Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system
Abstract We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a pr...
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Nature Portfolio
2024-03-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-55947-0 |
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| author | Marine Le Boulch Eric Jacquet Naïma Nhiri Maya Shmulevitz Philippe H. Jaïs |
| author_facet | Marine Le Boulch Eric Jacquet Naïma Nhiri Maya Shmulevitz Philippe H. Jaïs |
| author_sort | Marine Le Boulch |
| collection | DOAJ |
| description | Abstract We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3′UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5–3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy. |
| first_indexed | 2024-03-07T15:03:01Z |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-07T15:03:01Z |
| publishDate | 2024-03-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj.art-17601f11857d42479a9305bebdb3dc1c2024-03-05T19:02:22ZengNature PortfolioScientific Reports2045-23222024-03-0114112110.1038/s41598-024-55947-0Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 systemMarine Le Boulch0Eric Jacquet1Naïma Nhiri2Maya Shmulevitz3Philippe H. Jaïs4Eukarÿs SASInstitut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-SaclayInstitut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-SaclayMedical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of AlbertaEukarÿs SASAbstract We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3′UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5–3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.https://doi.org/10.1038/s41598-024-55947-0C3P3mRNAPolyadenylationPoly(A) polymeraseArtificial expression system |
| spellingShingle | Marine Le Boulch Eric Jacquet Naïma Nhiri Maya Shmulevitz Philippe H. Jaïs Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system Scientific Reports C3P3 mRNA Polyadenylation Poly(A) polymerase Artificial expression system |
| title | Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system |
| title_full | Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system |
| title_fullStr | Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system |
| title_full_unstemmed | Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system |
| title_short | Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system |
| title_sort | rational design of an artificial tethered enzyme for non templated post transcriptional mrna polyadenylation by the second generation of the c3p3 system |
| topic | C3P3 mRNA Polyadenylation Poly(A) polymerase Artificial expression system |
| url | https://doi.org/10.1038/s41598-024-55947-0 |
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