李娟蔡政剛Cheng-Kang Tsai生物醫學科學學系碩士班2026-03-189999-12-312016https://handle.ncl.edu.tw/11296/gn5zx3https://scholars.csmu.edu.tw/handle/123456789/5938在脊椎動物中，蛋白質精胺酸甲基轉移酶 (protein arginine methyltransferase； PRMT) PRMT8 與 PRMT1序列有很高的同源性，主要差異在蛋白質 N 端的部位。PRMT8 是 PRMTs家族中唯一成員具有組織特異性，只表現在腦和神經細胞中。然而 PRMT8 N 端序列在人類和斑馬魚卻不盡相同，在人類富含有獨特的脯胺酸序列，斑馬魚則具有富含麩醯胺酸序列，以及富含脯胺酸和組胺酸的區域。因此我們想進一步探討 PRMT8 N端的功能，並且分析PRMT8 N端在不同物種間的結合蛋白。我們利用酵母雙雜交實驗 (yeast two-hybrid screen; Y2H)，以PRMT8 N端的序列為誘餌來針對成年人類腦部 cDNA 基因庫進行分析，鑑定出10個新的PRMT8 N端特異性的結合蛋白，這些結合蛋白分別與細胞黏著、細胞代謝過程、RNA 結合，轉錄調控、肌動蛋白絲結合、有絲分裂紡錘體的組成和衰老相關。之後也使用藥物3-AT系列性稀釋分析來檢查結合蛋白之間結合的強弱。在跨物種中的分析，也發現到 SDHB 和 TMEFF2 這兩個人類 PRMT8 N 端 (H8N) 的結合蛋白，同時會與斑馬魚PRMT8 N 端（ZF8N）相互結合。當斑馬魚 PRMT8 富含麩醯胺酸的區域被踢除時，DCTN6 和 CACYBP這兩個 H8N 的結合蛋白，就可與 ZF8N 結合。我們也確定了 H8N 與 SDHB 的結合區是在 C 端的134個氨基酸。此外我們也利用共免疫沉澱法來驗證細胞內 H8N 與結合蛋白 (例如: SYNE1、CACYBP和CRIP1a) 之間的結合作用。在本篇研究中，最後我們也使用特異性結合實驗、酵母雙雜交實驗和共免疫沉澱法等，來研究過去曾鑑定出的ZF8N和其結合蛋白 (包含 Epd、ZFYwhaba、INA、actin、 tubulin 和 GAPDH) 的相互作用。由本篇研究結果中，發現了 PRMT8 的結合蛋白，並且對於PRMT8的功能有更具體的了解。Protein arginine methyltransferase (PRMT) 8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence. Its expression is limited in brain and is the only PRMT with tissue-restricted expression pattern. The N-terminus of PRMT8 contains a unique proline-rich sequence in human and a unique glutamine-rich (Q-rich) sequence before a proline- and histidine-rich region in zebrafish. Therefore, we are interested in the function of the N-terminus of PRMT8 and would like to analyze the interacting partners of this segment cross species. We performed the yeast two-hybrid (Y2H) screen for specific interacting proteins of the N-terminus of human PRMT8 (H8N) against a human brain cDNA library. We identified ten proteins as potential PRMT8 N-terminal interactors that had not previously been reported to interact with PRMT8. These diverse binding partners of H8N are associated with cell adhesion, cellular metabolic process, RNA binding, transcriptional regulation, actin filament binding, mitotic spindle organization and aging. Based on the Y2H system, we performed 3-AT spot dilute assay to examine the binding strength between PRMT8 and PRMT8 interactors. We also conducted a Y2H-based analysis in cross species and showed that two H8N interactors, SDHB and TMEFF2, can interact with the N-terminal 96 amino acid of zebrafish Prmt8 (ZF8N). Besides, two more H8N interactors DCTN6 and CACYBP interacted with ZF8N when a unique glutamine-rich (Q-rich) sequence before the proline and histidine-rich region in zebrafish PRMT8 was deleted (∆ polyQ). In addition, we identified that the N-terminus of human PRMT8 interact with SDHB at the C-terminal 134 amino acids. Moreover, we used co-immunoprecipitation assay to validate specific interaction between H8N and the putative interactors such as SYNE1, CACYBP and CNRIP1a. In this study, we finally validated the interaction of a few putative ZF8N interacting proteins identified by my previous pulldown experiments that are non-overlapping with H8N interactors. We used the pull-down assay and the Y2H analysis to study the interactions between ZF8N and two putative interactors Epd and ZFYwhaba. Co-immunoprecipitation assay was also used to examine the interaction of other specific interactors including INA, actin, tubulin, and GAPDH. Results of this study should extend the interactome of PRMT8 and provide more specific understanding of the function of PRMT8.蛋白質精胺酸甲基轉移&#37238N-terminus of protein arginine methyltransferase PRMT8thesis