演化上高度保留的蛋白質精氨酸甲基轉移酶PRMT1在鳥類中缺失之分析

蛋白質精胺酸甲基轉移酶(PRMT)所扮演的轉譯後修飾包含調控細胞內多種生理反應，如訊息傳遞、轉錄調節、DNA修補和RNA運輸。多數脊椎動物具有9種prmt基因，但鳥類卻缺乏prmt1、2、4和 6。其中，prmt1在真核生物中有著極高的保留度和分佈，且先前的研究也指出prmt1的剔除會導致小鼠胚胎的死亡。但鳥類中並沒有prmt1，反而是prmt1旁系同源基因prmt8存在鳥類中。而鳥類在演化中經歷過大量且大片段的染色體缺失，而我們在研究中也確定了prmt1和其鄰近的基因皆在鳥類中有消失的現象。同時我們也分析了在UCSC的脊椎動物中prmt1和prmt8上下游各11個基因，依照演化上出現的順序，由蛋白質BLAST將物種做兩兩比較來取得同源基因，並將這些基因依照他們所在染色體上的位置進行排序。在這些兩兩排序比較的分析中，在演化上prmt8 周圍的同源基因和基因簇保留的比例較prmt1來得高。多數脊椎動物的第一型PRMT成員 (PRMT1、PRMT2、PRMT3、PRMT4、PRMT6及PRMT8)會催化精胺酸形成非對稱甲基精胺酸(ADMA)的型態，而鳥類的第一型精胺酸甲基轉移酶成員相較於其他脊椎動物來的少，因此我們也測試鳥類中非對稱甲基精胺酸程度是否有受到影響。本研究中使用雞和老鼠的腦部、肝臟和肌肉來進行蛋白萃取。經由西方點墨法並利用非對稱甲基精胺酸抗體偵測的結果顯示，和老鼠相比，在雞的組織當中，非對稱甲基精胺酸的程度並沒有減少。我們進而推測prmt1在鳥類中的缺失可能會由與其同源prmt8來產生補償的現象。但經過RT-PCR的分析顯示，prmt8的mRNA在雞體內也表現出了腦部專一性，因此prmt8並沒有在鳥類中彌補prmt1的功能。我們也將雞和老鼠的腦部以不同的離心速度將組織蛋白萃取液分fraction來觀察蛋白質精胺酸甲基轉移酶的分佈。而結果顯示了雞和老鼠腦部中第一型蛋白質精胺酸甲基轉移酶在次細胞中的表現分佈有著極高的相似性。而我們也將這些離心出來的fraction進行in vitro methylation的分析，並觀察這些fraction的對於受質的甲基化能力。我們的研究結果提供了關於prmt1及其周邊基因在鳥類演化中缺失現象及染色體重組的情形。雖然鳥類缺乏了許多的第一型精胺酸甲基轉移酶的成員，卻依然能維持其正常的生理功能，因此我們可以將鳥類視為天然多精胺酸甲基轉移酶剔除的模式生物，並且幫助我們多加了解蛋白質精胺酸甲基轉移酶在物種演化中如何保留，以及剩下蛋白質精胺酸甲基轉移酶的成員在鳥類中是否有不同於先前研究的功能。

Protein arginine methyltransferases (PRMTs) preform posttranslational modification involved in transcriptional regulation, RNA processing and many different cellular processes. Nine prmt genes are conserved in vertebrates, while prmt1, 2, 4 and 6 are not present in avian. Among these prmt1 is the most conserved and widely distributed prmt gene in eukaryotes, and its deletion is embryonic lethal in mouse. No prmt1 but only its vertebrate paralogue prmt8 could be identified in birds. As avian underwent large segmental chromosomal deletions and gene loss during their evolution, we first determined whether in birds the prmt1 gene was specifically eliminated alone or with neighboring chromosomal segments. We obtained 11 downstream and upstream genes of prmt1 and prmt8 in typical vertebrate species as shown in the UCSC genome browser, collected their homologous genes in other species by BLAST, and then recorded and compared their chromosome localizations. In pair-wise species comparison, orthologous genes near prmt8 have higher conserved level and more gene clusters than prmt1. As most vertebrate type I PRMTs (PRMT1, 2, 3, 4, 6, and 8) that catalyze the formation of asymmetric dimethylarginine (ADMA) are missing in avian, we are interested in whether the ADMA level in birds may be lower than that in other vertebrates. We prepared brain, liver and muscle extracts from chicken and mouse. The ADMA levels were not reduced in different chicken tissues as shown by western blot analyses with an ADMA-specific antibody. We then suspected that loss of prmt1 may be compensated by its paralogue prmt8. However, as shown by RT-PCR analyses, prmt8 mRNA expression was still brain-specific in chicken. It is thus unlikely that prmt8 can complement the loss other type I PRMTs. We also fractionated mouse and chicken brain tissue to examine the PRMT distribution pattern. The result reveals subcellular distribution of type I PRMTs in chicken and mouse have high similarity. Furthermore, in vitro methylation revealed that chicken brain still has similar capability to catalyze arginine methylation as mouse brain extract. Our study provides information about chromosome rearrangement and syntenic blocks deletion near prmt1 in avian evolution. Chicken can be considered as a natural multiple prmt-deleted biological system to further elucidate the critical function of protein arginine methylation conserved and not conserved in vertebrates.