@phdthesis{oai:sucra.repo.nii.ac.jp:00010308,
 author = {KHALID, KHAN},
 month = {},
 note = {77 p., 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., 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, 主指導教員 : 吉田稔, text, application/pdf},
 school = {埼玉大学},
 title = {Chemical Biology of Splicing Inhibitors},
 year = {2014},
 yomi = {カリド, カーン}
}