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

Abstract

ABSTRACTThe 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 bacteriumEscherichia coliwith 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 leader 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 thein vitrobinding kinetics andin vivofunctionality of a series of RNA mutants. Moreover, the dynamic response of the system was well recapitulated by a bottom-up mathematical model. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby paving the way for engineering more complex circuits in prokaryotes by combining transcription and translation control with proteins.