2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38783Protein arginine methylation is an intensively studied posttranslational modification involved in various cellular functions such as signal transduction, protein subcellular localization, RNA processing and export, transcriptional regulation, protein–protein interactions, and even embryogenesis. RNA binding proteins (RBPs) are important for different RNA processing events. RGG domain present in many RNA binding proteins is the first identified protein arginine methylation site. Many RBPs containing the RGG domain have been reported for this modification and the effects of the modification have been illustrated in some cases. However, even more proteins with the RGG domain have not been characterized for the modification. In our previous proteomic studies, through different proteomic or bioinformatics analyses, we have identified a few RBPs that are candidate arginine methylated proteins.We have obtained evidences of the modification and would like to further pursue the physiological roles of the modification in this study.We will focus on two of these proteins--CNBP and PAIRBP1. By now eleven protein arginine methyltransferase (PRMT) genes responsible for the modification have been identified in mammals. From our analyses, the PRMT genes in mammals have their orthologues in zebrafish. Zebrafish is a model organism that is amenable to genetic manipulations and has been widely used in developmental studies.We have used morpholino-oliogonucleotides to specifically inhibit the expression of the gene for the predominant and ubiquitously expressed type I PRMT enzyme PRMT1 in zebrafish.We observed abnormal phenotypes and confirmed reduced protein arginine methylation of a few proteins in the morphants. Thus in this three-year project, we will use cellular systems as well as zebrafish as the model system to study arginine methylation of specific RNA binding proteins.We are going to characterize protein arginine methylation of specific RBPs (eg. CNBP and PAIRBP1) and their involvements of the protein functions via cellular systems.We will determine the methylation sites in CNBP or PAIRBP1 and the specific PRMT responsible for the modification.We will examine the subcellular localizations of the proteins under different methylation conditions. We will detect whether there are differential RNA or protein binding activity of the proteins due to their methylation status and further the effects of the modification on downstream processes.We will also determine the effect of arginine methylation of the specific RNA binding proteins in zebrafish embryogenesis.We will determine the expression pattern of the RNA binding protein in embryogenesis and the function of the protein in zebrafish as well as analyze the modification in zebrafish. Finally we will investigate the zebrafish proteomically and identify specific methylarginine containing RBPs at early embryonic stages.We will continue proteomic analyses of the PRMT morphants for the specific methylaccepting substrates of PRMT in zebrafish and detailed analyses of methylation status of specific methylarginine containing RBPs during zebrafish embryonic developments.蛋白質精胺酸甲基化為一常見的轉譯後修飾，參與細胞中多種反應，如：訊息傳遞， 調節RNA 的轉錄、成熟與運送，蛋白質的交互作用及胚胎發育等。RNA 結合蛋白(RBPs) 在RNA 的成熟過程中扮演重要的角色，過去研究已知許多RBPs 中的RGG 區域含甲基 精胺酸，部分蛋白中此修飾的生理效應也已分析，然而大部分RBPs 中此修飾對蛋白質 功能的影響仍未釐清。在過去研究中，我們已發現數個可能會受到精胺酸甲基化修飾的 RBPs，也得到初步甲基化的證據。因此本計劃想針對其中CNBP 和PAIRBP1 二種蛋白 進行精胺酸甲基化修飾後的生理活性分析。目前在哺乳類中已發現有11 種蛋白質精胺 酸甲基轉移酶(PRMT)且發育的模式動物斑馬魚均有其同源基因。PRMT1 為廣泛存在的 甲基轉移酶，本實驗室已研究斑馬魚中此基因抑制後對蛋白質甲基化以及胚胎發育的影 響。因此這三年計劃中，我們將透過細胞培養方式確定CNBP 和PAIRBP1 蛋白被甲基 化的位置、會受何種甲基轉移酶作用、在不同甲基化的環境下於細胞內的分布情形、觀 察在不同甲基化修飾的狀態下會與RNA 或蛋白進行結合室會有差異，進一步研究對下 游路徑的影響。此外也將透過班馬魚模式觀察RNA 結合蛋白在胚胎發育中的表現，研 究其受到胺基酸甲基化修飾後，對發育過程的影響。並利用蛋白質體研究早期胚胎含有 甲基精胺酸的RBPs，同時也繼續利用在胚胎發育過程中抑制甲基轉移酶來研究特定甲 基化RBPs 的角色。Project