Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy
Abstract
:1. Introduction
2. Material and Methods
2.1. Cell Culture
2.2. Lentivirus Generation, Lentiviral Transfection
2.3. CRISPR CAS9 Knockout/Knockin Kit
2.4. FACs Single Cell Sorting
2.5. RNA Isolation and cDNA Synthesis
2.6. qPCR Assay Validation
2.7. Real-Time qPCR Standard Curve Analysis
2.8. Western Blot Analysis
2.9. Immunofluorescence Staining
2.10. Transfection and Luciferase Assay (Plasmids)
2.11. Luciferase Assay with mRNA Transfection
2.12. Proliferation Analysis
2.13. Protein Synthesis Assay (OPP Labeling)
2.14. BisANS Assay
2.15. Northern Blot Analysis
2.16. Ratio Analysis of Multiple Precursors (RAMP)
2.17. Polysomal Profiling
2.18. Carbonylation Assay
2.19. Statistical Analysis
3. Results
3.1. Generation of Knockout and Knockdown Cells
3.2. Reduced TTDN1 and RNF113A Impact on the RNA Polymerase I Transcription Factor UBF
3.3. rRNA Transcription and Ribosomal Subunits Are Differentially Affected in the TTDN1 Knockout and RNF113A Knockdown Cells
3.4. The Accuracy of the Translation Process at the Ribosome Is Decreased in TTDN1 and RNF113A Cells
3.5. Elevated Misfolded Proteins and a Reduced Protein Synthesis in the Affected Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Catalog | Source Clone ID | Sequence in 5′-3′ | |
---|---|---|---|
RNF113A-shRNA1 | V3SH11240-226743083 | V3SVHS00_6680731 | CAATGGCGTCTTCAATCCA |
RNF113A-shRNA2 | V3SH11240-230736642 | V3SVHS00_10674292 | GCGAAAGAATTGATTGCTA |
RNF113A-shRNA3 | V3SH11240-230369254 | V3SVHS00_10306903 | CCCGAGCATCTACGTGCCA |
Scrambled control | SO-2952232G | SVC17010402 | - |
Reagents | Well 1 | Well 2 | Well 3 |
---|---|---|---|
Donor | 1 µg | 1 µg | 1 µg |
gRNA (1 µg each) | gRNA1 | gRNA2 | Scramble Control |
Opti-MEM | 250 µL | 250 µL | 250 µL |
TurboFectin | 6 µL | 6 µL | 6 µL |
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Zhu, G.; Khalid, F.; Zhang, D.; Cao, Z.; Maity, P.; Kestler, H.A.; Orioli, D.; Scharffetter-Kochanek, K.; Iben, S. Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy. Cells 2023, 12, 1877. https://doi.org/10.3390/cells12141877
Zhu G, Khalid F, Zhang D, Cao Z, Maity P, Kestler HA, Orioli D, Scharffetter-Kochanek K, Iben S. Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy. Cells. 2023; 12(14):1877. https://doi.org/10.3390/cells12141877
Chicago/Turabian StyleZhu, Gaojie, Fatima Khalid, Danhui Zhang, Zhouli Cao, Pallab Maity, Hans A. Kestler, Donata Orioli, Karin Scharffetter-Kochanek, and Sebastian Iben. 2023. "Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy" Cells 12, no. 14: 1877. https://doi.org/10.3390/cells12141877