Interaction of the C9orf72-Amyotrophic Lateral Sclerosis-Related Proline–Arginine Dipeptide Repeat Protein with the RNA-Binding Protein NOVA1 Causes Decreased Expression of UNC13A Due to Enhanced Inclusion of Cryptic Exons, Which Is Reversed by Betulin Treatment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals, Media, and Neuron-like Cell Line Maintenance
2.2. Processing of Small RNA Interference in SH-SY5Y and SK-N-DZ Cells
2.3. Western Blotting of SH-SY5Y and SK-N-DZ Cells
2.4. Total RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) of SH-SY5Y and SK-N-DZ Cells
2.5. Recombinant Plasmid Construction and Transfection
2.6. Yeast Two-Hybrid Screening of PR50-Associated Protein
2.7. Yeast Two-Hybrid Assay for PR50 and NOVA1
2.8. Co-Immunoprecipitation Analysis of 293T Cells
2.9. Immunofluorescence Staining of PR50-Expressing SH-SY5Y Cells
2.10. Screening Inhibitors of the Interaction between GA50 and NOVA1 Using a Yeast Two-Hybrid-Based Growth Assay
2.11. Treatment with Betulin and Cell Viability Analysis of SH-SY5Y and SK-N-DZ Cells
2.12. Statistical Methods Used in This Study
3. Results
3.1. PR-DPR Expression Promotes the Inclusion of Cryptic Exon (CE) in UNC13A mRNA in Neuronal Cell Lines, Resulting in a Decrease in the Expression of UNC13A Protein
3.2. RNA-Binding Protein NOVA1 Is a Specific Interaction Partner of PR-DPR
3.3. PR-DPR Interacts with the Amino Acid Fragment at the C Terminus of NOVA1
3.4. CE Inclusion of UNC13A mRNA in Neuronal Cell Lines Will Be Significantly Increased by Knockdown of NOVA1, Resulting in Downregulation of UNC13A Protein Expression
3.5. Betulin (BT) Can Directly Interfere with the Interaction between PR-DPR and NOVA1 in the Yeast Two-Hybrid Model
3.6. BT treatment Can Significantly Inhibit the CE Inclusion of UNC13 A mRNA in Neuronal Cell Lines to Avoid Downregulation of UNC13 A Protein Expression
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fu, R.-H.; Chen, H.-J.; Hong, S.-Y. Interaction of the C9orf72-Amyotrophic Lateral Sclerosis-Related Proline–Arginine Dipeptide Repeat Protein with the RNA-Binding Protein NOVA1 Causes Decreased Expression of UNC13A Due to Enhanced Inclusion of Cryptic Exons, Which Is Reversed by Betulin Treatment. Cells 2023, 12, 2476. https://doi.org/10.3390/cells12202476
Fu R-H, Chen H-J, Hong S-Y. Interaction of the C9orf72-Amyotrophic Lateral Sclerosis-Related Proline–Arginine Dipeptide Repeat Protein with the RNA-Binding Protein NOVA1 Causes Decreased Expression of UNC13A Due to Enhanced Inclusion of Cryptic Exons, Which Is Reversed by Betulin Treatment. Cells. 2023; 12(20):2476. https://doi.org/10.3390/cells12202476
Chicago/Turabian StyleFu, Ru-Huei, Hui-Jye Chen, and Syuan-Yu Hong. 2023. "Interaction of the C9orf72-Amyotrophic Lateral Sclerosis-Related Proline–Arginine Dipeptide Repeat Protein with the RNA-Binding Protein NOVA1 Causes Decreased Expression of UNC13A Due to Enhanced Inclusion of Cryptic Exons, Which Is Reversed by Betulin Treatment" Cells 12, no. 20: 2476. https://doi.org/10.3390/cells12202476
APA StyleFu, R.-H., Chen, H.-J., & Hong, S.-Y. (2023). Interaction of the C9orf72-Amyotrophic Lateral Sclerosis-Related Proline–Arginine Dipeptide Repeat Protein with the RNA-Binding Protein NOVA1 Causes Decreased Expression of UNC13A Due to Enhanced Inclusion of Cryptic Exons, Which Is Reversed by Betulin Treatment. Cells, 12(20), 2476. https://doi.org/10.3390/cells12202476