在哺乳類動物中有 9 個不同的PRMT基因 ( PRMT1-9 )。 PRMT1是在真核生物裡最廣泛且高度保留的PRMT。 PRMT8是PRMT1在脊椎動物中特有的異種同源基因，它們主要區別在於PRMT8包含一個約 60-90 個胺基酸所組成額外的N-端。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；然而在其它的報導指出PRMT8表現於細胞核，其N端有不同的轉譯起始點。先前實驗室的研究當中提到prmt1以及prmt8的序列非常相似，在斑馬魚以及人類當中皆具有高度的保留性，並以斑馬魚為模式生物系統對prmt1以及prmt8探討其在胚胎內的表現以及功能。為要了解PRMT8蛋白質在斑馬魚胚胎內的表現，我們試著純化斑馬魚PRMT8 N端的特異性抗體；在無法順利取得自製抗體之後，再利用市售抗體探討PRMT8以及PRMT1蛋白在老鼠腦的分佈。我利用不同的離心速率，將老鼠的腦蛋白分成S0~P4，發現PRMT8大多分部在細胞核部分的P1，而PRMT1主要在細胞質的S3部分或核糖體 / 細胞膜相關的S4部分，這個結果顯示大多數PRMT1和PRMT8分佈在於大腦細胞的不同位置。我們收集來自不同脊椎動物的PRMT8以及不同物種的PRMT1的genomic DNA和cDNA序列，經由BLAST比對或從Ensembl預測序列來構建基因結構圖。由於選擇性剪接 ( alternative splicing ) 的關係，我們在人類以及老鼠中觀察到有不同PRMT1的variant，而大多數有不同的N-端。然而人類PRMT1 variant 4和老鼠的PRMT1 variant 3，它們的exon 6和exon 7缺失，是在先前的研究中沒有被報導過的。最後透過序列比對，發現PRMT8在哺乳動物、有袋動物、鳥類、兩棲類和魚類的N端是主要的變異區；一種新的PRMT8 exon1 variant 2可以在NCBI資料庫找到，是先前的文獻還沒報導過並且存在於靈長類當中。進一步分析PRMT1和PRMT8的基因組結構和轉錄的調節，將有助於我們追查PRMT8的演化起源，並提供關鍵訊息以備將來的實驗設計。
There are nine different PRMT genes in mammals ( PRMT1-9 ). PRMT1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-specific paralogue of the PRMT1 and the major difference is that PRMT8 contains an extra N-terminus of about 60-90 amino acids in different species. PRMT8 was reported to be distributed in brain and localizes to the plasma membrane via N-terminal myristoylation. However, another study reported nuclear localization of PRMT8 and suggested a different N-terminal translational start site. Our laboratory has shown that the sequences of prmt1 and prmt8 are very similar and highly conserved in zebrafish and humans and thus used zebrafish as a model system to analyze prmt1 and prmt8 during embryogenesis. To understand the distribution of PRMT8 protein in zebrafish embryos, we tried to purified zebrafish PRMT8 N terminal-specific antibodies. After failing to obtain useful antibodies, we analyzed the PRMT1 and PRMT8 protein distribution by a commercially available antibody. Mouse brain extracts were fractionated into S0 ~ P4 by differential centrifugation. PRMT8 was mostly detected in the nuclear fraction P1 while PRMT1 was mainly detected in the cytosolic S3 fraction or the ribosome / plasma membrane-associated S4 fractions. The results indicate that most PRMT1 and PRMT8 are localized in different subcellular fractions of brain. We then collected the PRMT8 as well as PRMT1 genomic and cDNA sequences from different vertebrate species and constructed the gene structure diagram through BLAST ( Basic Local Alignment Search Tools ) or from the Ensembl prediction. Due to alternative splicing, we observed different PRMT1 variants in human and mice and most variants have different N-termini. However, we found human PRMT1 variant 4 and mouse PRMT1 variant 3 with missing exon 6 and exon 7 that have not been reported in previous publications. Finally, through sequence alignments, we showed the major variable region of PRMT 8 is at the N-terminus in mammals, marsupials, birds, amphibians and fish. A new PRMT8 exon 1 variant 2 in primates that have not been reported in previous publication can be identified in the NCBI database. Further analyses of the genomic structures and the putative transcriptional regulatory sequences of PRMT1 and PRMT8 will help to trace the evolution origin of PRMT8 and provide critical information for future experimental designs.
