Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria
The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expr...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2024-02-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/91777 |
| _version_ | 1797304949963489280 |
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| author | Roswitha Dolcemascolo María Heras-Hernández Lucas Goiriz Roser Montagud-Martínez Alejandro Requena-Menéndez Raúl Ruiz Anna Pérez-Ràfols R Anahí Higuera-Rodríguez Guillermo Pérez-Ropero Wim F Vranken Tommaso Martelli Wolfgang Kaiser Jos Buijs Guillermo Rodrigo |
| author_facet | Roswitha Dolcemascolo María Heras-Hernández Lucas Goiriz Roser Montagud-Martínez Alejandro Requena-Menéndez Raúl Ruiz Anna Pérez-Ràfols R Anahí Higuera-Rodríguez Guillermo Pérez-Ropero Wim F Vranken Tommaso Martelli Wolfgang Kaiser Jos Buijs Guillermo Rodrigo |
| author_sort | Roswitha Dolcemascolo |
| collection | DOAJ |
| description | The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control. |
| first_indexed | 2024-03-08T00:18:48Z |
| format | Article |
| id | doaj.art-2f7dd1c6b8fa456f82d0226daa76c65a |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-08T00:18:48Z |
| publishDate | 2024-02-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-2f7dd1c6b8fa456f82d0226daa76c65a2024-02-16T14:47:22ZengeLife Sciences Publications LtdeLife2050-084X2024-02-011210.7554/eLife.91777Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteriaRoswitha Dolcemascolo0María Heras-Hernández1Lucas Goiriz2Roser Montagud-Martínez3Alejandro Requena-Menéndez4Raúl Ruiz5Anna Pérez-Ràfols6R Anahí Higuera-Rodríguez7Guillermo Pérez-Ropero8Wim F Vranken9https://orcid.org/0000-0001-7470-4324Tommaso Martelli10Wolfgang Kaiser11Jos Buijs12Guillermo Rodrigo13https://orcid.org/0000-0002-1871-9617Institute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, Spain; Department of Biotechnology, Polytechnic University of Valencia, Valencia, SpainInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, SpainInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, Spain; Department of Applied Mathematics, Polytechnic University of Valencia, Valencia, SpainInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, Spain; Department of Biotechnology, Polytechnic University of Valencia, Valencia, SpainInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, SpainInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, SpainGiotto Biotech SRL, Sesto Fiorentino, Italy; Magnetic Resonance Center (CERM), Department of Chemistry Ugo Schiff, Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (CIRMMP), University of Florence, Sesto Fiorentino, ItalyDynamic Biosensors GmbH, Planegg, Germany; Department of Physics, Technical University of Munich, Garching, GermanyRidgeview Instruments AB, Uppsala, Sweden; Department of Chemistry – BMC, Uppsala University, Uppsala, SwedenStructural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium; Interuniversity Institute of Bioinformatics in Brussels, Université Libre de Bruxelles – Vrije Universiteit Brussel, Brussels, BelgiumGiotto Biotech SRL, Sesto Fiorentino, ItalyDynamic Biosensors GmbH, Planegg, GermanyRidgeview Instruments AB, Uppsala, Sweden; Department of Immunology, Genetics, and Pathology, Uppsala University, Uppsala, SwedenInstitute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, Paterna, SpainThe RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.https://elifesciences.org/articles/91777binding kineticsdynamic systems and modellinggenetic circuitspost-transcriptional regulationRNA recognition motifsynthetic biology |
| spellingShingle | Roswitha Dolcemascolo María Heras-Hernández Lucas Goiriz Roser Montagud-Martínez Alejandro Requena-Menéndez Raúl Ruiz Anna Pérez-Ràfols R Anahí Higuera-Rodríguez Guillermo Pérez-Ropero Wim F Vranken Tommaso Martelli Wolfgang Kaiser Jos Buijs Guillermo Rodrigo Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria eLife binding kinetics dynamic systems and modelling genetic circuits post-transcriptional regulation RNA recognition motif synthetic biology |
| title | Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria |
| title_full | Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria |
| title_fullStr | Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria |
| title_full_unstemmed | Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria |
| title_short | Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria |
| title_sort | repurposing the mammalian rna binding protein musashi 1 as an allosteric translation repressor in bacteria |
| topic | binding kinetics dynamic systems and modelling genetic circuits post-transcriptional regulation RNA recognition motif synthetic biology |
| url | https://elifesciences.org/articles/91777 |
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