Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction of the Intermediate Plasmids Containing Two PPR Repeats for the Golden Gate Assembly of the Designer PPR Protein Gene
2.2. Construction of the Expression Vector
2.3. Golden Gate Assembly of Designer PPR Gene
2.4. Expression and Purification of Recombinant MS2 Protein
2.5. RNA-Binding Protein ELISA (RBP-ELISA)
2.6. Electrophoresis Mobility Shift Assay (EMSA)
2.7. Gel Filtration Assay
2.8. Transfections
2.9. RNA Extraction, Reverse-Transcription, and RT-PCR
2.10. Observation by Fluorescent Microscopy
2.11. Construction of Stable Cells and Dox Induction
3. Results
3.1. The Construction Method for the Designer PPR Proteins That Bind to Target RNA Sequences
3.1.1. Scaffolds of the Designer PPRs
3.1.2. Establishment of a High-Throughput Cloning System for Designer PPRs
3.1.3. Establishment of a High-Throughput Cloning System for Designer PPRs
3.1.4. Evaluation of the RNA Binding Affinities and Selectivities of the Designer PPR Proteins
3.1.5. Evaluation of the Versatility of the Designer PPR Proteins of the PPR2.0 Scaffold
3.2. Target Splicing Control Using Artificially Constructed PPR Proteins
3.2.1. Enhancement of Exon Skipping of a Reporter Gene by Designer PPR Proteins in HEK293T Cells
3.2.2. Splicing Control of Endogenous mRNA by Designer PPR Proteins in HEK293T Cells
4. Discussion
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Number of PPR Motifs | Binding Affinity 1 (KD) | Sequence of PPR Scaffold | Reference |
---|---|---|---|---|
PPR2.0_A | 18 | This study | VVTYTTLIDGLCKAGDVDEALELFKEMRSKGVKPN | This study |
PPR2.0_C | 18 | This study | VVTYNTLIDGLCKSGKIEEALKLFKEMEEKGITPS | This study |
PPR2.0_G | 18 | This study | VVTYTTLIDGLCKAGKVDEALELFDEMKERGIKPD | This study |
PPR2.0_U | 18 | This study | VVTYNTLIDGLCKAGRLDEAEELLEEMEEKGIKPD | This study |
dPPR SCD | 10 | 90 nM | VVTYXTLIDGLCKAGKLDEALKLFEEMVEKGIKPX | [26,27,28] |
11 | 7.5 nM | VVTYXTLISGLGKAGRLEEALELFEEMKEKGIVPX | ||
14 | 16 nM | VVTYXTLIDGLAKAGRLEEALQLFQEMKEKGVKPX | ||
cPPR | 8 | 370 nM | VVTYTTLIDAFCRKGRLDEALSLFSEMKSKGIKPN | [25] |
synthPPR | 4 | No data | VVTYNTLISGFCKAGRLEEAMSLFSEMKSKGLVPS | [29] |
MCD_A | 14 | 18 nM | VVTYTILIDALCKAGRLEEALSLFSEMKEIGIKPD | [28] |
MCD_C | VVTYNILIKGLCKAGKLEEALSLLSEMVEKGIQPD | |||
MCD_G | VVTYNTLIDGLCKSGKIEEALKLFKEMEEKGITPS | |||
MCD_U | VVTYTTLIDGLCKAGKVDEALELFDEMKERGIKPD |
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Yagi, Y.; Teramoto, T.; Kaieda, S.; Imai, T.; Sasaki, T.; Yagi, M.; Maekawa, N.; Nakamura, T. Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells. Cells 2022, 11, 3529. https://doi.org/10.3390/cells11223529
Yagi Y, Teramoto T, Kaieda S, Imai T, Sasaki T, Yagi M, Maekawa N, Nakamura T. Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells. Cells. 2022; 11(22):3529. https://doi.org/10.3390/cells11223529
Chicago/Turabian StyleYagi, Yusuke, Takamasa Teramoto, Shuji Kaieda, Takayoshi Imai, Tadamasa Sasaki, Maiko Yagi, Nana Maekawa, and Takahiro Nakamura. 2022. "Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells" Cells 11, no. 22: 3529. https://doi.org/10.3390/cells11223529
APA StyleYagi, Y., Teramoto, T., Kaieda, S., Imai, T., Sasaki, T., Yagi, M., Maekawa, N., & Nakamura, T. (2022). Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells. Cells, 11(22), 3529. https://doi.org/10.3390/cells11223529