1 Introduction ................................................. 1
1.1 p53 protein structure ................................... 1
1.2 Regulation of p53 levels and activity ................... 3
1.3 p53's activation outcome; p53-responsive genes .......... 6
1.4 p53 and cancer. Treatments avenues ...................... 7
1.5 Cancer. (Non)-oncogene addiction and the concept of
synthetic lethality ..................................... 8
1.6 Systematic approaches. Genome scale RNAi screens ....... 12
1.7 SMN complex. snRNP biogenesis .......................... 14
1.7.1 Spinal muscular atrophy (SMA) and SMN complex ... 15
1.7.2 SMN complex functions in snRNP biogenesis ....... 17
1.8 Rational and Aims of thesis ............................ 22
2 Results ..................................................... 23
2.1 Genome-scale Screen .................................... 23
2.1.1 Preface ......................................... 23
2.1.2 Screen assay .................................... 27
2.1.3 Assay evaluation ................................ 29
2.1.4 Screen flow ..................................... 30
2.1.5 Data analysis ................................... 31
2.2 Hits validation ........................................ 35
2.3 Description of the genes whose knockdown decreased
the WT/KO ratio ........................................ 37
2.4 Description of the genes whose knock down increases
the WT/KO ratio ........................................ 39
2.5 Gene characterization strategies ....................... 42
2.6 Characterization of STRAP/UNRIP RNAi phenotype ......... 44
2.6.1 Background ...................................... 44
2.6.2 UNRIP RNAi phenotype. Time-lapse analysis ....... 45
2.6.3 Investigation of TGFβ signaling upon UNRIP
depletion ....................................... 48
2.6.4 Stable UNRIP-LAP ВАС cell lines.
Immunoprecipitation (IP) and Mass-Spectrometry
(MS) analysis ................................... 52
2.6.5 Quantitative MS analysis via SILAC .............. 57
2.6.6 SMN complex behavior upon UNRIP depletion ....... 61
2.6.7 SMN localization rescue experiment .............. 65
2.6.8 Investigation of possible molecular mechanisms
underlying the SMN phenotype .................... 66
3 Discussion .................................................. 73
3.1 Genome-scale screen for synthetic lethal interactions
with TP53 loss-of-function ............................. 73
3.1.1 Screen improvements ............................. 74
3.1.2 Data analysis ................................... 75
3.2 Screen hits ............................................ 75
3.2.1 Hits validation ................................. 75
3.2.2 Hits whose knockdown lead to decrease of the
WT cell number (p53 modulators) ................. 76
3.2.3 Hits whose knockdown lead to decrease of
the TP53KO cell number (synthetic lethal to
TP53 loss-of-mnction) ........................... 77
3.3 Detailed investigation of UNRIP RNAi phenotype ....... 79
3.3.1 TGFβ-signaling upon UNRIP depletion ............. 79
3.3.2 UNRIP RNAi influence over the SMN complex ....... 80
4 Materials and Methods ....................................... 89
4.1 General solutions, buffers and media ................... 89
4.1.1 Materials ....................................... 89
4.1.2 Buffer recipes .................................. 89
4.1.3 Bacteria medium recipes ......................... 90
4.2 DNA cloning ............................................ 91
4.2.1 Purification of plasmid DNA ..................... 91
4.2.2 Restriction enzyme digestion .................... 91
4.2.3 DNA gel electrophoresis ......................... 92
4.2.4 Polymerase Chain reaction (PCR) ................. 92
4.2.5 Ligation of DNA ................................. 92
4.3 ВАС trasngeneomics ..................................... 93
4.4 СеЛ Culture ............................................ 95
4.4.1 Materials ....................................... 95
4.4.2 Cell culturing .................................. 95
4.4.3 Cell splitting .................................. 95
4.4.4 Generation of stable cell lines ................. 96
4.4.5 Cell freezing ................................... 96
4.4.6 Cell thawing .................................... 97
4.5 esiRNA ................................................. 98
4.5.1 esiRNA library preparation ...................... 98
4.5.2 esiRNA transfection ............................. 98
4.6 Performing the genome-scale screen .................... 101
4.6.1 Materials ...................................... 101
4.6.2 Screening procedure ............................ 101
4.6.3 Data analysis .................................. 102
4.7 Examination of gene knockdown on mRNA level ........... 104
4.7.1 Isolation of total mRNA and complementary DNA
synthesis ...................................... 104
4.7.2 Quantitative RT-PCR ............................ 104
