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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 涂世隆(Shih-Long Tu) | |
| dc.contributor.author | Chien-Chang Wang | en |
| dc.contributor.author | 王健彰 | zh_TW |
| dc.date.accessioned | 2021-06-17T07:01:08Z | - |
| dc.date.available | 2021-01-20 | |
| dc.date.copyright | 2021-01-20 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2021-01-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72572 | - |
| dc.description.abstract | 我們以小立碗藓（Physcomitrella patens）為模式植物，研究植物在照到光之後所引發信使核糖核酸前驅物(pre-mRNA)選擇性剪接(alternative splicing, AS)的現象。選擇性剪接可將相同的信使核糖核酸前驅物剪接成不同的信使核糖核酸，此機制可以改變蛋白質組成來調控生長發育各階段的反應。實驗室先前的研究發現植物照到光之後發生頻率最高的選擇性剪接，是內插子保留(intron retention, IR)的現象，而且有很多內插子保留的調控發生在核糖體蛋白(ribosomal proteins)的信使核糖核酸前驅物上。由於過去曾有動物系統的文獻指出轉錄的過程中，信使核糖核酸選擇性剪接伴隨著組蛋白修飾(histone modification)同時進行。我參考動物系統的文獻並加以測試，以暸解植物中信使核糖核酸選擇性剪接的分子調控機制。我的研究發現，小立碗藓照光之後在受光調控而保留內插子的基因座上，組蛋白3第36號離氨酸被加上三個甲基 （H3K36me3)的機制受到調控；小立碗藓的MRG1 (MORF-Related Gene 1, PpMRG1) 蛋白會辨識H3K36me3並參與許多核糖體蛋白信使核糖核酸前驅物的剪接，選擇性保留信使核糖核酸前驅物的內插子。我們也嘗試找出MRG1對植物向光性的影響，發現PpMRG1的突變株對紅光誘導的向光性不敏感。這些結果結合動物系統的研究，我推測在紅光的處理後，H3K36me3的累積在個別基因座的變化，間接影響許多內插子保留現象發生在核糖體蛋白的信使核糖核酸前驅物上，可能使得最終核糖體蛋白結構改變，或使大小次單元的核糖體蛋白重組，而對特定現有的信使核糖核酸進行轉譯，使植物可以快速的對光線的變化作出反應。 | zh_TW |
| dc.description.abstract | Plants perceive dynamic light conditions and optimize their growth and development accordingly by mainly regulating gene expression at multiple layers. Alternative splicing (AS), a widespread mechanism in eukaryotes that post-transcriptionally generates two or more mRNAs from the same pre-mRNA, is rapidly controlled by light. However, detailed mechanism of light-regulated AS is still not clear. In this study, I demonstrate that histone 3 lysine 36 trimethylation (H3K36me3) rapidly and differentially responds to light at specific gene loci with light-regulated intron retention (IR) of their transcripts in the moss Physcomitrella patens. However, the level of H3K36me3 following exposure to light is inversely related to that of IR events. P. patens MORF-Related Gene 1 (PpMRG1), a chromatin adaptor, bound with higher affinity to H3K36me3 in light conditions than in darkness and was differentially targeted to gene loci showing light-responsive IR. Transcriptome analysis indicated that PpMRG1 functions in light-mediated splicing regulation. We also found PpMRG1 associated with a hnRNP-K like splicing regulator to potentially control AS. Furthermore, PpMRG1 was also involved in red-light-mediated phototropic responses. my results suggest that light regulates histone methylation, which leads to alterations of AS patterns. The chromatin adaptor PpMRG1 potentially participates in light-mediated AS, revealing that chromatin-coupled regulation of pre-mRNA splicing is an important aspect of the plant’s response to environmental changes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T07:01:08Z (GMT). No. of bitstreams: 1 U0001-1301202111321200.pdf: 10897404 bytes, checksum: 75523c3bfd9856dc5b50eaaa052b7e8d (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 1. 