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RNA scaffolds the Golgi ribbon by forming condensates with GM130

Nature Cell Biology volume 26, pages 1139–1153 (2024)Cite this article

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Abstract

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid–liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

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Fig. 1: GM130 is in a complex with the RBPs FXR1, G3BP1 and PABPC1.
Fig. 2: Association of RBPs with the N-terminal domain of GM130 is RNA dependent.
Fig. 3: Acute degradation of RNA or GM130 disrupts lateral connections of the Golgi ribbon.
Fig. 4: Oxidative stress displaces RNA from GM130 and causes Golgi fragmentation.
Fig. 5: GM130 forms RNA-dependent liquid-like condensates in cells.
Fig. 6: The N-terminal domain of GM130 forms RNA-dependent condensates.
Fig. 7: Condensates of N122-Di and RNA are sufficient to link Golgi membranes in vitro.

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All of the data that support the findings of this study are available within the article and its supplementary materials. Source data are provided with this paper.

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Acknowledgements

We thank J.-H. Wei for suggestions and comments, H. Guo for suggestions, the University of Texas Southwestern Proteomics Core Facility for mass spectrometry analysis and the University of Texas Southwestern Live Cell Imaging Core Facility for imaging support. This work was supported by grants from the National Institutes of Health (GM096070) and Welch Foundation (I-1910) to J.S.

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  1. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Yijun Zhang & Joachim Seemann

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  1. Yijun Zhang
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Y.Z. and J.S. conceived of the project, designed and performed the experiments, analysed the data, prepared the figures and wrote the manuscript.

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Correspondence to Joachim Seemann.

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Extended data

Extended Data Fig. 1 GM130 is in a complex with the RNA-binding proteins FXR1, G3BP1 and PABPC1.

a, Volcano plot showing proteins enriched in GRASP65 pulldowns compared to GRASP55 (RC65/55) identified by mass spectrometry. Flag-tagged GRASP55 and GRASP65 were immunoprecipitated from RC55 or RC65 cell lysates and associated proteins were analyzed by mass spectrometry. The abundance of each interactor in n = 2 independent experiments was normalized to total and analyzed by multiple t-tests and plotted. GRASP55 and GRASP65 (blue), representative interactors of GRASP55 (green), representative interactors of GRASP65 (red) are highlighted. Statistical analysis was performed using multiple unpaired two-tailed t-tests with threshold set to 0.05 for multiple comparisons. b-c, Immunoblots of cells transfected with GM130 siRNA are shown in (b) and FXR1 siRNA shown in (c). d, Relative labeling density of PLA puncta in cells transfected with control siRNA or GM130 siRNA. Relative labeling density was calculated per cell by normalizing labeling density at the Golgi to non-Golgi area. n = 133 cells from 2 independent experiments were analyzed per condition. Statistical significance was assessed using unpaired two-tailed Student’s t-tests. Error bars represent mean ± SD. **** P < 0.0001. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 The second lysine cluster of GM130 is important for RNA-dependent complex formation with FXR1, G3BP1, and PABPC1.

a, RNase A treatment digests RNA from GM130 immunoprecipitants. GM130 was immunoprecipitated from SV589 lysates, treated with 40 U/ml RNase inhibitor (RNasin) or 20 µg/ml RNase A as in Fig. 2a and RNA was extracted. 250 ng total RNA from SV589 cells, 250 ng RNA from RNasin (RNase inhibitor) treated samples and equal volume fractions from other conditions were separated on an agarose gel. b, Sequence of the N-terminal domain of GM130 (N74) with the two lysine/arginine (K/R) clusters highlighted. c, The second lysine cluster of N74 is important for RNA binding. Strep-tagged N74 WT, 1A (the first K/R cluster mutated to A), 2A (the second K cluster mutated to A) and AA (both K/R clusters changed to A) were incubated with SV589 cell lysates, pulled down and analyzed by immunoblotting or Coomassie blue staining. d, Quantitation of (c). The intensity of each protein was normalized to WT. n = 2 independent experiments. e, RNA mediates association of N74 and G3BP1. N74-strep was incubated with G3BP1-his and indicated concentrations of total RNA. N74-strep was then pulled down and analyzed by SDS-PAGE and Coomassie blue staining. G3BP1-his was pulled down by N74-strep with increasing RNA concentrations. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Acute degradation of GM130 does not affect the Golgi area or stacking.

