Bidirectional photoriboswitch for translational regulation in mammalian cells, Hu et al.
Description
This study presents a bidirectional photoriboswitch platform that mimics orthogonal RNA-binding proteins (RBPs)-mediated translational regulation of multiple mRNAs during biological events. The research focuses on the design, functionality, and applications of the bidirectional photoriboswitch. The experiments involve: 1. Evaluation of exisiting light-sensitive proteins and split inteins. The dimerization efficiency of light-sensitive protein pairs was measured through fluorescent co-localization and single-cell intensity profiling in cell. The self-splicing of split intein pairs was assessed by localization shift of fluorescent signals in cell. 2. Development of a Light-OFF system. A rigid spacer was introduced to spatially segregate the split intein upon photo-dimerization of the light-sensitive protein, thereby creating a Light-OFF system. This system was validated through fluorescent co-localization analysis and single-cell intensity profiling. 3. Development of a bidirectional light-sensing protein reconstitution system. The Light-OFF system was combined with an existing Light-ON system to enable bidirectional control. Functional protein reconstitution was validated while confirming the orthogonality of the two systems. 4. Integration of the designs for split RBP-mediated translational regulation. Model bidirectional photoriboswitches were constructed by coupling the Light-ON/OFF systems with split RBPs. These systems were used to regulate the translation of aptamer-bearing mRNAs. Bidirectionality and orthogonality were confirmed through reporter fluorescence expression in cells, demonstrating precise and hierarchical translational control. Notable findings of this study include: 1. High efficiency of the Light-OFF system. The modular Light-OFF system showed that blue light-induced pMag-nMag dimerization effectively blocked split intein assemby, enabling robust translational inhibition under light. 2. Orthogonal control of translation by split RBPs. The bidirectional photoriboswitches demonstrated effective translational regulation of two mRNAs through light-mediated RBP reconstitution. PCP was reconstituted to suppress EGFP-switch expression under light, whereas MCP was reconstituted to suppress iRFP-switch expression in the dark. This differential regulation could potentially be consistent and reversible across multiple cycles. 3. Protein struture simulation-assisted design of split RBPs. While protein structure simulation of flexible domains has been considered less accurate than for rigid domains, successful engineering of functional split RBPs using AlphaFold3 highlights the expanded potential of structure simulation in protein engineering. This bidirectional photoriboswitch platform offers broad potential applications, including synthetic biology circuits for dynamic gene therapy, tools for investigating molecular mechanisms of translational control, and fine-tuning expression of therapeutic mRNAs for personalized treatments.
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Institutions
- City University of Hong KongHong Kong
Categories
Funders
- Bridge Gap Fund, HKUSTGrant ID: BGF.2025.023
- General Research Fund, Research Grants Council of Hong KongGrant ID: #16300521