Journal: bioRxiv

Article Title: Synthetic Cytosolic Splicing Enables Programmable mRNA-Encoded Receptors

doi: 10.1101/2025.04.27.650769

Figure Lengend Snippet: a, Schematic representation of the mRNA splicing reporter. The reporter consists of the XBP1 mRNA splicing region (nucleotides 410-633) inserted upstream of the out-of-frame coding sequence of the fluorescent protein mScarlet. Upon removal of the cytosolic intron by IRE1, the translation of mScarlet is restored, providing a fluorescent readout of the receptor splicing activity. b, Splicing reporter output in HEK293 cells treated with the ER-stress inducer tunicamycin. The bar chart shows the mScarlet normalized units (norm. u.) for biological triplicates, n=3. Individual data points are overlaid in gray, with error bars indicating the standard deviation. An unpaired two-tailed t-test was performed, and asterisks indicate p < 0.0001 (****). c, Semi-quantitative RT-PCR gel showing the unspliced (top, 375 base pairs) and spliced (bottom, 349 base pairs) forms of the splicing reporter in HEK293 cells treated with tunicamycin for 0 (control), 2, or 6 hours. d, Fluorescence microscopy images of HEK293 or HeLa IRE1 knockout (KO) cells transfected with the mRNA splicing reporter and treated with tunicamycin to induce ER stress. Increased red fluorescence indicates splicing activity of the IRE1 receptor. Scale bars represent 250 μm. e, Splicing reporter output in HeLa IRE1 KO cells upon addition of tunicamycin. The bar graph illustrates the normalized units of mScarlet (norm. u.) for three biological replicates (n=3). Gray points denote individual data, with error bars indicating the standard deviation. f, Splicing reporter induction with the human IRE1 cytosolic fragment fused with constitutive self-assembly domains in HeLa IRE1 KO cells. The fluorescent mScarlet (norm. u.) signal increases with dimerization stoichiometry for biological duplicates (n=2). The dashed line indicates the output of a genetically encoded, constitutively spliced fluorescent reporter driven by the EF1alpha promoter. g, Schematic of the inducible heterodimerization system using FKBP and FRB domains fused to IRE1 cytosolic regions. The addition of the small molecule A/C induces heterodimerization, activating the IRE1 splicing domains and inducing the production of mScarlet by the reporter. h, Dose-response curve showing the normalized mScarlet fluorescence in response to varying concentrations of the heterodimerizer A/C. Each data point shown is the average of biological triplicates (n=3), with error bars indicating the standard deviation.

Article Snippet: The reagents used to induce receptor sensing were A/C heterodimerizer (Takara Bio; cat# 635079), the B/B homodimerizer (Takara Bio; cat# 635059), recombinant human TNF-α (R&D cat# 210-TA-020), and recombinant human IL-1β (R&D cat# 201-LB-010).

Techniques: Sequencing, Activity Assay, Standard Deviation, Two Tailed Test, Quantitative RT-PCR, Control, Fluorescence, Microscopy, Knock-Out, Transfection