a, Acute degradation of GM130 disjoints the Golgi ribbon. NRK cells stably expressing TRIM21 and the Golgi marker NAGTI-GFP were microinjected with anti-GM130 IgG to degrade GM130 or anti-Flag IgG as control together with fluorescent dextran as injection marker. Injected cells were then subjected to FRAP analysis of NAGTI-GFP. Representative images at indicated times are shown. White boxes indicate the photobleached area of the Golgi. b, c, d, NRK cells stably expressing TRIM21 were injected with anti-GM130 to degrade GM130 together with fluorescent dextran as an injection marker. After 2 hours cells were immunostained for the TGN marker TGN38 (red), the cis-Golgi protein Golgin-84 (green) and labeled for DNA (blue). White lines on the top left non-injected cell and injected cell on the bottom indicate the line scan of the fluorescence intensities shown in (c) and (d). e, Microinjected anti-GM130 antibodies label the Golgi marked by NAGTI-GFP. NRK/NAGTI-GFP cells not expressing TRIM21 were injected with anti-GM130 antibodies or control IgG. Injected cells were stained with secondary antibodies to label injected IgG. f, NRK/NAGTI-GFP cells not expressing TRIM21 were injected with anti-GM130 antibodies or control IgG, together with fluorescent dextran as injection marker and then subjected to FRAP of NAGTI-GFP. Representative images at indicated times are shown. White boxes indicate the photobleached area of the Golgi. g, SV589 cells were co-transfected with siG3BP1/2_#1 or siG3BP1/2_#2 for 72 h, treated with 250 µM SA for 1 h, and immunolabeled for FXR1 (red), GM130 (green) and stained for DNA (blue). h, Quantitation of the Golgi area marked by GM130 from (g). n = 250 cells per condition from 3 independent experiments. * P < 0.05, **** P < 0.0001. i, Knockdown of G3BP1/2 does not affect the lateral linking of Golgi ribbon. SV589/NAGTI-GFP cells were co-transfected with siG3BP1/2_#1 or siG3BP1/2_#2 for 72 h, and then subjected to FRAP. Representative images at indicated time points are shown, with white boxes showing the photobleached area of the Golgi. Statistical significance of all comparisons was assessed using unpaired two-tailed Student’s t-tests. All error bars represent mean ± SD. All scale bars, 10 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Translation inhibition does not disrupt the Golgi ribbon.

a, SA treatment dissociates RNA from GM130, and binding is restored after SA washout. SV589 cells were treated with 250 µM SA for 1 h (SA), or SA was washed out and cells were incubated for an additional 16 h (SA w/o). GM130 was immunoprecipitated from cell lysates, followed by RNA extraction. 250 ng total RNA from SV589 cells, 250 ng RNA from control treated samples and equal volume fractions from other conditions were separated on an agarose gel. b, Translation inhibition by cycloheximide (CHX) or emetine compacts the Golgi. SV589 cells were treated with 50 µg/ml CHX or emetine for 1 h, followed by 250 µM SA for another 1 h. Cells were immunolabeled for FXR1 (red), GM130 (green) and stained for DNA (blue). c, Quantitation of the GM130-labelled Golgi area from (b). N = 170 cells per condition from 3 independent experiments. **** P < 0.0001. d, Cycloheximide (CHX) or emetine enhances the lateral linking of Golgi ribbon. SV589/NAGTI-GFP cells treated with 50 µg/ml CHX or emetine for 1 h were subjected to FRAP. Representative images at indicated time points are shown, with white boxes showing the photobleached area of the Golgi. e, Quantitation of FRAP from (d). control: n = 29 cells, CHX: n = 30, emetine: n = 31 from 3 independent experiments. Statistical significance of all comparisons was assessed using unpaired two-tailed Student’s t-tests. Error bars for FRAP data represent mean ± SEM, all other error bars represent mean ± SD. All scale bars, 10 µm. Source numerical data and unprocessed gels are available in source data.

Source data

Extended Data Fig. 5 GM130 condensates in cells are highly dynamic.

a-f, GM130 condensates do not co-localize with nuclear speckles. SV589 cells transiently overexpressing full-length GM130 were labeled for GM130 (green), DNA (blue) and the nuclear speckle proteins SON (red) in a or SC-35 (red) in d. Two representative images are shown in a and d. White lines in a and d indicate the line scan of the fluorescence intensities shown in b, c and e, f, respectively. g, GM130-GFP condensates were subjected to FRAP analysis. Representative images are shown at the indicated time points, with a white box marking the photobleached area of the condensate. h, Quantitation of (g). n = 18 cells from two independent experiments. Error bars represent mean ± SEM. i, Representative time-lapse images of GM130-GFP at indicated time points. Arrowheads indicate fusion of GM130 condensates. All scale bars, 10 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 Validation of N122 oligomers and RNA dependence of N122 condensates.

