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

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

Onderzoeksoutput: Articlepeer review

Samenvatting

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.

Originele taal-2English
Aantal pagina's29
TijdschrifteLife
Volume13
DOI's
StatusPublished - 16 feb 2024

Bibliografische nota

© 2023, Dolcemascolo, Heras-Hernández, Goiriz et al.

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