Fig. 3. A versatile OT organelle for selective and efficient orthogonal translation.

(A to C) Confocal images of cells transfected with constructs encoding PylRS (left column) or the OT^K2::P1 organelle (right column) for different protein pairs. SCO and tRNA^Pyl were present in all cases. (A) HEK293T cells were transfected with NUP153::GFP^149TAG::ms2 and VIM^116TAG::mOrange. (B) VIM^116TAG::mOrange::ms2 and NUP153::GFP^149TAG transfected in COS-7 cells. More representative examples for (A) using COS-7 cells and for (B) using HEK293T cells are shown in fig. S5. Shown from top to bottom are Vimentin^116TAG::mOrange (magenta, characteristic cytoskeletal stain), Nup153::GFP^149TAG (yellow, characteristic nuclear envelope stain), overlay with Hoechst (cyan, nuclear stain), magnified images of representative cells (red boxes), and line profiles for the mOrange and GFP channel (red line, magenta and yellow curves, respectively), to highlight that only the ms2-tagged mRNA yields its respective expressed protein if the OT^K2::P1 organelle is present. Scale bars, 20 mm. (C) HEK293T cells were transfected with INSR^676TAG::mOrange::ms2 and NUP153::GFP^149TAG. Shown from top to bottom are INSR^676TAG::mOrange (magenta, characteristic plasma membrane stain), Nup153::GFP^149TAG (yellow), overlay with Hoechst (cyan), magnified images of representative cells (red boxes), and line profiles for the mOrange and GFP channel (red line, magenta and yellow profiles, respectively), demonstrating selective translation of insr^676TAG::mOrange::ms2 mRNA by the OT^K2::P1 organelle. Scale bars, 20 μm.