The NMD Pathway Regulates GABARAPL1 mRNA during the EMT
Abstract
:1. Introduction
2. Materials and Methods
2.1. Antibodies, Vectors, Chemicals and siRNAs
2.2. Cell Culture and Transfection
2.3. Western Blotting
2.4. RNA Extraction and RT-qPCR
2.5. RNA-FISH
2.6. Immunohistochemistry and Immunostaining Assessment
2.7. RNA-Immunoprecipitation (RIP)
2.8. Statistical Analysis
3. Results
3.1. The EMT was Associated with A Loss of UPF1 Expression as well as GABARAPL1 Overexpression
3.2. GABARAPL1 Transcript Increased upon NMD Inhibition
3.3. UPF1 Was Associated with GABARAPL1 mRNAs
3.4. The 3′UTR of GABARAPL1 mRNAs Contributed to Its Degradation by The NMD
3.5. eIF4A3 Regulated GABARAPL1 mRNA Degradation via Its 3′UTR
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Target Sequence |
---|---|
siUPF1 | 5′-GATGCAGTTCCGCTCCATT-3′ |
siSMG1 | 5′-GTGTATGTGCGCCAAAGTA-3′ |
sieIF4A3 | 5′-AGACATGACTAAAGTGGAA-3′ |
siSTAU1 | 5′-CCUAUAACUACAACAUGAG-3′ |
Gene | Forward Primer | Reverse Primer |
---|---|---|
18S | 5′-GTAACCCGTGAACCCCATT-3′ | 5′-CCATCCAATCGGTAGTAGCG-3′ |
H3B2 | 5′-GCTAGCTGGATGTCTTTTGG-3′ | 5′-GTGGTAAAGCACCCAGGAA-3′ |
UPF1 | 5′-CGGAAAGCAGCAGTGTACCA-3′ | 5′-GGCAGAAGGGAGTGTGTCTGA-3′ |
UPF2 | 5′-GAAGATGAGGAAGCCAGCAC-3′ | 5′-CCCGTACCAACTTCTTCCTG-3′ |
UPF3A | 5′-GAAAAGAGCGACTGGCAAAC-3′ | 5′-TGCCTTGCAGATCCTCCTG-3′ |
UPF3B | 5′-AAGGGGGAAGATAAGCAGGA-3′ | 5′-GTTCCTGAAGCTGCTCCTTG-3′ |
SMG1 | 5′-CAAGCGATGTCAGCAGATGT-3′ | 5′-TGCTGACAAAAGCCATTCAG-3′ |
GABARAPL1 | 5′-CCCTCCCTTGGTTATCATCCA-3′ | 5′-ACTCCCACCCCACAAAATCC-3′ |
GABARAP | 5′-GCCTTTCCCATCCTGCTGTA-3′ | 5′-GGAAGGGATTGCTGGGTTCT-3′ |
GABARAPL2 | 5′-TAGTGGCCACAATGACCAGA-3′ | 5′-TGAACACAGCTTTGGTCCAG-3′ |
LC3B | 5′-CGGAAAGCAGCAGTGTACCA-3′ | 5′-GGCAGAAGGGAGTGTGTCTGA-3′ |
ATF4 | 5′-CCAAGCACTTCAAACCTCATG-3′ | 5′-ATCCATTTTCTCCAACATCCAATC-3′ |
ATG5 | 5′-AGGATCAGAATGCAGGGAAC-3′ | 5′-AAGGCCTGACACTGGTTTTG-3′ |
SC35 | 5′-CGGTGTCCTCTTAAGAAAATGATGTA-3′ | 5′-CTGCTACACAACTGCGCCTTTT-3′ |
SF3B5 | 5′-ACCGCTACACCATCCATAGC-3′ | 5′-AGGCTGAAGCATCTTTTCCA-3′ |
GFP | 5′-TCCTGGTCGAGCTGGACGG-3′ | 5′-CAGCTTGCCGTAGGTGGCAT-3′ |
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Baudu, T.; Parratte, C.; Perez, V.; Ancion, M.; Millevoi, S.; Hervouet, E.; Peigney, A.; Peixoto, P.; Overs, A.; Herfs, M.; et al. The NMD Pathway Regulates GABARAPL1 mRNA during the EMT. Biomedicines 2021, 9, 1302. https://doi.org/10.3390/biomedicines9101302
Baudu T, Parratte C, Perez V, Ancion M, Millevoi S, Hervouet E, Peigney A, Peixoto P, Overs A, Herfs M, et al. The NMD Pathway Regulates GABARAPL1 mRNA during the EMT. Biomedicines. 2021; 9(10):1302. https://doi.org/10.3390/biomedicines9101302
Chicago/Turabian StyleBaudu, Timothée, Chloé Parratte, Valérie Perez, Marie Ancion, Stefania Millevoi, Eric Hervouet, Anne Peigney, Paul Peixoto, Alexis Overs, Michael Herfs, and et al. 2021. "The NMD Pathway Regulates GABARAPL1 mRNA during the EMT" Biomedicines 9, no. 10: 1302. https://doi.org/10.3390/biomedicines9101302
APA StyleBaudu, T., Parratte, C., Perez, V., Ancion, M., Millevoi, S., Hervouet, E., Peigney, A., Peixoto, P., Overs, A., Herfs, M., Fraichard, A., Guittaut, M., & Baguet, A. (2021). The NMD Pathway Regulates GABARAPL1 mRNA during the EMT. Biomedicines, 9(10), 1302. https://doi.org/10.3390/biomedicines9101302