sea urchin ChAT in HeLa cells. This in vitro system will allow us to investigate whether A-to-I RNA editing affects the enzymatic activity of ChAT. This study will provide insights into whether ADAR1 regulates neuronal activity during nervous system development by fine-tuning the activity of cholinergic neurons via modulating chat.