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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 譚婉玉(Woan-Yuh Tarn) | |
dc.contributor.author | Bin-Tse Lin | en |
dc.contributor.author | 林秉澤 | zh_TW |
dc.date.accessioned | 2021-06-17T07:02:42Z | - |
dc.date.available | 2019-08-26 | |
dc.date.copyright | 2019-08-26 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-07-30 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72647 | - |
dc.description.abstract | 前驅傳訊核醣核酸(precursor message RNA, pre-mRNA)的剪接發生在轉錄時期,並受到trans-acting factors與cis-regulatory elements之交互調控。核醣核酸結合蛋白Y14是外顯子接合複合體(exon junction complex)之核心成員,但Y14本身可以影響pre-mRNA剪接。我發現Y14的第166和168胺基酸位點的SA(serine-to-alanine)突變(Y14-SA)能修復因Y14缺失所造成的錯誤選擇性剪接(alternative splicing);我也發現RNA聚合酶或拓樸異構酶抑制劑有類似功能。我亦觀察到正常和突變的Y14對基因的啟動子(promoter)有不同的親和性,因此Y14-SA是否透過轉錄機制來調控mRNA剪接仍需近一步調查。另外,我們亦探討Y14是否藉由影響小核核醣核酸(snRNA)U6上第43個鹼基(腺苷)第六位的氮(N)元素上的甲基化修飾(N6-methyladenosine,m6A)來調控pre-mRNA剪接。我們發現Y14跟核醣核酸去甲基酶ALKBH5和核醣核酸甲基酶METTL16結合,Y14在體外(in vitro)會抑制ALKBH5的活性。Y14表現量減少會使U6之m6A減少,但是藉由ALKBH5的大量表現或METTL16的表現減少所造成的U6之m6A減少,都不會產生選擇性剪接。儘管如此,U6的m6A是否對pre-mRNA剪接有任何影響仍是不確定的。我們仍持續研究Y14是否可藉由參與轉錄機制或U6的m6A來調控pre-mRNA之剪接。 | zh_TW |
dc.description.abstract | Splicing occurs co-transcriptionally and is regulated by trans-acting factors (RNA binding proteins) via binding to cis-regulatory RNA elements. Y14 is a core factor of the exon junction complex (EJC), which functions in the post-splicing events, but recent studies have revealed that deletion of Y14 generated aberrant alternative splicing products. I unexpectedly discovered that the C-terminally truncated Y14 or phospho-deficient mutant (SA), but not wild type Y14, reversed Y14 knockdown-induced splicing changes. Besides, inhibition of RNA polymerase II and topoisomerase I & II prevented Y14 knockdown-induced splicing changes on a different scale. Next, we evaluated how the Y14-SA mutant exerts the dominant effect. We here observed that both wild-type and mutant (SA) Y14 can bind the promoter regions of its target genes, indicating a potential role of Y14 in coordinating transcription and splicing, which is independent of Y14’s phosphorylation. Moreover, my colleagues and I found that Y14 interacted with both the RNA demethylase ALKBH5 and RNA methyltransferase METTL16. Depletion of Y14 reduced m6A modification on U6 snRNA. However, depletion of METTL16 or overexpression of ALKBH5, although minimally affected m6A modification of U6 snRNA, had no effect on alternative splicing of Y14 target genes. At present, whether m6A modification of U6 snRNA is essential for splicing or involved in splicing regulation is yet unclear. Besides, we found that Y14 inhibited the demethylase activity of ALKBH5 in vitro. Whether the interaction between Y14 and m6A modifying enzymes has any biological functions remains to be examined. Investigation of Y14’s role in splicing regulation is still ongoing. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:02:42Z (GMT). No. of bitstreams: 1 ntu-108-R06448012-1.pdf: 2208557 bytes, checksum: 7bc121a14d29a59c296549ba2e2aefe5 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | Contents I
List of Figures/ Appendixes II Acknowledgements III Chinese Abstract IV English Abstract V Introduction 1.The splicing machinery 1 2.Role of the exon junction complex (EJC6) in mRNA metabolism 4 3.Role of the EJC and its peripheral factors in pre-mRNA splicing 6 4.Impacts promoter proximal pausing 8 5.Role of N6-methyladenosine modification in splicing 10 Results 1.Non-phosphorylated Y14 restores splicing changes caused by Y14 depletion 15 2.Y14 may coordinate with the transcription machinery in splicing regulation 17 3.Weak 5’ splice site accounts for Y14 loss-induced intron retention 18 4.Y14-mediated m6A modification of U6 snRNA is not critical for splicing regulation 19 5.Y14 interacts with m6A modifying enzymes 22 6.Y14 inhibits the demethylation activity of ALKBH5 in vitro 24 7.Novel m6A-containing small RNAs 25 Discussion 27 Experimental Procedures 37 References 52 Figures 76 Appendixes 89 | |
dc.language.iso | en | |
dc.title | Y14調控RNA剪接之分子機制研究 | zh_TW |
dc.title | Investigation toward the molecular mechanism underlying Y14-mediated splicing regulation | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李芳仁(Fang-Jen Lee),蔡欣祐(Hsin-Yue Tsai) | |
dc.subject.keyword | 外顯子接合複合體,選擇性剪接,轉錄,小核核醣核酸U6,鹼基(腺?)第六位的氮(N)甲基化修飾, | zh_TW |
dc.subject.keyword | exon junction complex,alternative splicing,transcription,small nuclear RNA U6,N6-methyladenosine modification, | en |
dc.relation.page | 91 | |
dc.identifier.doi | 10.6342/NTU201902205 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-07-31 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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