a,10 µg of GM130 N122, N122-Di and N122-Tet at indicated concentrations were incubated with the crosslinker disuccinimidyl suberate (DSS) and then analyzed by SDS-PAGE and Coomassie blue staining. b, RNase A prevents LLPS of N122-Di and total RNA. LLPS was performed with N122-Di and 100 µg/ml total RNA with or without 1 mg/ml RNase A. DIC images of droplets were acquired within 5 min after LLPS induction. Scale bar, 10 µm. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 7 N122-PEG condensates do not organize Golgi membranes in vitro.

a, Fluorescently labeled rat liver Golgi (RLG) membranes were mixed with N122-Di (containing 5% fluor 488-labeled N122-Di) at different concentrations without RNA. Before imaging, the membranes were allowed to sediment for 30 min. The boxed areas are shown enlarged in the right panels. Scale bar, 20 µm. b, c, d, Condensates formed by N122-Di and PEG engulf Golgi membranes. N122-Di (containing 5% fluor 488-labeled N122-Di) was mixed with 10% PEG 3350 in (b) or with PEG and RLG membranes in (c). Before imaging, the membranes were allowed to sediment for 30 min. The boxed areas are shown enlarged. The white line marks the line scan shown in (d). Scale bar, 10 µm. e, Golgi membranes were mixed with N122-Di (containing 5% fluor 488-labeled N122-Di) and 100 µg/ml total RNA on a shaker for 45 min and transferred to a 96-well glass-bottom plate. Images were captured 30 min later. Representative images were shown. The boxed areas are shown enlarged with the brightness of N122-Di increased to show that it can form weak condensates and link the RLG membranes. The masks applied for quantitation of RLG cluster sizes are shown in the right panels. Scale bar, 50 µm. f, g, Condensates formed by N122-Di and PEG do not cluster Golgi membranes. RLG membranes were mixed with 10% PEG and with or without N122-Di (containing 5% fluor 488-labeled N122-Di) on a shaker for 45 min and transferred to a 96-well glass-bottom plate. Images were captured 30 min later. Representative images are shown in (f). The masks applied for quantitation of RLG cluster size are shown in the right panels in (f). RLG clusters were categorized by size and the percentage of total RLG elements in each size range is shown in (g). n = 9 images from 3 independent experiments. Scale bar, 50 µm. **** P < 0.0001. Statistical significance was assessed using unpaired two-tailed Student’s t-tests. Error bars represent mean ± SD. Source numerical data are available in source data.

Source data

Extended Data Fig. 8 The GM130-RNA complex stabilizes BFA-induced tubules.

a, Microinjection of RNase A prevents the BFA-induced re-distribution of NAGTI-GFP into the ER. DEPC-inactivated RNase A (DEPC/RNase) or RNase A was injected into SV589/NAGTI-GFP cells together with fluorescent dextran as injection marker. After 1 h, cells were treated with Brefeldin A (BFA) for 10 min and stained for GM130 (red). The boxed areas are shown enlarged in right panel. b, Quantitation of (a). Mock-treated, DEPC/RNase A-injected: n = 280 cells, mock-treated, RNase A-injected: n = 184, BFA-treated, DEPC/RNase A-injected: n = 161, BFA-treated, RNase A-injected: n = 289 from 3 independent experiments. **** P < 0.0001. c, GM130 knockdown prevents the re-distribution of NAGTI-GFP into the ER. SV589/NAGTI-GFP cells were transfected with control siRNA or siRNA against GM130. Cells were then treated with BFA for 10 min or 30 min and stained for GM130 and DNA (blue). d, Quantitation of (c). n = 3 independent experiments. *** P < 0.001, **** P < 0.0001. e, Arsenite (SA) treatment prevents the re-distribution of NAGTI-GFP into the ER. SV589/NAGTI-GFP cells were treated with 400 µM SA for 1 h, followed by the BFA treatment for indicated times. Cells were fixed and labelled for GM130 and DNA (blue). f, Quantitation of (e). n = 3 independent experiments. **** P < 0.0001. Statistical significance of all comparisons was assessed using unpaired two-tailed Student’s t-tests. All error bars represent mean ± SD. Scale bar, 10 µm. Source numerical data are available in source data.

Source data

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Supplementary Table 1. Semi-quantitative mass spectrometry results showing the proteins in complex with GRASP55-Flag (RC55) or GRASP65-Flag (RC65). Table 2. Oligonucleotides and siRNA information. Table 3. Reagents and resources, including antibodies, bacterial strains, chemicals and recombinant proteins, commercial kits, cell lines, recombinant DNA and the software used in this study.

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Zhang, Y., Seemann, J. RNA scaffolds the Golgi ribbon by forming condensates with GM130. Nat Cell Biol 26, 1139–1153 (2024). https://doi.org/10.1038/s41556-024-01447-2

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