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. 2022 Jan 12;8(2):eabk2141. doi: 10.1126/sciadv.abk2141

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Fig. 1. — (A) Translation inhibition reveals that colocalization of PEX14 mRNA and peroxisomes is translation independent. The maximum intensity projections of confocal images of smRNA-FISH assay of sfGFP-PEX14 (TAMRA, red) following 1-hour incubation with CHX are shown. Peroxisomes in cyan (CFP-PTS1); yellow arrowheads highlight colocalization between PEX14 mRNA and peroxisomes; yeast periphery in dashed white line. (B) Quantification of (A). Random colocalization threshold calculated by mRNA of Golgi SEC7 (n = 150; **P < 0.05, unpaired t test). n.s., not significant. (C) Schematic description of interference with PEX14 translation. sfGFP-PEX14 under the NOP1 promoter (NOP1pr) was integrated into the HO (homothallic switching endonuclease) locus locus either with the start methionine (+ATG) or without (−ATG). (D) Representative smFISH micrographs of +ATG and −ATG strains described above. Mature peroxisomes in cyan (CFP-PTS1), PEX14 mRNA in magenta (TAMRA), and Pex14 protein in green (GFP); yellow arrowheads indicate colocalization. Percentage of peroxisomes colocalizing with sfGFP-PEX14 mRNA (n = 50; P > 0.05, unpaired t test). (E) Representative micrograph from time-lapse experiments of strains following induction of GALpr-PEX19. PEX14-MS2L in green (MS binding coat protein–GFP) and peroxisomes in magenta (mCherry-PTS1). Percentage of mRNA granules colocalizing with peroxisomes. n = 100. (F) Same as (E) but visualizing PEX11-MS2L mRNA (green). (G) Micrograph showing PEX11-MS2L mRNA (green) colocalized with pre-peroxisomal vesicle (PPV) in magenta (Pex14-mCherry) in the absence of Pex19 (GALpr-PEX19 grown on glucose). Percentage of mRNA granules colocalizing with PPVs. Scale bar, 5 μm. (H) Graphical summary of Fig. 1—three experimental methodologies suggest that mRNA localization to peroxisomes is independent of translation.