Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation
Abstract
:1. Introduction
2. Materials and Methods
2.1. The Small Interfering RNAs (siRNA) Description and Lipid Nanoparticles (LNP) Formulation
2.2. Cell Culture and Transfection
2.3. RNA Isolation and RT-qPCR
2.4. Anti-m6A Immunoprecipitation
2.5. Formaldehyde-Crosslinked RNA-Immunoprecipitation (RIP)
2.6. Nascent RNA Purification and cDNA Preparation
2.7. Separation of Nuclear and Cytoplasmic Cell Fractions
2.8. Fluorescent in Situ Hybridization (FISH)
2.9. Estimation of rRNA Precursors’ Lifetime
2.10. Western Blotting
2.11. Ligation of DNA Probes Using RNA Template
2.12. Polysome Profiling
2.13. Bioinformatic Analysis
2.14. Statistical Analysis of the Experimental Data
3. Results
3.1. Mettl3 Methyltransferase Binds to 47S Pre-rRNA and Modifies Adenosine 196
3.2. Knockdown of Mettl3 Methyltransferase Increases Rates of Pre-rRNA Maturation
3.3. Influence of Mettl3 Methyltransferase Knockdown on Pre-rRNA Processing Factors
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sergeeva, O.; Sergeev, P.; Melnikov, P.; Prikazchikova, T.; Dontsova, O.; Zatsepin, T. Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation. Cells 2020, 9, 1061. https://doi.org/10.3390/cells9041061
Sergeeva O, Sergeev P, Melnikov P, Prikazchikova T, Dontsova O, Zatsepin T. Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation. Cells. 2020; 9(4):1061. https://doi.org/10.3390/cells9041061
Chicago/Turabian StyleSergeeva, Olga, Philipp Sergeev, Pavel Melnikov, Tatiana Prikazchikova, Olga Dontsova, and Timofei Zatsepin. 2020. "Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation" Cells 9, no. 4: 1061. https://doi.org/10.3390/cells9041061
APA StyleSergeeva, O., Sergeev, P., Melnikov, P., Prikazchikova, T., Dontsova, O., & Zatsepin, T. (2020). Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation. Cells, 9(4), 1061. https://doi.org/10.3390/cells9041061