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

  1. Institute for Integrative Systems Biology (I2SysBio), CSIC – University of Valencia, 46980 Paterna, Spain
  2. Department of Biotechnology, Polytechnic University of Valencia, 46022 Valencia, Spain
  3. Department of Applied Mathematics, Polytechnic University of Valencia, 46022 Valencia, Spain
  4. Giotto Biotech SRL, 50019 Sesto Fiorentino, Italy
  5. Magnetic Resonance Center (CERM), Department of Chemistry Ugo Schiff, Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (CIRMMP), University of Florence, 50019 Sesto Fiorentino, Italy
  6. Dynamic Biosensors GmbH, 82152 Planegg, Germany
  7. Department of Physics, Technical University of Munich, 85748 Garching, Germany
  8. Ridgeview Instruments AB, 75237 Uppsala, Sweden
  9. Department of Chemistry – BMC, Uppsala University, 75123 Uppsala, Sweden
  10. Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium
  11. Interuniversity Institute of Bioinformatics in Brussels, Université Libre de Bruxelles – Vrije Universiteit Brussel, 1050 Brussels, Belgium
  12. Department of Immunology, Genetics, and Pathology, Uppsala University, 75185 Uppsala, Sweden

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, and public reviews.

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Editors

  • Reviewing Editor
    Joseph Wade
    Department of Biomedical Sciences, School of Public Health University at Albany, Albany, United States of America
  • Senior Editor
    Christian Landry
    Université Laval, Québec, Canada

Reviewer #1 (Public Review):

The authors develop reporter constructs in E. coli where gene expression, presumably translation, is repressed by MSI-1. This is a potentially useful tool for synthetic biologists, with the advantage over transcriptional regulation that one gene in an operon could be targeted. That being said, an important caveat of translational regulation that is not addressed in the manuscript is the potential for downstream effects on RNA stability and/or transcription termination. The authors' MSI-1-regulated reporter constructs could also be useful for mechanistic studies of MSI-1.

The author's initial construct design led to only weak regulation by MSI-1, presumably because the MSI-1 binding sites were not suitably positioned to repress translation initiation. A more rationally designed construct led to considerably greater repression. One weakness of the paper is that the authors did not use their redesigned construct that is more strongly repressed to demonstrate allosteric regulation by oleic acid using a comparable assay (e.g., flow cytometry) to that used in other experiments. The potential for allosteric regulation is a major strength of the MSI-1 system, so this is a significant gap. Similarly, the authors use the weakly regulated constructs to assess the effect of MSI-1 binding site mutations and for their mathematical modeling; these experiments would be better suited to the more strongly regulated construct.

Reviewer #2 (Public Review):

Summary:
Dolcemascolo and colleagues describe the use of the mammalian RNA-binding protein Musashi-1 (MSI-1) to implement translational regulation systems in E. coli. They perform detailed in vitro studies of MSI-1 and its binding to different RNA sequences. They provide compelling evidence of the effectiveness of the regulatory system in multiple circuits using different mRNA sequence motifs. They harness allosteric inhibition of MSI-1 by omega-9 monounsaturated fatty acids to demonstrate a fatty-acid-responsive circuit in E. coli.

Strengths:
The experimental results are compelling and the characterization of the binding between MSI-1 and different RNA sequences is thorough and performed via multiple complementary techniques. Several new useful circuit components are demonstrated.

Weaknesses:
MSI-1 provides 8.6-fold downregulation of sfGFP with an optimized mRNA sequence. In some applications, a larger degree of repression may be required.

Reviewer #3 (Public Review):

Summary:
In this work, the authors co-opt the RRM-binding protein Musashi-1 to act as a translational repressor. The novelty of the work is in the adoption of the allosteric RRM protein Musashi-1 into a translational reporter and the demonstration that RRM proteins, which are ubiquitous in eukaryotic systems, but rare in prokaryotic ones, may act effectively as post-translational regulators in E. coli. The extent of repression achieved by the best design presented in this work is not substantially improved compared to other synthetic regulatory schemes developed for E. coli, even those that similarly regulate translation (eg. native PP7 repression is approximately 10-fold, Lim et al. J. Biol. Chem. 2001 276:22507-22513). Furthermore, the mechanism of regulation is not established due to missing key experiments. The work would be of broader interest if the allosteric properties of Musashi-1 were more effective in the context of regulation. Unfortunately, the authors do not demonstrate that fatty acids can completely de-repress expression in the experimental system used for most of their assays, nor do they use this ability in their provided application (NIMPLY gate).

Strengths:
The first major achievement of this work is the demonstration that a eukaryotic RRM protein may be used to post-transcriptionally regulate expression in bacteria. In my limited literature search, this appears to be the first engineering attempt to design an RBP to directly regulate translation in E. coli, although engineered control of translation via other approaches including alterations to RNA structure or via trans-acting sRNAs have been previously described (for review see Vigar and Wieden Biochim Biophys. Acta Gen. Subj. 2017, 1861:3060-3069). Additionally, several viral systems (e.g. MS2 and PP7) have been directly co-opted to work in a similar fashion in the past (utilized recently in Nguyen et al. ACS Synthetic Biol 2022, 11:1710-1718).

The second achievement of this work is the demonstration that the allosteric regulation of Musashi-1 binding can be utilized to modulate the regulatory activity. However, the liquid culture demonstration (Suppl. Fig 8) shows that this is not a very effective switch, with de-repressed reporter activity showing substantial change but not approaching un-repressed activity. This effect is stronger when colonies are grown on a solid medium (Fig. 5).

Weaknesses:
In this work, the authors codon optimize the mouse Musashi-1 coding sequence for expression in E. coli and demonstrate using an sfGFP reporter that an engineered Musashi-1 binding site near the translational start site is sufficient to enable a modest reduction in reporter gene expression. The authors postulate that the reduction in expression due to inhibition of ribosome translocation along the transcript (lines 134/135), as an expression of a control transcript (mScarlet) driven by the same promoter (Plac) but without the Musashi-1 recognition site does not demonstrate the same repression. However, the situation could be more complex. Other possibilities include inhibition of translation initiation rather than elongation, as well as accelerated mRNA decay of transcripts that are not actively translated. The authors do not present any measurements of sfGFP mRNA levels.

In subsequent sections of the work, the authors create a series of point mutations to assess RNA-protein binding and assess these via both a sfGFP reporter and in vitro binding assays (switchSENSE). Ultimately, it is difficult to fully rationalize and interpret the behavior of these mutants in the context provided. The authors do identify a relationship between equilibrium constant (1/KD) and fold-repression. However, it is not clear from the narrative why this relationship should exist. Fold-repression is one measure of regulator efficacy, but it is an indirect measure determined from unrepressed and repressed expression. It is not clear why unrepressed expression (in the absence of the protein) is expected to be a function of the equilibrium constant.

Subsequent rational redesign of the Musashi-1 binding sequence to produce three alternative designs shows that fold-repression may be improved to approximately 8.6-fold. However, the rationalization of why the best design (red3) achieves this increase based on either the extensive modelling or in vitro measured binding constants is not well articulated. Furthermore, this extent of regulation is approximately that which can be achieved from the PP7 system with its native components (Lim et al. J. Biol. Chem. 2001 276:22507-22513).

The application provided for this regulator (NIMPLY gate), is not an inherently novel regulatory paradigm, and it does not capitalize on the allosteric properties of Musashi-1, but rather treats Musashi-1 as a non-allosteric component of a regulatory circuit.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation