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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 譚婉玉 | zh_TW |
| dc.contributor.advisor | Woan-Yuh Tarn | en |
| dc.contributor.author | 洪珮瑜 | zh_TW |
| dc.contributor.author | Pei-Yu Hung | en |
| dc.date.accessioned | 2025-09-09T16:05:49Z | - |
| dc.date.available | 2025-09-10 | - |
| dc.date.copyright | 2025-09-09 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-07-29 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99343 | - |
| dc.description.abstract | 生物分子在時間與空間上的凝聚行為已被視為細胞於DNA雙股斷裂（DSBs）修復過程中的關鍵調控機制。多功能RNA結合蛋白Y14/RBM8A可於DNA損傷部位，透過與RNA介導的交互作用促進非同源性末端接合（NHEJ）的DNA修復途徑。Y14具有帶電的內在無序區域（intrinsically disordered regions, IDRs），提供其進行液-液相分離（liquid-liquid phase separation, LLPS）的結構基礎。其C端RS重複序列經SR蛋白激酶SRPK1磷酸化後，可在鎂離子存在的條件下產生相分離現象。磷酸化後的Y14（pY14）可形成動態凝聚物，並透過不同的交互作用方式與DNA損傷反應（DNA damage response, DDR）因子（如Ku70/80）及核酸分子（包括長鏈非編碼RNA與DNA）共同聚集。非磷酸化Y14與聚（ADP-核糖）（PAR）聚合物的結合親和力高於磷酸化的 Y14，表明 Y14 在被 SRPK1 磷酸化之前就被募集到 DNA 損傷處；隨後磷酸化的Y14在鎂離子存在下形成凝聚物。綜合本研究結果，Y14經磷酸化後所引導的靜電驅動型相分離機制，與鎂離子協同調控，在DNA損傷位置上有助於修復因子的時空性募集與組織。 | zh_TW |
| dc.description.abstract | Spatiotemporal condensation of biomolecules has emerged as a critical mechanism for coordinating the DNA repair process at double-strand breaks (DSBs). The multifunctional RNA-binding protein Y14/RBM8A facilitates non-homologous end joining (NHEJ) through RNA-guided interactions at damage sites. Structurally, Y14 contains charged intrinsically disordered regions (IDRs) that provide the basis for its liquid-liquid phase separation (LLPS). SR protein kinase-1 (SRPK1)-mediated phosphorylation of C-terminal RS dipeptides enables Y14 to undergo magnesium-dependent LLPS in vitro. Phosphorylated Y14 (pY14) condensates accommodate DNA damage response (DDR) factors such as Ku70/80 and nucleic acids such as lncRNA HOTAIRM1. Non-phosphorylated Y14 bound poly(ADP-ribose) (PAR) polymers with a higher affinity than phosphorylated Y14, suggesting that Y14 is recruited to DNA lesions prior to its phosphorylation by SRPK1. Y14 forms condensates after phosphorylation with Mg2+ present. Together, our findings suggested electrostatic interaction-mediated LLPS for recruiting DNA repair factors to Y14 condensates at DNA damage sites. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-09T16:05:49Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-09T16:05:49Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 摘要 iii Abstract iv Table of contents v Introduction 1 DNA repair is activated to maintain genome integrity 1 Liquid-liquid phase separation enables dynamic biochemical organization 3 The role of liquid-liquid phase separation in DNA damage repair 5 Nucleic acids modulate liquid-liquid phase separation behavior 6 Multifunctional RNA processing factor Y14 participates in DNA damage repair and undergoes liquid-liquid phase separation 9 Aims 11 Results 12 SRPK1 phosphorylates Y14 in cells 12 Mg2+ promotes phospho-Y14 to undergo LLPS 14 NHEJ factors engage charged IDRs to partition into Y14-driven condensates 18 Nucleic acid partitioning into pY14 droplets 20 Y14 does not distinguish RNA m6A modifications 22 Y14 interacts with PAR 24 Discussion 26 The effects of DDR kinases on Y14 phosphorylation are indirect. 26 pY14 droplets exhibit greater stability than Y14 under Mg2+/PEG conditions. 27 pY14 co-phase separates with RNA and NHEJ factors via electrostatic interactions. 28 Limitations of the study 30 Materials and methods 31 Cell culture and drug treatment 31 Phos-tag gel analysis 31 Protein purification of non-tagged Y14 32 In vitro phosphorylation of recombinant Y14 33 Mass Spectrometry 34 Liquid-liquid phase separation assay 34 Sedimentation assay 35 Turbidity assay 35 Fluorescence recovery after photobleaching (FRAP) 36 Far Western 37 PAR binding assay 37 In vitro transcription 38 Electrophoretic Mobility Shift Assay (EMSA) 40 Figures 41 Fig. 1 SRPK1 phosphorylates Y14 in vivo 41 Fig. 2 Mg2+ promotes LLPS of pY14 43 Fig. 3 Analysis of the interaction between pY14 and IDR peptides using Far Western blot 45 Fig. 4 Co-phase separation of nucleic acids with pY14 47 Fig. 5 Y14 interacts with PAR 49 Appendix 50 Appendix 1 In vitro phosphorylation of Y14 51 Appendix 2 Non-tagged recombinant Y14 purification 52 Appendix 3 Mg2+ promotes LLPS of pY14 55 Appendix 4 Electrostatic interaction drives Ku70 partitioning into pY14 condensates 57 Appendix 5 EMSA shows that m6A modification does not affect the binding between Y14 and lncRNA HM1-3 60 Appendix 6 EMSA shows that m6A modification does not affect Y14 and RNA binding 62 Reference 63 | - |
| dc.language.iso | en | - |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | 液態-液態相分離 | zh_TW |
| dc.subject | DNA損傷修復 | zh_TW |
| dc.subject | 靜電作用 | zh_TW |
| dc.subject | 二價陽離子 | zh_TW |
| dc.subject | liquid-liquid phase separation | en |
| dc.subject | phosphorylation | en |
| dc.subject | DNA damage repair | en |
| dc.subject | electrostatic interaction | en |
| dc.subject | divalent cation | en |
| dc.title | 磷酸化Y14與核酸之液態-液態相分離現象之研究 | zh_TW |
| dc.title | Characterization of liquid-liquid phase separation of phosphorylated Y14 and nucleic acids | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.coadvisor | 蔡欣祐 | zh_TW |
| dc.contributor.coadvisor | Hsin-Yue Tsai | en |
| dc.contributor.oralexamcommittee | 陳瑞華;林倩伶 | zh_TW |
| dc.contributor.oralexamcommittee | Ruey-Hwa Chen;Chien-Ling Lin | en |
| dc.subject.keyword | 液態-液態相分離,磷酸化,二價陽離子,靜電作用,DNA損傷修復, | zh_TW |
| dc.subject.keyword | liquid-liquid phase separation,phosphorylation,divalent cation,electrostatic interaction,DNA damage repair, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU202502570 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-07-29 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 分子醫學研究所 | - |
| dc.date.embargo-lift | 2025-09-10 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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| ntu-113-2.pdf | 2.97 MB | Adobe PDF | 檢視/開啟 |
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