4.8 Luciferase luminescence assay ......................... 106
4.8.1 Materials ...................................... 106
4.8.2 Constructs transfection ........................ 106
4.8.3 Luciferase luminescence measurement ............ 106
4.9 Immunoprecipitation with anti-GFP antibody ............ 108
4.9.1 Materials and solutions ........................ 108
4.9.2 Preparation of cell extract .................... 109
4.9.3 Coupling of anti-GFP antibody to G Sepharose
beads .......................................... 110
4.9.4 Immunoprecipitation protocol ................... 110
4.9.5 Analysis of immuno-purified complexes .......... 11l
4.9.6 Stable isotope labeling with amino acids in
cell culture (SILAC) ........................... 114
4.10 Immunoprecipitation of RNPs from tissue cultured
cells ................................................. 116
4.10.1 Materials and solutions ........................ 116
4.10.2 Cell labeling .................................. 116
4.10.3 RNP immunoprecipitation ........................ 117
4.10.4 Denaturing PAGE ................................ 118
4.10.5 Autoradiogram .................................. 118
4.11 Imaging ............................................... 119
4.11.1 Immunofluorescent staining ..................... 119
4.11.2 Live-cell imaging .............................. 120
4.12 Cell fractionation ................................... 121
5 Supplemental information ................................... 122
6 References ................................................. 135
7 Acknowledgements ........................................... 145
8 Declaration ................................................ 147
Figure 1. p53 structure and p53 pathway ........................ 4
Figure 2. snRNP biogenesis pathway ............................ 19
Figure 3. p53 pathway activation in HCT116 WT cells ........... 25
Figure 4. Screen flow ......................................... 28
Figure 5. Plate correction procedure .......................... 34
Figure 6. Genome-scale data distribution and hit selection .... 36
Figure 7. Schematic representation of UNRIP's domain
structure ........................................... 45
Figure 8. The UNRIP RNAi phenotype ............................ 46
Figure 9. UNRIP depletion influence overTGFp signaling ........ 51
Figure 10. UNRIP-LAP immunoprecipitation ....................... 54
Figure 11. Quantitative mass-spectrometry of UNRIP complexex
via SILAC ........................................... 51
Figure 12. SMN complex localization upon UNRIP depletion ....... 59
Figure 13. SMN localization rescue experiment .................. 63
Figure 14. SMN1 and SmB levels upon UNRIP depletion ............ 66
Figure 15. snRNP assembly rate upon UNRIP depletion ............ 69
Figure 16. SMN1 nucleo-cytoplasmic distribution upon UNRIP
depletion ........................................... 71
Figure 17. Model of the mechanism underlying UNRIP depletion
phenotype ........................................... 85
Table 1. Enrichment analysis for the genes whose knockdown
decrease the WT/KO ratio ............................ 38
Table 2. Brief summary for the genes whose knockdown
increased the WT/KO ratio ........................... 40
Table 3. Summary of MS results for UNRIP-LAP immune
complexes ........................................... 56
Table 4. Summary of the used antibiotics ..................... 90
Table 5. Summary of the used plasmids ........................ 91
Table 6. UNRIP constructs used in ET cloning ................. 93
Table 7. Tagging and checking primers used for UNRIP ET
cloning ............................................. 94
Table 8. Table for scaling up or down transfection volumes ... 99
Table 9. Primers used in RT-PCR reactions ................... 105
Supplemental table 1. Table of genes whose knock down leads
to decreased viability of HCT116 WT cells versus TP53KO
cells ...................................................... 122
Supplemental table 2 Table of gene-specific primers for
primary esiRNA production of the genes that decrease
WT/KO ratio ................................................ 124
Supplemental table 3. Table of gene-specific primers for
secondary esiRNA production of the genes that decrease
WT/KO ratio ................................................ 126
Supplemental table 4. Table of gene-specific primers for
primary esiRNA production of the genes that increase
WT/KO ratio ................................................ 128
Supplemental table 5. Table of gene-specific primers for
secondary esiRNA production of the genes that increase
WT/KO ratio ................................................ 128
Supplemental table 6. Table summarizing the SILAC ratios ...... 128
Supplemental table 7. Table of the used antibodies ............ 133
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