中文摘要 1 2. Abstract 2 3. Introduction 3 4. Results 6 4.1. H3K36 is trimethylated in response to RL 6 4.2. IR events and H3K36me3 levels have opposite responses to RL 8 4.3. PpMRG1 differentially associates with H3K36me3 in response to RL 8 4.4. Loss of MRG1 affects RL-responsive IR 10 4.5. PpMRG1 positively regulates phototropic responses in P. patens 12 4.6. A FLK like protein interacts with PpMRG1 13 5. Discussion 14 6. Materials and Methods 18 6.1. Plant materials and growth conditions 18 6.2. Plasmid construction 18 6.3. CRISPR-cas9 mediated mutation 19 6.4. Transformation and generation of knockout and overexpression lines 20 6.5. RT-qPCR analysis 20 6.6. Chromatin immunoprecipitation (ChIP) 21 6.7. Immunoprecipitation 21 6.8. RNA-Seq and Data Analysis 22 6.9. Phototropism Assay 23 6.10. Yeast Two-Hybrid experiment 23 6.11. Accession numbers 24 7. Figures 25 Figure 1. H3K36 trimethylation responds to light. 25 Figure 2. Validation of H3K36me3 enrichment on additional genes by using ChIP-qPCR. 26 Figure 3. Validation of H3K36me3 enrichment on additional genes by using ChIP-qPCR. 27 Figure 4. Light-dependent changes in H3K36me3 enrichment and IR level have opposite patterns. 28 Figure 5. Association of PpMRG1 with histone lysine trimethylations. 29 Figure 6. MRG1 associates with H3K36me3 under light. 30 Figure 7. Validation of PpMRG1 enrichment on additional genes by using ChIP-qPCR. 31 Figure 8. PpMRG1 is involved in RL-regulated IR. 32 Figure 9. Validation of IR pattern in the WT and ppmrg1. 33 Figure 10. Validation of IR pattern in the WT and ppmrg1. 37 Figure 11. PpMRG1 is involved in regulating RL-mediated AltD/A, ES. 38 Figure 12. PpMRG1 functions in RL-dependent phototropic response. 39 Figure 13. Yeast two-hybrid assay to identify PpMRG1 interacting proteins. 40 Figure 14. Sequence alignment of FLKs. 41 Figure 15. Proposed model for chromatin-coupled regulation of AS. 42 Supplemental Figure S1. H3K36 trimethylation responds to light. 43 Supplemental Figure S2. Generation of c-Myc-PpMRG1 transgenic plants by homozygous recombination. 44 Supplemental Figure S3. RL-dependent association of PpMRG1 with H3K36me3. 45 Supplemental Figure S4. Generation of ppmrg1 knock mutant by homozygous recombination. 46 Supplemental Figure S5. Validation of H3K36me3 and PpMRG1 enrichment on additional genes by using ChIP-qPCR. 47 Supplemental Figure S6. Phototropism in the WT and ppmrg1. 49 8. Table 50 Supplemental Table S1. Read mapping statistics of RNA-seq data. 50 Supplemental Table S2. Functional enrichment of IR genes regulated by PpMRG1. 51 Supplemental Table S3. Distribution of bending angles in the WT and ppmrg1. 52 Supplemental Table S4. IDs and annotations of genes used in this study. 53 9. Literature Cited 54 | |
| dc.language.iso | en | |
| dc.subject | 選擇性剪接 | zh_TW |
| dc.subject | 小立碗藓 | zh_TW |
| dc.subject | 內插子保留 | zh_TW |
| dc.subject | MRG1蛋白 | zh_TW |
| dc.subject | 組蛋白修飾 | zh_TW |
| dc.subject | Physcomitrella patens | en |
| dc.subject | alternative splicing | en |
| dc.subject | intron retention | en |
| dc.subject | MRG protein | en |
| dc.subject | H3K36me3 | en |
| dc.title | PpMRG1藉由辨識H3K36me3參與光調控mRNA選擇性剪接 | zh_TW |
| dc.title | PpMRG1 Recognizes H3K36me3 for the Regulation of Light-Responsive Alternative Splicing in Physcomitrella patens | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 謝旭亮(Hsu-Liang Hsieh) | |
| dc.contributor.oralexamcommittee | 吳素幸(Shu-Hsing Wu),陸重安(Chung-An Lu),陳柏仰(Pao-Yang Chen) | |
| dc.subject.keyword | 小立碗藓,選擇性剪接,內插子保留,MRG1蛋白,組蛋白修飾, | zh_TW |
| dc.subject.keyword | Physcomitrella patens,alternative splicing,intron retention,MRG protein,H3K36me3, | en |
| dc.relation.page | 59 | |
| dc.identifier.doi | 10.6342/NTU202100051 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2021-01-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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