Journal: Cell Research

Article Title: Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells

doi: 10.1038/s41422-023-00896-y

Figure Lengend Snippet: a Translational regulation by protein–protein interaction (PPI)-mediated STIF reconstitution. Bipartite STIF systems with NSP3 and RBP domains split into two independent proteins allow flexible incorporation of different PPI systems to regulate conditional STIF assembly and translation of RBP-specific mRNA. b Compatibility of STIF-mediated translational regulation with split-complementation approaches. Left, HEK-293 cells were co-transfected with a SEAP expression vector containing 24 tandem MCP-specific aptamer repeats (pSL468) and different combinations of constitutive expression vectors for MCP-NSP3 (pSL674) and (DocS) 3 -NSP3 (pSL86). Right, HEK-293 cells were co-transfected with a SEAP expression vector containing bacteriophage λ N-Peptide (λN)-repressible HHR placed upstream of poly(A) (pMX116) and constitutive expression vectors for λN-Coh2 (pSL334) and DocS-mCherry (pSL1099). For (–) conditions, pcDNA3.1(+) was transfected instead of expression vectors. SEAP levels in culture supernatants were scored at 48 h post transfection. Data are shown as means ± SD; n = 4 independent experiments. c Regulation through trigger-inducible STIF reconstitution. For danoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-NS3a (pSL497) and DNCR-NSP3 (pLZ72). For abscisic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for ABI-MCP (pPW22) and (PYL) 3 -NSP3 (pPW4). For gibberellic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-GID (pPW23) and GAI-NSP3 (pPW2). For grazoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-NS3a (pSL497) and GNCR-NSP3 (pYF3). For rapamycin-inducible SEAP translation, HEK-293 cells were co-transfected with a SEAP expression vector containing poly(A)-surrogates with 16 tandem MCP-specific MS2-box repeats (pSL516) and constitutive expression vectors for MCP-FRB (pSL1097) and FKBP-NSP3 (pSL1098). For blue light-inducible SEAP translation, HEK-293 cells were co-transfected with pSL516 and constitutive expression vectors for MCP-CIB1 (pSL1096) and Cry2-NSP3 (pSL71). For red light-inducible SEAP translation, HEK-293 cells were co-transfected with pSL516 and constitutive expression vectors for MCP-(Aff6 V18FΔN ) 4 (pSL917) and DrBPhP-NSP3 (pSL901). SEAP levels in culture supernatants were scored at 48 h after addition of the corresponding inducers (danoprevir, 0.5 µM; abscisic acid, 100 µM; gibberellic acid, 100 µM; grazoprevir, 0.5 µM; rapalog, 0.1 µM) or at 48 h after exposure to blue light (450 nm; ON, 30 s at 5 mW/cm 2 ; OFF, 30 s) or at 24 h after exposure to red light (660 nm, constantly 1 W/m 2 ). Data are shown as means ± SD; n = 4 independent experiments. d Genetically encoded signaling-specific sensors employing phosphorylation-dependent STIF reconstitution. HEK-293 cells were co-transfected with a constitutive FLuc expression vector (pYW99), an expression vector for NanoLuc-mRNA containing MCP-specific poly(A)-surrogates (P hCMV -NanoLuc-P2A-mCherry-(MS2-box) 24 -HHR-pA, pSL683) and different combinations of constitutive expression vectors for MCP-(pE59) 2 (pSL637) and (ERK2) 2 -NSP3 (pSL189) before cultivation in cell culture medium containing 2% fetal bovine serum (FBS) (v/v). Luciferase levels in culture supernatants were quantified at 48 h after the addition of 0 or 100 ng/mL recombinant human EGF. For (–) conditions, pcDNA3.1(+) was transfected instead of expression vectors. Data are presented as means ± SD of relative luciferase activity (NanoLuc/FLuc); n = 3 individual experiments. e Genetically encoded protein sensors employing protein association-dependent STIF reconstitution. HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-(Coh2) 3 (pSL1080), (Coh2) 3 -NSP3 (pSL243) and (DocS) 3 (different amounts of pSL244). SEAP levels in culture supernatants were scored at 48 h post transfection. Data are shown as means ± SD of n = 4 independent experiments. Bars represent means ± SD, and filled circles show individual results; numbers above bars are fold changes ( b – e ). ns, not significant; ** P < 0.01.

Article Snippet: Danoprevir (Cat# HY-10238; stock solution 10 mM in DMSO) and gibberellic acid (Cat# HY-N1964; stock solution 100 mM in DMSO) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Cell Culture, Luciferase, Recombinant, Activity Assay