Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian Cells

Fabiana Calandra ¹, Velia Siciliano ²

⁰Synthetic and Systems Biology lab for Biomedicine, Istituto Italiano di Tecnologia-IIT, Naples, Italy.

Methods in Molecular Biology +

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March 5, 2024

View abstract on PubMed

Summary

Researchers engineered protease-responsive RNA-binding proteins (RBPs) to enhance synthetic gene circuits in mammalian cells. This expands the toolkit for creating sophisticated, multilayered cellular regulation for biomedical applications.

Area Of Science

Synthetic biology
Molecular and cellular biology
Biotechnology

Background

Genetically encoded sensor-actuator circuits reprogram cellular functions using input-output signaling.
Posttranscriptional gene regulation, utilizing elements like RNA-binding proteins (RBPs), is crucial for advanced biomedical applications.
Existing synthetic circuits faced limitations in regulatory orthogonality, hindering complex network construction.

Purpose Of The Study

To engineer novel protease-responsive RNA-binding proteins (RBPs) for expanding the synthetic biology toolbox.
To enhance the capabilities of posttranscriptional gene regulation in mammalian cells.
To enable the creation of sophisticated, multilayered regulatory networks in synthetic circuits.

Main Methods

Engineering of specific RNA-binding proteins (L7Ae and MS2-cNOT7) to be responsive to protease activity.
Testing the functionality and responsiveness of engineered RBPs in mammalian cell systems.
Evaluating the potential for creating orthogonal and multilayered regulatory networks using these engineered RBPs.

Main Results

Successful engineering and testing of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7).
Demonstrated expansion of the available regulatory parts for synthetic circuits in mammalian cells.
Established a foundation for building more complex and sophisticated gene regulatory networks.

Conclusions

Protease-responsive RBPs offer a novel mechanism for controlling gene expression posttranscriptionally.
The engineered RBPs enhance the orthogonality and layering capabilities of synthetic gene circuits.
This work provides valuable tools for advancing synthetic biology in biomedical research and applications.

Keywords:

Mammalian synthetic biology Protein engineering Protein–RNA regulation Protein–protein interaction RNA-binding proteins Tobacco etch virus protease