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Thus, splicing is a key mechanism for regular gene expression and interference with splicing will affect gene expression. Deregulation or inhibition of splicing will result in production of non-functional or even deleterious proteins. Recently, FR901464 and its methylated derivative Spliceostatin A (SSA) were reported for the first time to inhibit pre-mRNA splicing by binding non-covalently to the SF3b sub-complex in the U2 snRNP. SSA is chemically more stable than its parent molecule FR901464, which was originally isolated from a fermentation broth of Pseudomonas sp. During screening of microbial metabolites, FR901464 was found to activate viral promoters including those of the SV40 and the cytomegalovirus (CMV). Previous studies have shown that SSA leads to transcriptional repression of about 20% of expressed genes, while enhancing transcription of only a small subset. Among the most highly up-regulated genes was Interleukin 8 (IL-8). Since IL-8 and CMV promoters are activated by a similar set of transcription factors, I used luciferase reporter constructs of each promoter for a comparative study.\nIL-8 is a well-studied CXC family chemokine that activates and recruits leukocytes to the site of inflammation. Interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-α) and phorbol 12-myristate 13-acetate (PMA) are well known inducers of IL-8. These inducers can enhance the transcriptional stimulatory activity of viral promoters (SV40 and CMV) as well and are also able to activate cellular signaling kinases. Previous investigations suggested that IL-8 production is regulated by extracellular signal-regulated protein kinase (ERK) and transcription factor NF-kB in response to TNFα, IL-1β or PMA. To better understand the underlying mechanism we investigated both possible upstream signaling pathways as well as likely transcription factors responsible for IL-8 induction. The mitogen-activated protein kinases (MAPKs) are important signaling kinases that activate a variety of transcription factors through phosphorylation. Here I report that SSA treatment induces ERK activation and that SSA treatment leads to enhancement of NF-kB activity.", "subitem_description_type": "Abstract"}]}, "item_113_description_24": {"attribute_name": "目次", "attribute_value_mlt": [{"subitem_description": "Chapter 1: Introduction\n 1.1 What is splicing?........................................................11\n 1.2 Discovery of SSA and its derivatives…………………12\n 1.3 Other molecules targeting pre-mRNA Splicing………13\n 1.4 FR901464 induces ER stress…………………………14\n 1.5 SSA-induced gene activation……………………………15\n 1.6 Aim of this study…………………………………………16\n Chapter 2: SSA enhances the transcriptional activation of IL-8 and CMV\n promoters\n 2.1 Introduction……………………………………………………20\n 2.2 Material and methods………………………………………22\n 2.2.1 Materials and cell lines……………………………22\n 2.2.2 Antibodies and immunoblotting……………………22\n 2.2.3 qPCR analysis………………………………………22\n 2.2.4 Expression and knockdown………………………23\n 2.3 Results………………………………………………………24\n 2.3.1 Effect of SSA on IL-8 and CMV promoter activity.24\n 2.3.2 Transcriptional activation in a time and dose-dependent\n manner……………………………………………25\n 2.3.3 Effect of splicing inhibitors and splicing inhibition on\n gene activation……………………………………25\n 2.4 Conclusion……………………………………………………27\n Chapter 3: Role of ERK pathway in IL-8 and CMV promoters Up-regulation\n 3.1 Introduction…………………………………………………31\n 3.1.1 ERK pathway activation……………………………31\n 3.1.2 Abnormal activation of ERK leads to disease\n development…………………………………………31\n 3.1.3 Role in controlling apoptosis………………………32\n 3.1.4 Role in transcriptional activation…………………32\n 3.1.5 SSA and ERK pathway……………………………33\n 3.2 Material and methods………………………………………34\n 3.2.1 Materials, cell lines and antibodies………………34\n 3.2.2 Antibodies and immunoblotting……………………34\n 3.2.3 qPCR analysis……………………………………35\n 3.2.4 Expression and knockdown………………………35\n 3.3 Results…………………………………………………………36\n 3.3.1 SSA affects ERK activation………………………36\n 3.3.2 Effect of SAP knockdown on CMV promoter\n activity…………………………………………………37\n 3.4 Conclusion……………………………………………………38\n Chapter 4: SSA affects NF-kB pathway\n 4.1 Introduction……………………………………………………41\n 4.2 Material and methods………………………………………43\n 4.2.1 Materials, cell lines and antibodies……………43\n 4.2.2 Antibodies and immunoblotting………………43\n 4.2.3 Immunofluorescence microscopy……………43\n 4.2.4 Expression and knockdown……………………44\n 4.3 Results…………………………………………………………45\n 4.3.1 Transcription factor point mutants reveals an\n essential role of NF-kB in the regulation of IL-8\n and CMV promoters…………………………………45\n 4.3.2 SSA modulates the transcriptional activity through\n p65-NF-kB……………………………………………46\n 4.4 Conclusion……………………………………………………48\n Chapter 5: SSA induces ER stress\n 5.1 Introduction…………………………………………………51\n 5.1.1 what is ER stress?..............................................51\n 5.1.2 Cellular responses combating with ER stress…51\n 5.1.3 FR901464 as an ER stress inducer……………53\n 5.2 Material and methods………………………………………55\n 5.2.1 Materials, cell lines and antibodies………………55\n 5.2.2 In vitro binding assay ………………………………55\n 5.2.3 RNA interference…………………………………55\n 5.3 Results………………………………………………………56\n 5.3.1 FR induces UPR…………………………………56\n 5.3.2 Identification of SSA binding proteins and activation\n of JNK………………………………………………57\n 5.3.3 SSA-induced IL-8 promoter activation occurs\n independent of TMX………………………………58\n 5.4 Conclusion……………………………………………………59\n Overview…………………………………………………………………66\n References\n References………………………………………………………67", "subitem_description_type": "Other"}]}, "item_113_description_25": {"attribute_name": "注記", "attribute_value_mlt": [{"subitem_description": "主指導教員 : 吉田稔", "subitem_description_type": "Other"}]}, "item_113_description_33": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"subitem_description": "text", "subitem_description_type": "Other"}]}, "item_113_description_34": {"attribute_name": "フォーマット", "attribute_value_mlt": [{"subitem_description": 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Chemical Biology of Splicing Inhibitors
https://doi.org/10.24561/00010302
https://doi.org/10.24561/000103025562c15d-603f-40ff-8b26-d1f8d3539ec1
名前 / ファイル | ライセンス | アクション |
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GD0000525.pdf (5.2 MB)
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||||||
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公開日 | 2015-02-04 | |||||||||
タイトル | ||||||||||
言語 | en | |||||||||
タイトル | Chemical Biology of Splicing Inhibitors | |||||||||
言語 | ||||||||||
言語 | eng | |||||||||
資源タイプ | ||||||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||||
資源タイプ | doctoral thesis | |||||||||
ID登録 | ||||||||||
ID登録 | 10.24561/00010302 | |||||||||
ID登録タイプ | JaLC | |||||||||
アクセス権 | ||||||||||
アクセス権 | open access | |||||||||
アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||||
タイトル(別言語) | ||||||||||
その他のタイトル | スプライシング阻害剤に関する化学生物学的研究 | |||||||||
著者 |
KHALID, KHAN
× KHALID, KHAN
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著者 所属 | ||||||||||
埼玉大学大学院理工学研究科(博士後期課程)理工学専攻 | ||||||||||
著者 所属(別言語) | ||||||||||
Graduate School of Science and Engineering, Saitama University | ||||||||||
書誌 | ||||||||||
収録物名 | 博士論文(埼玉大学大学院理工学研究科(博士後期課程)) | |||||||||
書誌情報 |
発行日 2014 |
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出版者名 | ||||||||||
出版者 | 埼玉大学大学院理工学研究科 | |||||||||
出版者名(別言語) | ||||||||||
出版者 | Graduate School of Science and Engineering, Saitama University | |||||||||
形態 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | 77 p. | |||||||||
学位授与番号 | ||||||||||
学位授与番号 | 甲第935号 | |||||||||
学位授与年月日 | ||||||||||
学位授与年月日 | 2014-03-24 | |||||||||
学位名 | ||||||||||
学位名 | 博士(理学) | |||||||||
学位授与機関 | ||||||||||
学位授与機関識別子Scheme | kakenhi | |||||||||
学位授与機関識別子 | 12401 | |||||||||
学位授与機関名 | 埼玉大学 | |||||||||
抄録 | ||||||||||
内容記述タイプ | Abstract | |||||||||
内容記述 | Splicing of primary transcripts (pre-mRNA) removes non-coding intronic sequences and joins exonic sequences to make a mature mRNA that can be transported from the nucleus into the cytoplasm and translated into protein. Thus, splicing is a key mechanism for regular gene expression and interference with splicing will affect gene expression. Deregulation or inhibition of splicing will result in production of non-functional or even deleterious proteins. Recently, FR901464 and its methylated derivative Spliceostatin A (SSA) were reported for the first time to inhibit pre-mRNA splicing by binding non-covalently to the SF3b sub-complex in the U2 snRNP. SSA is chemically more stable than its parent molecule FR901464, which was originally isolated from a fermentation broth of Pseudomonas sp. During screening of microbial metabolites, FR901464 was found to activate viral promoters including those of the SV40 and the cytomegalovirus (CMV). Previous studies have shown that SSA leads to transcriptional repression of about 20% of expressed genes, while enhancing transcription of only a small subset. Among the most highly up-regulated genes was Interleukin 8 (IL-8). Since IL-8 and CMV promoters are activated by a similar set of transcription factors, I used luciferase reporter constructs of each promoter for a comparative study. IL-8 is a well-studied CXC family chemokine that activates and recruits leukocytes to the site of