Regulation of entry into replicative senescence : global spliceosomal deregulation and mitoribosome-linked telomere deprotection
- 주제(키워드) Cellular senescence , Alternative splicing , Splicesome , Mitochondrial dysfunction , Mitoribosome
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 윤계순
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031073
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
목차
Ⅰ. INTRODUCTION 1
Ⅱ. Part 1. Global spliceosome activity regulate entry into cellular senescence 18
A. INTRODUCTION 19
B. METRIALS AND METHODS 22
1. Cell culture, cell growth and development of cell senescence 22
2. Data processing and selection of senescence-associated genes 22
3. Acquisition of spliceosomal machinery gene list 24
4. Flow cytometric cell cycle analysis 24
5. SA-β-gal activity staining 25
6. Transfection of siRNAs into cells 26
7. Western blot analysis 26
8. Detection of splice variants by reverse transcription-polymerase chain reaction (RT-PCR) 27
9. Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) assay 28
10. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 29
11. Statistical analysis 29
C. RESULTS 30
1. Progressive global deregulation of spliceosomal genes during cellular senescence 30
2. Identification of a senescence-associated spliceosomal gene signature 34
3. Spliceosomal regulation triggers senescence 40
4. Sp1 is a key TF that regulates senescence-associated spliceosomal gene expression 45
D. DISCUSSION 53
Ⅲ. Part 2. Mitoribosomal defect is a primary regulator of cell senescence via telomere deprotection 60
A. INTRODUCTION 61
B. METRIALS AND METHODS 63
1. Cell culture, cell growth and development of cell senescence 63
2. Data processing and selection of senescence-associated genes 64
3. Determination of mitochondrial ROS level and monitoring cell size and cell granularity 65
4. SA-β-gal activity staining 65
5. Transfection of siRNAs into cells 66
6. Western blot analysis 66
7. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 66
8. Statistical analysis 67
C. RESULTS 68
1. Global deregulation of mitoribosomal gene expression is an early event of replicative senescence 68
2. Identification of a senescence-associated mitoribosomal gene signature 71
3. Mitoribosomal perturbation triggers senescence 74
4. Decreased TPP1 expression is another early event to drive cellular senescence 78
5. Mitoribosomal deregulation is an upstream event of transcriptional suppression of TPP1 80
D. DISCUSSION 82
Ⅳ. CONCLUSION 88
Ⅴ. REFERENCE 89
