Figure S2.
STAU1 condensate recruits MTOR mRNA to enhance its translation. (A) Left: Representative images showing co-localization of mCherry-STAU1 5KE (red) with RNA (green) within STAU1 5KE condensate (dashed box) in COS7 cells. RNA was stained with SYTO 12 Green-Fluorescent Nucleic Acid Stain. Scale bars, 10 μm; inset, 2 μm. Right: Line plot of STAU1 and RNA fluorescence intensities in indicated box regions. (B)MTOR mRNA levels in COS7 cells transfected with GFP-STAU1 WT or LLPS-deficient 5KE mutant or the empty vector (Mock) were analyzed by qRT-PCR. RNA expression levels were normalized to GAPDH. (C) Representative EMSA assay image of MTOR 5′UTR RNA (20 ng) incubated with increasing amounts of STAU1 WT or 5KE mutant in 500 mM NaCl. (D) Representative EMSA assay image of OSKAR 3′UTR RNA (20 ng) incubated with or without STAU1 (1 μM). (E) Representative images showing the droplets of Cy5-STAU1 (5 μM, magenta) could not enrich OSKAR 3′UTR (40 ng, green) stained by SYTO12. (F) Representative DIC images of STAU1 droplets with the increasing amounts of MTOR 5′UTR RNA. Images were acquired at ∼3 min after injecting the mixture into the chamber. Scale bar, 2 μm. (G) Representative Western blotting images of COS7 cells co-transfected with MTOR-5′UTR-LUC reporter and PEGFP-C3, EGFP-STAU1 WT or mutants. (H) EMSA assay image of MTOR 5′UTR RNA (20 ng) incubated with 0.4 μM of STAU1 WT or HKΔRBD4 mutant. (I) Upper: Representative images of STAU1 and STAU1 HKΔRBD4 under DIC microscope at 10 μM. Scale bar: 15 μm. Lower: Scatter plot representing the droplet size of STAU1 and STAU1 HKΔRBD4 at 10 μM. >150 droplets counted for each batch. Data are shown as mean ± SEM; ns, not significant. (J) Representative confocal images of exogenously expressed GFP-STAU1 HKΔRBD4 in COS7 cells. Scale bar: 10 μm. (K) Statistical data of GFP-STAU1 and GFP-STAU1 HKΔRBD4 puncta formation. Data were collected from three or four independent experimental cell culture batches, >300 cells counted for each batch. Specimens’ statistics are presented as mean ± SD; ns, not significant. (L) Upper: Representative DIC images of the reaction mixtures with STAU1 HKΔRBD4 mutant after the in vitro translation assay. Protein concentrations of STAU1 HKΔRBD4 are indicated on the top of the panel. Lower: Statistical data for the relative luminescence of different samples. All statistic data are presented as the mean ± SD of three independent experiments; ns, not significant. Source data are available for this figure: SourceData FS2.

STAU1 condensate recruits MTOR mRNA to enhance its translation. (A) Left: Representative images showing co-localization of mCherry-STAU1 5KE (red) with RNA (green) within STAU1 5KE condensate (dashed box) in COS7 cells. RNA was stained with SYTO 12 Green-Fluorescent Nucleic Acid Stain. Scale bars, 10 μm; inset, 2 μm. Right: Line plot of STAU1 and RNA fluorescence intensities in indicated box regions. (B)MTOR mRNA levels in COS7 cells transfected with GFP-STAU1 WT or LLPS-deficient 5KE mutant or the empty vector (Mock) were analyzed by qRT-PCR. RNA expression levels were normalized to GAPDH. (C) Representative EMSA assay image of MTOR 5′UTR RNA (20 ng) incubated with increasing amounts of STAU1 WT or 5KE mutant in 500 mM NaCl. (D) Representative EMSA assay image of OSKAR 3′UTR RNA (20 ng) incubated with or without STAU1 (1 μM). (E) Representative images showing the droplets of Cy5-STAU1 (5 μM, magenta) could not enrich OSKAR 3′UTR (40 ng, green) stained by SYTO12. (F) Representative DIC images of STAU1 droplets with the increasing amounts of MTOR 5′UTR RNA. Images were acquired at ∼3 min after injecting the mixture into the chamber. Scale bar, 2 μm. (G) Representative Western blotting images of COS7 cells co-transfected with MTOR-5′UTR-LUC reporter and PEGFP-C3, EGFP-STAU1 WT or mutants. (H) EMSA assay image of MTOR 5′UTR RNA (20 ng) incubated with 0.4 μM of STAU1 WT or HKΔRBD4 mutant. (I) Upper: Representative images of STAU1 and STAU1 HKΔRBD4 under DIC microscope at 10 μM. Scale bar: 15 μm. Lower: Scatter plot representing the droplet size of STAU1 and STAU1 HKΔRBD4 at 10 μM. >150 droplets counted for each batch. Data are shown as mean ± SEM; ns, not significant. (J) Representative confocal images of exogenously expressed GFP-STAU1 HKΔRBD4 in COS7 cells. Scale bar: 10 μm. (K) Statistical data of GFP-STAU1 and GFP-STAU1 HKΔRBD4 puncta formation. Data were collected from three or four independent experimental cell culture batches, >300 cells counted for each batch. Specimens’ statistics are presented as mean ± SD; ns, not significant. (L) Upper: Representative DIC images of the reaction mixtures with STAU1 HKΔRBD4 mutant after the in vitro translation assay. Protein concentrations of STAU1 HKΔRBD4 are indicated on the top of the panel. Lower: Statistical data for the relative luminescence of different samples. All statistic data are presented as the mean ± SD of three independent experiments; ns, not significant. Source data are available for this figure: SourceData FS2.

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