Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

Author:

Dolcemascolo Roswitha12,Heras-Hernández María1,Goiriz Lucas13,Montagud-Martínez Roser12,Requena-Menéndez Alejandro1,Ruiz Raúl1,Pérez-Ràfols Anna45,Higuera-Rodríguez R Anahí67,Pérez-Ropero Guillermo89,Vranken Wim F1011ORCID,Martelli Tommaso4,Kaiser Wolfgang6,Buijs Jos812,Rodrigo Guillermo1ORCID

Affiliation:

1. Institute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia

2. Department of Biotechnology, Polytechnic University of Valencia

3. Department of Applied Mathematics, Polytechnic University of Valencia

4. Giotto Biotech SRL

5. Magnetic Resonance Center (CERM), Department of Chemistry Ugo Schiff, Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (CIRMMP), University of Florence

6. Dynamic Biosensors GmbH

7. Department of Physics, Technical University of Munich

8. Ridgeview Instruments AB

9. Department of Chemistry – BMC, Uppsala University

10. Structural Biology Brussels, Vrije Universiteit Brussel

11. Interuniversity Institute of Bioinformatics in Brussels, Université Libre de Bruxelles – Vrije Universiteit Brussel

12. Department of Immunology, Genetics, and Pathology, Uppsala University

Abstract

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.

Funder

European Commission

Ministerio de Ciencia e Innovación

Generalitat Valenciana

Publisher

eLife Sciences Publications, Ltd

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