探討DHX8磷酸化在RNA加工及基因穩定性中的功能

Abstract

DHX8是一種首次在酵母菌中發現的DExH-box RNA解旋酶，在前體mRNA剪接的過程後期幫助mRNA從剪接體中脫離。研究表明，沒有DHX8表現的斑馬魚出現胚胎發育缺陷並死亡。然而，DHX8在人類中的確切功能仍然不清楚。 DNA損傷反應 (DDR) 在DNA損傷後被啟動，並由DDR激酶ATR、ATM和DNA-PK介導，它們調控下游生化路徑並促進DNA修復系統，其中包括同源重組修復(HR)。有研究發現若在人類癌細胞中敲除DHX8可能會影響HR的效率 ; 我們實驗室同仁之前的研究也表明，當癌細胞缺乏DHX8時會使細胞存活率降低，並且Chk1及RPA這兩個關鍵的ATR下游受質的磷酸化也會下降。這表明DHX8可能可以調控DDR及DNA修復系統。先前的蛋白質組學研究辨認了DHX8在紫外線照射後出現的兩個磷酸化位點 (S460 & T554) ，表明了DHX8的磷酸化可能在DDR或在正常情況下起作用。因此，我使用專一性抗體確認了DHX8的磷酸化位點，並發現S460的磷酸化是不會受DNA損傷而升高的，這表明DHX8的磷酸化可能涉及維持其正常功能。 於是在對正常細胞功能的研究中，我發現S460和T554的磷酸化對癌細胞的生長存活率並不是非常重要。根據數據庫預測的結果，我確認了S460的磷酸化是由細胞周期依賴性激酶 (CDKs) 調節的。考慮到細胞周期的調節在DDR中的重要性，我推測DHX8可能在有無DNA損傷的情況下皆能參與細胞周期的調節。 此外，基於DHX8與酵母菌中剪切活性的關聯，我發現抑制DHX8中S460的磷酸化不會影響特定基因的選擇性剪接 (Alternative splicing) 。然而抑制DHX8中S460和T554的磷酸化會影響其與RNA Pol II、PRP19和PRPF8等蛋白質的相互作用，而這些蛋白質參與了前體mRNA的剪切和RNA的加工。 綜上所述，DHX8的磷酸化在正常情況下可能在RNA的加工或其他生理功能中短暫地發揮作用。雖然取得了一些線索，但仍無法很好的闡明DHX8的生理功能。此外，DHX8在DNA損傷後調節DDR的相關機制仍需要進一步的研究。

DHX8, a DExH-box RNA helicase first discovered in yeasts, helps mRNA detach from the spliceosome at the end of the pre-mRNA splicing process. Studies have shown that the loss of DHX8 in zebrafish led to embryo development defects and lethality. However, the exact function of DHX8 in humans remains largely unknown. DNA damage response (DDR) is triggered after DNA damage and mediated by DDR kinases ATR, ATM, and DNA-PK, which regulate the downstream pathways that facilitate the DNA repair systems, including homologous recombination (HR). In human cancer cells, it was discovered that the loss of DHX8 might affect the efficiency of HR. Our previous research demonstrated that cancer cells lacking DHX8 exhibited reduced cell viability and decreased phosphorylation of the two critical ATR substrates, Chk1 and RPA. This suggests that DHX8 is likely to regulate the DDR and DNA repair. Proteomic data from previous studies identified two phosphorylation sites of DHX8 (S460 & T554) after exposure to UV radiation, indicating a potential role of DHX8 phosphorylation in DDR or normal function. Therefore, I confirmed the phosphorylation of DHX8 using specific antibodies and found that pS460 occurs independently of DNA damage, suggesting that the phosphorylation of DHX8 could be involved in its normal function. In my research on normal cellular function, I have discovered that phosphorylation at S460 and T554 is not essential for cancer cell viability. Based on the database prediction, I have confirmed that phosphorylation at S460 is regulated by Cyclin-dependent kinases (CDKs). Considering the significance of cell cycle regulation in DDR, DHX8 might be involved in cell cycle regulation with or without DNA damage. Furthermore, based on its association with splicing activities in yeast, I discovered that inhibiting DHX8 phosphorylation at S460 did not affect the alternative splicing of specific genes. However, inhibiting DHX8 pS460 and pT554 did affect its interaction with proteins such as RNA Pol II, PRP19, and PRPF8, which are involved in pre-mRNA splicing and RNA processing. DHX8 phosphorylation may be transient in RNA processing or other physiological functions under normal conditions. Although some clues were found, these findings still failed to elaborate on the exact function of DHX8. Besides, the function of DHX8 in regulating DDR after DNA damage requires further investigation.