王怡鈞WANG, YI-CHUN許睿綸HSU, JUI-LUNJUI-LUNHSU生物醫學科學學系碩士班2026-03-182028-08-1820252025-07-17https://handle.ncl.edu.tw/11296/m3m632https://scholars.csmu.edu.tw/handle/123456789/5993核糖體 RNA（rRNA）的轉錄後修飾對於維持核糖體結構完整性與轉譯效率具有重要影響，特別是假尿苷化與 2′-O-甲基化這兩種修飾主要依賴小核仁 RNA（snoRNA）及其相關蛋白複合體（snoRNP）執行。Glycine-Arginine-Rich（GAR）區域作為一種含有大量甘胺酸與精胺酸重複序列的結構，在多種核仁蛋白中高度保守存在，並與 RNA 結合、液–液相分離（LLPS）及 snoRNP 組裝功能密切相關。GAR1 是 H/ACA box snoRNP 的核心成員之一，能促進 rRNA 假尿苷化修飾的準確性與效率。 本研究以 GAR1 為模型，結合 FBL（fibrillarin）作為比較對象，探討 GAR 區域在不同物種中的保守性與演化變化，並進一步分析 GAR 區域在基因層級上的密碼子使用偏好與可能形成 RNA G-四鏈體（rG4）結構的潛力。結果顯示，GAR1 蛋白在酵母、線蟲、果蠅、斑馬魚及人類等多種模式生物中皆保留典型 N 端與 C 端 GAR 區域，其中脊椎動物 GAR1 基因多由 7 exon 組成，N-GAR 區域主要由大部分的 exon 2編碼，C-GAR 區域則主要由exon 5的中後部分及全部的exon 6編碼 ，並顯現出在一個exon以上編碼的GAR 區域長度相對穩定下，各exon 長度在不同物種中具補償的現象。 在密碼子使用偏好方面，GAR1 的 GAR 區域中精胺酸密碼子偏好使用 AG 開頭組合，Gly 則偏好 GGA，整體趨勢使 GAR 區域多為 G-rich 而非 CG-rich，與 FBL 中 GAR 區域密碼子偏好在部分物種（如爬蟲類）明顯偏向 CG 開頭而易形成 CG-rich 序列的情況形成對比。G4 結構預測顯示，多數脊椎動物 GAR1 的 N-GAR 與 C-GAR 區域皆具備形成 rG4 的潛力，驗證 GAR 區域序列與 rG4 結構之間可能存在調控與功能上的聯繫。 本研究不僅證實 GAR1 的 GAR 區域在跨物種間結構與功能上的高度保守性，也指出其在基因序列層面上可能透過密碼子偏好選擇，調控 rG4 結構形成，進一步深化對核仁蛋白 GAR 區域在演化與功能調控上的理解。Post-transcriptional modifications of ribosomal RNA (rRNA) are crucial for maintaining ribosome structural integrity and translational efficiency, particularly pseudouridylation and 2′-O-methylation, which are primarily mediated by small nucleolar RNAs (snoRNAs) and their associated protein complexes (snoRNPs). The Glycine-Arginine-Rich (GAR) domain, characterized by repetitive glycine and arginine sequences, is highly conserved among various nucleolar proteins and plays key roles in RNA binding, liquid–liquid phase separation (LLPS), and snoRNP assembly. GAR1, a core component of H/ACA box snoRNPs, contributes to the accuracy and efficiency of rRNA pseudouridylation.In this study, GAR1 was used as a model, with fibrillarin (FBL) as a comparative reference, to investigate the conservation and evolutionary changes of the GAR domain across species. We further analyzed codon usage bias at the gene level and the potential of the GAR domain to form RNA G-quadruplex (rG4) structures. Our results revealed that GAR1 proteins from various model organisms—including yeast, C. elegans, Drosophila, zebrafish, and humans—retain both N-terminal and C-terminal GAR domains. In vertebrates, the GAR1 gene typically comprises seven exons: the N-GAR domain is encoded by the majority of exon 2, excluding its terminal residues, while the C-GAR domain is encoded by the latter part of exon 5 and all of exon 6. Although the GAR domains span more than one exon, their overall lengths remain relatively conserved, with compensatory variations observed in individual exon lengths across species.Regarding codon usage, the arginine residues within the GAR domain of GAR1 show a preference for AG-starting codons, while glycine residues preferentially use GGA, resulting in a G-rich rather than CG-rich sequence profile. This contrasts with FBL, whose GAR domain in some species (e.g., reptiles) displays a marked preference for CG-starting codons, forming CG-rich sequences. G4 structure predictions further revealed that both the N-GAR and C-GAR domains of vertebrate GAR1 possess high potential for rG4 formation, supporting a possible regulatory or functional link between GAR sequences and rG4 structures.Overall, this study confirms the structural and functional conservation of the GAR domain in GAR1 across species and highlights the potential role of codon usage bias in regulating rG4 formation at the gene sequence level. These findings enhance our understanding of the evolutionary and regulatory significance of GAR domains in nucleolar proteins.脊椎生物GAR1GAR domainthesis