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22대 대한민국 국회 국회정보길라잡이 국회의원검색
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소장정보 검색

국회도서관 홈으로 정보검색 소장정보 검색
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Title Page

Contents

ABBREVIATIONS 9

ABSTRACT 10

INTRODUCTION 12

MATERIALS AND METHODS 19

Zebrafish husbandry 19

Generation of fam50a KO zebrafish 19

Transient suppression of fam50a and embryo microinjections 20

Molecular cloning of FAM50A plasmids and in vitro transcription 21

Zebrafish phenotyping 22

Whole-mount in situ hybridization (WISH). 22

Whole-mount immunostaining. 22

Craniofacial phenotyping. 23

Whole-mount TUNEL assay. 24

Mitotic cell cycle progression. 25

RNAseq of fam50a mutant zebrafish 25

RNA preparation. 25

Sequencing library preparation. 26

RNA-seq data analysis. 26

Results 28

fam50a KO zebrafish display patient-relevant phenotypes 28

In vivo complementation studies in zebrafish indicate that human FAM50A variants are hypomorphic 43

Transcriptomic analysis of fam50a zebrafish models reveal discrete biological groups of diminished function 50

fam50a KO transcriptomes are enriched for mRNA miss-splicing events 60

FAM50A interacts directly with spliceosome U5 and C complex proteins 66

Discussion 70

REFERENCES 74

SUMMARY (IN KOREAN) 85

List of Figures

Figure 1. Genes with identified mutations that cause syndromal XLID with chromosomal band location. 15

Figure 2. Progress in identifying XLID syndromes and associated genes, 1988-2017. 16

Figure 3. Photographs of available affected males. 17

Figure 4. Clinical manifestations in affected males. 18

Figure 5. FAM50A is conserved across eukaryotic phyla and missense variants are located in conserved regions. 32

Figure 6. fam50a is expressed during early zebrafish development and was targeted using CRISPR/Cas9 to generate fam50a KO zebrafish. 34

Figure 7. Generation of zebrafish fam50a mutants using CRISPR/Cas9 technology. 35

Figure 8. fam50a KO zebrafish larvae display central nervous system and craniofacial patterning defects. 37

Figure 9. In situ expression of cell proliferation markers in fam50a KO zebrafish larvae is depleted at 3 days post-fertilization (dpf). 38

Figure 10. fam50a KO zebrafish larvae display central nervous system and craniofacial patterning defects. 40

Figure 11. fam50a KO zebrafish larvae display normal blood vessel patterning up to 2.5 days post-fertilization (dpf). 42

Figure 12. fam50a is targeted efficiently and specifically by a splice-blocking morpholino (MO). 46

Figure 13. fam50a suppression in zebrafish larvae disrupts cartilage patterning and in vivo complementation assays indicate that FAM50A missense variants... 48

Figure 14. In vivo complementation assays using fam50a KO zebrafish indicate that FAM50A missense variants confer a partial loss of function. 49

Figure 15. FAM50A protein localizes to the nucleus in mammalian cells. 54

Figure 16. RNA-seq analysis revealed disruption of functionally relevant pathways and mRNA splicing defects in fam50a KO zebrafish. 55

Figure 17. RNA-seq analysis revealed disruption of functionally relevant pathways and mRNA splicing defects in fam50a KO zebrafish. 57

Figure 18. Expression pattern of major spliceosome effectors is upregulated in fam50a KO zebrafish. 58

Figure 19. Validation of additional genes whose transcript levels were altered in RNAseq analysis of fam50a KO zebrafish. 59

Figure 20. fam50a suppression in zebrafish results in apoptosis and altered cell cycle progression. 64

Figure 21. Phenotypic analysis of fam50a KO zebrafish in a tp53 KO background. 65

Figure 22. Co-immunoprecipitation assays to validate FAM50A interaction with U5 and C complex proteins. 69

초록보기

 1991년 처음 보고된 발달장애/지적장애 관련 Armfield syndrome은 남성에서만 발견되는 X-염색체 연관으로 추정되어 왔다. 그동안의 환자 가계도 분석 및 exome sequencing을 통해 Armfield syndrome의 원인유전자가 Xq28 locus에 8 Mb 근처에 존재하는 것으로 유전자 지도를 좁혀왔으며, 최근에서야 미지의 FAM50A 유전자에서 돌연변이를 발견하여 Armfield syndrome 원인의 후보유전자로 검토하게 되었다. 후보유전자의 생물학적인 검증을 위하여 CRISPR-Cas9 시스템으로 fam50a 유전자 녹아웃 (knockout, KO) 제브라피쉬를 제작하였다. fam50a KO 제브라피쉬는 Armfield syndrome 환자의 발달장애와 유사하게 안면기형 및 비정상적인 신경발생 표현형을 보임을 확인하였다. FAM50A 관련 분자신호전달 규명 및 기능 연구를 위해 fam50a KO 제브라피쉬에서 RNA-seq analysis를 수행한 결과 신경발달에 주요한 다수의 전사체들의 발현변화가 관찰되었고, 특히 mRNA의 3' alternative splicing events의 비정상적인 변화를 확인하였다. 이를 통해 FAM50A가 생체 내에서 spliceosome의 구성과 기능에 관여할 것으로 예측하였고, co-immunoprecipitation assays를 통한 단백질체 분석으로 FAM50A 단백질과 spliceosome C complex 단백질들 사이의 직접적인 결합을 확인하였다. 본 연구를 통하여 최종적으로 FAM50A 유전자가 Armfield syndrome의 원인유전자임을 규명하게 되었으며, mRNA splicing complex의 단백질들이 발달장애 및 지적장애의 주요 원인이 될 수 있다는 새로운 spliceopathy 개념을 처음으로 도입하게 되었다.

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