TY - JOUR
T1 - Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria
AU - Dolcemascolo, Roswitha
AU - Heras-Hernández, María
AU - Goiriz, Lucas
AU - Montagud-Martínez, Roser
AU - Requena-Menéndez, Alejandro
AU - Ruiz, Raúl
AU - Pérez-Ràfols, Anna
AU - Higuera-Rodríguez, R Anahí
AU - Pérez-Ropero, Guillermo
AU - Vranken, Wim F
AU - Martelli, Tommaso
AU - Kaiser, Wolfgang
AU - Buijs, Jos
AU - Rodrigo, Guillermo
N1 - © 2023, Dolcemascolo, Heras-Hernández, Goiriz et al.
PY - 2024/2/16
Y1 - 2024/2/16
N2 - 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.
AB - 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.
KW - Animals
KW - Nerve Tissue Proteins/metabolism
KW - RNA-Binding Proteins/metabolism
KW - RNA/metabolism
KW - RNA, Messenger/metabolism
KW - Escherichia coli/genetics
KW - Mammals/genetics
UR - http://www.scopus.com/inward/record.url?scp=85185233889&partnerID=8YFLogxK
U2 - 10.7554/eLife.91777
DO - 10.7554/eLife.91777
M3 - Article
C2 - 38363283
VL - 13
JO - eLife
JF - eLife
SN - 2050-084X
ER -