膜穿孔蛋白毒素（PFT）是常見細菌的細胞毒性蛋白，主要作用於破壞宿主的細胞膜而導致死亡。而為了抵禦膜穿孔毒素，細胞會活化下游信號（例如自噬作用）以調控宿主對於毒素的忍受度。因此，在先前實驗室的研究中，我們發現給予線蟲Cry5B膜穿孔毒素後，會造成腸道細胞的轉錄因子HLH-30從細胞質轉移至細胞核中，並啟動自噬作用來抵抗毒素所造成的傷害。HLH-30在哺乳類動中，同源基因為TFEB。在我們高通量基因靜默篩選的實驗中，發現在抑制掉第七型蛋白質精氨酸甲基轉移酶(prmt-7)後，會導致由Cry5B毒素所引起的HLH-30入核現象有所下降，並且也造成自噬作用中的生物標記LGG-1表現量也有所降低。而為了反覆證明prmt-7在調控HLH-30入核現象中扮演重要角色，我們通過將只在腸道表現的prmt-7質體回補到整個prmt-7功能喪失的線蟲體內，發現腸道細胞的HLH-30入核現象會有所恢復。另外，我們也將prmt-7酵素失活質體回補到整個prmt-7功能喪失的線蟲體內，也發現該株線蟲的HLH-30入核表現並沒有所改變，證明了prmt-7在調控HLH-30入核現象中有一定的作用。
為了證明prmt-7是在細胞質去進行HLH-30入核的調控。在Fluorescence recovery after photo-bleaching(FRAP)分析影像結果中，表明PRMT-7可能是在細胞質而不是在細胞核中去調控HLH-30入核現象。除此之外，當prmt-7突變株的xpo-1（編碼出核蛋白）和ima-2（編碼入核蛋白）被靜默時，HLH-30入核表現在xpo-1實驗組中有所增加，但HLH-30入核表現在ima-2組與對照組的相比中，並沒有很大差異性。這些結果再次證實線蟲受到膜穿孔毒素後，腸道細胞的PRMT-7可能在是細胞質中調節HLH-30，並促使其移動到細胞核中。另外，我們也證明了prmt-7參與了mTOR訊息傳遞路徑以調節HLH-30入核現象。接著，我們通過單一點突變方式將不同的HLH-30甲基化位點進行突變，並確定了HLH-30上第29、36和38位點上的精氨酸，可能是參與藉由膜穿孔毒素所誘發HLH-30入核表現的潛在甲基化位點。最後，我們還發現重組活化基因蛋白(Rag proteins)和mTOR調節相關蛋白(Raptor protein)可能會影響HLH-30入核現象。綜合以上的結論，我們的遺傳分析證明了第七型精氨酸甲基轉移酶(PRMT-7)是如何調控由膜穿孔毒素所誘導HLH-30入核現象的機制。

The pore-forming toxins (PFTs) are most common bacterial cytotoxic proteins causing damage to the plasma membrane of their host cells. Hosts activate downstream signal such as autophagy to protect from PFTs assaults. In our previous study, we discovered that the transcription factor HLH-30 is required for the activation of autophagy to defend Cry5B, a PFT produced by B. thuringiensis, in C. elegans. From our genetic suppression screening, we found that the prmt-7 (protein arginine methyltransferase 7) mutant worms suppressed HLH-30 nuclear localization (HLH-30 translocates from cytosol to nucleus) after Cry5B treatment. Moreover, we found that prmt-7 involved in the HLH-30-regulated autophagy activation and pore-repair in the PFT targeted cells. To reconfirm that prmt-7 plays an important role in regulating HLH-30 nuclear localization, we generated a prmt-7 rescued strain by injecting prmt-7 expressing plasmid to prmt-7 loss-of function mutants. We found that HLH-30 nuclear localization was restored to wild type. Moreover, we also generated a PRMT-7 enzyme inactivation mutant and found that the nuclear localization of HLH-30 in the mutant worm was decreased. Furthermore, our FRAP (Fluorescence recovery after photobleaching) assay results suggested that the function of PRMT-7 could be in the cytoplasm but not in the nucleus to regulate HLH-30 nuclear localization. Additionally, when prmt-7 mutants subjected to xpo-1 (encodes for the nuclear export protein) and ima-2 (encodes for the nuclear import protein) RNAi, HLH-30 nuclear localization was increased in xpo-1 RNAi group but showed no difference in ima-2 group compared to that of RNAi control group. These results reconfirmed that PRMT-7 might regulate HLH-30 in the cytoplasm to facilitate HLH-30 translocating into nucleus after Cry5B treatment. Besides, we demonstrated that prmt-7 participated in the mTOR signaling pathway and regulated mTOR-mediated HLH-30 nuclear localization. Moreover, we mutated several HLH-30 methylation sites via site-directed mutagenesis and identify the arginine 29, 36 and 38 of HLH-30, could be the potentially methylation sites of HLH-30 in regulating its nucleus translocation under Cry5B intoxication. Finally, we also found that Rags and DAF-15 might affect HLH-30 nuclear localization. In summary, our genetic analysis demonstrated the mechanism of how the arginine methyltransferase PRMT-7 can regulate PFT-induced HLH-30 nuclear localization.

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