inflammation. Interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-α) and phorbol 12-myristate 13-acetate (PMA) are well known inducers of IL-8. These inducers can enhance the transcriptional stimulatory activity of viral promoters (SV40 and CMV) as well and are also able to activate cellular signaling kinases. Previous investigations suggested that IL-8 production is regulated by extracellular signal-regulated protein kinase (ERK) and transcription factor NF-kB in response to TNFα, IL-1β or PMA. To better understand the underlying mechanism we investigated both possible upstream signaling pathways as well as likely transcription factors responsible for IL-8 induction. The mitogen-activated protein kinases (MAPKs) are important signaling kinases that activate a variety of transcription factors through phosphorylation. Here I report that SSA treatment induces ERK activation and that SSA treatment leads to enhancement of NF-kB activity. |
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目次 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | Chapter 1: Introduction 1.1 What is splicing?........................................................11 1.2 Discovery of SSA and its derivatives…………………12 1.3 Other molecules targeting pre-mRNA Splicing………13 1.4 FR901464 induces ER stress…………………………14 1.5 SSA-induced gene activation……………………………15 1.6 Aim of this study…………………………………………16 Chapter 2: SSA enhances the transcriptional activation of IL-8 and CMV promoters 2.1 Introduction……………………………………………………20 2.2 Material and methods………………………………………22 2.2.1 Materials and cell lines……………………………22 2.2.2 Antibodies and immunoblotting……………………22 2.2.3 qPCR analysis………………………………………22 2.2.4 Expression and knockdown………………………23 2.3 Results………………………………………………………24 2.3.1 Effect of SSA on IL-8 and CMV promoter activity.24 2.3.2 Transcriptional activation in a time and dose-dependent manner……………………………………………25 2.3.3 Effect of splicing inhibitors and splicing inhibition on gene activation……………………………………25 2.4 Conclusion……………………………………………………27 Chapter 3: Role of ERK pathway in IL-8 and CMV promoters Up-regulation 3.1 Introduction…………………………………………………31 3.1.1 ERK pathway activation……………………………31 3.1.2 Abnormal activation of ERK leads to disease development…………………………………………31 3.1.3 Role in controlling apoptosis………………………32 3.1.4 Role in transcriptional activation…………………32 3.1.5 SSA and ERK pathway……………………………33 3.2 Material and methods………………………………………34 3.2.1 Materials, cell lines and antibodies………………34 3.2.2 Antibodies and immunoblotting……………………34 3.2.3 qPCR analysis……………………………………35 3.2.4 Expression and knockdown………………………35 3.3 Results…………………………………………………………36 3.3.1 SSA affects ERK activation………………………36 3.3.2 Effect of SAP knockdown on CMV promoter activity…………………………………………………37 3.4 Conclusion……………………………………………………38 Chapter 4: SSA affects NF-kB pathway 4.1 Introduction……………………………………………………41 4.2 Material and methods………………………………………43 4.2.1 Materials, cell lines and antibodies……………43 4.2.2 Antibodies and immunoblotting………………43 4.2.3 Immunofluorescence microscopy……………43 4.2.4 Expression and knockdown……………………44 4.3 Results…………………………………………………………45 4.3.1 Transcription factor point mutants reveals an essential role of NF-kB in the regulation of IL-8 and CMV promoters…………………………………45 4.3.2 SSA modulates the transcriptional activity through p65-NF-kB……………………………………………46 4.4 Conclusion……………………………………………………48 Chapter 5: SSA induces ER stress 5.1 Introduction…………………………………………………51 5.1.1 what is ER stress?..............................................51 5.1.2 Cellular responses combating with ER stress…51 5.1.3 FR901464 as an ER stress inducer……………53 5.2 Material and methods………………………………………55 5.2.1 Materials, cell lines and antibodies………………55 5.2.2 In vitro binding assay ………………………………55 5.2.3 RNA interference…………………………………55 5.3 Results………………………………………………………56 5.3.1 FR induces UPR…………………………………56 5.3.2 Identification of SSA binding proteins and activation of JNK………………………………………………57 5.3.3 SSA-induced IL-8 promoter activation occurs independent of TMX………………………………58 5.4 Conclusion……………………………………………………59 Overview…………………………………………………………………66 References References………………………………………………………67 |
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注記 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | 主指導教員 : 吉田稔 | |||||||||
版 | ||||||||||
[出版社版] | ||||||||||
著者版フラグ | ||||||||||
出版タイプ | VoR | |||||||||
出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||||
資源タイプ | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | text | |||||||||
フォーマット | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | application/pdf | |||||||||
作成日 | ||||||||||
日付 | 2015-02-04 | |||||||||
日付タイプ | Created | |||||||||
アイテムID | ||||||||||
GD0000525 |