Figure S7.

GCE of boxB- and ms2-tagged mRNA in the presence of λ_N22- or MCP-based OT organelles, related to Figure 5

(A,C,F) FFC analysis of HEK293T cells expressing the double-recruitment reporter (EGFP^39TAG::boxB, mCherry^190TAG::ms2) together with tRNA^Pyl and the indicated GCE systems in the presence of the indicated ncAAs. Shown is the sum of at least three independent experiments.

(A) For these experiments, 400 ng of reporter and tRNA plasmid were co-transfected with 100 ng of the OT organelle construct and 300 ng mock plasmid. In the presence of MCP-based OT organelles, mCherry expression predominates, whereas in the presence of λ_N22-based OT organelles full-length EGFP is mostly produced.

(B) Bar graph showing the ratio of the mCherry signal to the EGFP signal corresponding to the conditions in panel B, normalized to a cytoplasmic GCE system (see A). The ratio is shown on a logarithmic scale, so that positive logarithmic values indicate a higher expression of mCherry compared to EGFP, while a negative logarithmic ratio corresponds to an excess of EGFP over mCherry, a ratio close to zero indicates that EGFP and mCherry are equally expressed.

(C) For these experiments, 400 ng of reporter and tRNA plasmid were co-transfected with 100 ng of each OT organelle construct and 200 ng mock plasmid. To our surprise, although all of our OT organelles obeyed all the orthogonality criteria tested, including orthogonality to the host machinery in the cytoplasm (A and B), unexpectedly, none of the combinations yielded two organelles that are orthogonal to each other when expressed in one cell. If OMMP^λN22,AF was combined with PMP^MCP,AA, full-length EGFP^39TAG and mCherry^190TAG expression was observed independently of the added ncAA. Similarly, if OMMP^λN22,AA was combined with ERMP^MCP,AF, substantial expression of both full-length EGFP^39TAG and mCherry^190TAG was observed under all conditions.

A possible explanation for this selectivity loss is that one or more of the organelle components “leaks” and shuttles between the two organelles by an unexpected mechanism.

mRNA recruitment is likely not the cause of selectivity loss as we demonstrated that it is selective between the different recruiting systems (Figure S6). We also demonstrated that the OMMP system is orthogonal to the PMP and ERMP systems (Figures 3 and S1) thus organelle assembly per se cannot cause selectivity loss.

Another explanation would be that after aminoacylation, the tRNA^Pyl (likely in a complex with elongation factors) could shuttle between organelles. Note that each organelle charges the same tRNA^Pyl with a different ncAA. However, to this end it would diffuse through the cytoplasm destroying selectivity to cytoplasmic translation which we specifically tested (Figure 2). Further we showed that tRNA^Pyl can be concentrated along a steep gradient in FISH stainings (Figure 4). Thus, diffusion of aminoacylated tRNA^Pyl is unlikely the cause of selectivity loss.

As PylRS is known to be active as a dimer (Kavran et al., 2007; Wan et al., 2014), another speculation was that dimerization prior to proper membrane localization might mistarget some synthetase molecules before they are sorted into their subcellular destination.

(D) Bar graph similar to the one in (B) corresponding to the conditions in panel C, normalized to a cytoplasmic GCE system (see panel A).

(E) To investigate this, we performed IF staining against two synthetase variants—one targeted to the PM or ERM and the other to the OMM (staining against MCP and λ_N22 corresponding to the conditions in panel C). We observed that the PM- or ERM-targeted synthetases indeed mix with the OMM-targeted synthetases. This is particularly pronounced for the PM-targeted synthetase, of which a substantial fraction relocalizes to the OMM. Top to bottom: MCP, λ_N22, mCherry, EGFP, and merged (MCP in yellow, λ_N22 in blue, mCherry in magenta, EGFP in green). Scale bars: 20 μm.

Gratifyingly, when making a dimeric PylRS construct in form of a PylRS::FUS::PylRS fusion, we obtained the desired mutually orthogonal film-like organelles. While further mechanistic understanding of this effect might be desirable and the goal of future studies, for the scope of this work it is important that using the dimeric system, two of our membrane associated, condensated film-like organelles function as designed and orthogonal to each other within the same cell.

(F) In these experiments, 400 ng of reporter and tRNA plasmid were co-transfected with 100 ng of the OT organelle construct and 300 ng mock plasmid. In the presence of MCP-based OT organelles, mCherry expression was predominant, whereas in the presence of λ_N22-based OT organelles, full-length EGFP is mostly produced.

(G) Bar graph similar to the one in (B) corresponding to the conditions in panel F, normalized to a cytoplasmic GCE system (see panel A).

For the analysis in (B,D,G) untransfected cells were not taken into account (determined by a minimum EGFP/mCherry fluorescence level as indicated by the lower left quadrant in the corresponding FFC dot plots). All bar graphs show the mean value of all experiments; error bars represent the SD.