Figure S6.

BoxB- and ms2-based mRNA recruitment systems fulfill the selective RNA recruitment criterion, related to Figure 3

To test if mRNAs tagged with boxB or ms2 loops are selectively recruited to their respective organelles, the two dual-color reporters were co-expressed with membrane-localized OT organelles that either had no RNA recruitment domain, MCP or λ_N22 peptides.

(A,B) Besides the indicated OT organelles and reporters, cells also expressed tRNA^Pyl and were incubated with the ncAA SCO-K. As a control, cells were transfected with a construct encoding a cytoplasmic PylRS variant. Shown is the sum of at least three independent experiments. (A) FFC analysis for the ms2 reporter reveals selective mCherry expression only in the presence of the MCP-based OT organelles. (B) Analogously, boxB-tagged mCherry is only selectively expressed in the presence of λ_N22-based OT organelles. PylRS^AF and PylRS^AA exhibit orthogonal substrate specificities. Note that the black line in the FFC dot plots indicates the EGFP signal cut-off used to identify transfected cells.

(C) Heatmaps showing the fold change in selectivity and relative efficiency corresponding to the FFC data shown in (A,B). For both the selectivity and the efficiency, the mean value of the three experiments is displayed.