Volume 21, Issue 13, Pages 3740-3753 (December 2017) Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum  Franka Voigt, Hui Zhang, Xianying A. Cui, Désirée Triebold, Ai Xin Liu, Jan Eglinger, Eliza S. Lee, Jeffrey A. Chao, Alexander F. Palazzo  Cell Reports  Volume 21, Issue 13, Pages 3740-3753 (December 2017) DOI: 10.1016/j.celrep.2017.12.008 Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 A Subset of mRNAs Encoding Cytosolic and Nuclear Proteins Is Resistant to Digitonin Extraction and Co-localizes with the ER (A) U2OS cells were treated with either digitonin or Triton X-100 (TX100) and deoxycholate (DOC), and extraction-resistant membranes were pelleted. Equal amounts of pellet (P) and supernatant (S) were separated by SDS-PAGE and immunoprobed for a cytosolic protein (α-tubulin), an ER-resident membrane protein (TRAPα), and a nuclear protein (Aly). The asterisk denotes a non-specific band. TRAPα is resistant to digitonin extraction but not Triton X-100 and deoxycholate. (B) Quantification of smFISH against indicated mRNA species in U2OS cells that either were fixed or were extracted with digitonin and then fixed. Graph indicates mean ± SEM of 3 independent experiments (12–40 cells each). (C) Quantification of smFISH against GAPDH mRNA and NORAD lncRNA in U2OS cells that were extracted with digitonin for indicated times. The number of foci in the cytosol normalized to the unextracted cells (y axis) was plotted against the extraction time (x axis). Each data point is the mean ± SEM of three independent experiments (15–40 cells each). (D) smFISH quantification of ER co-localization of GAPDH and TFRC mRNAs in U2OS or COS7 cells expressing GFP-KDEL or GFP-Sec61β. Graph indicates mean ± SEM (5 cells). (E and F) A U2OS cell (E) and a COS7 cell (F) co-stained for GFP-KDEL (red), GAPDH mRNA (green), and DAPI (blue). From the overlays (Ea, Fa), blown-up sections show the overlaid (Eb, Fb) and GFP-KDEL-only signals in more detail (Ec, Fc). Circles denote mRNAs that co-localize (white) and do not co-localize (magenta) with ER. Scale bars, 10 μm for (Fa) and 2 μm for (Fc). See also Table S1A. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 GAPDH mRNA Resistance to Digitonin Extraction Is Not Primarily due to Cytoskeleton Association (A) Quantification of smFISH against GAPDH mRNA in U2OS cells treated with 20 μM nocodazole or DMSO for 30 min and then either fixed (“Unextracted”) or digitonin extracted prior to fixation. (B and C) U2OS cells were either fixed (“Unextracted”) or extracted with digitonin or Triton X-100 (TX100) and deoxycholate (DOC) prior to fixation. Cells were stained with rhodamine-phalloidin to visualize F-actin and processed for smFISH with Stellaris probes against GAPDH mRNA. Scale bar in (B), 10 μm. (C) Quantification of the average integrated phalloidin intensity normalized to unextracted cells. (D and E) Quantification of smFISH against GAPDH mRNA (D) or TFRC mRNA (E) in U2OS cells either fixed or first extracted with digitonin or Triton X-100 and deoxycholate. Scale bar in (E), 10 μm. (F) Quantification of smFISH against GAPDH mRNA in untransfected and GFP-Sec61β-expressing U2OS cells either fixed or first extracted with digitonin. (G) smFISH against GAPDH mRNA in U2OS cells transfected with GFP-Sec61β and then extracted with digitonin. All four images show the same field of view. Individual GAPDH mRNA foci are circled in the bottom two panels. Overlay: the cell expressing GFP-Sec61β (red) has fewer GAPDH foci (green) than the untransfected neighboring cell. Scale bar, 5 μm. All experiments include at least 10–40 cells per condition. Graphs indicate mean ± SEM. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Resistance of GAPDH mRNA to Digitonin Extraction Is Dependent on Translation and Likely Requires the Initiating Ribosome (A) smFISH against GAPDH mRNAs in U2OS cells treated with PUR (200 μM), HHT (5 μM), or DMSO for 30 min. Cells were either fixed (“Unextracted”) or digitonin extracted before fixation. Scale bar, 10 μm. (B–F) Shown in (B): quantification of experiment shown in (A). Similar experiments were performed for HeLa (C), COS7 (D), MEF (E), and NIH 3T3 (F) cells. Graphs indicate mean ± SEM of three independent experiments (25–50 cells). (G) The fold decrease in the number of digitonin-resistant GAPDH mRNA foci after either PUR or HHT treatment in comparison to DMSO-treated cells. A lack of change corresponds to a “fold decrease” of 1. Data are the same as in (B)–(F) but recalculated as a fold change in foci in digitonin-extracted cells treated with DMSO in comparison to PUR or HHT. Each bar is the mean ± SEM of three independent experiments (25–50 cells). A lack of change corresponds to a fold decrease of 1. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Localization of a Small Fraction of Renilla and the Majority of Gaussia mRNAs to the ER Is Translation Dependent (A and B) Representative live-cell images of the Gaussia (A, red) and Renilla (B, blue) reporter constructs in HeLa cells expressing NLS-MCP-Halo and ER-Turq2. Scale bars, 5 μm. (C) Correlated diffusion and ER co-localization analysis of individual Gaussia (red) and Renilla (blue) mRNAs. y axis: instantaneous diffusion coefficients. x axis: cumulative ER localization index. Positive values indicate ER co-localization. (D) Same data as shown in (C), but only including tracks longer than 30 frames. Transcripts are defined as ER associated if they exhibit reduced mobility (D < 0.06 μm2 s−1) and positive ER localization (ER > 0). These thresholds include >95% of all Gaussia tracks longer than 30 frames. (E) Correlated diffusion and ER co-localization analysis for Gaussia and Renilla mRNAs in cells that were treated with PUR. (F) Graph of ER-bound mRNAs derived from quantification of data shown in C, D and E. Graph indicates mean ± SEM. px, pixels. See also Table S1C. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Gaussia mRNAs Can Return to the Cytosol after Translation on the ER (A) Shown in (A): representative live-cell image of the Gaussia TRICK reporter in a HeLa cell expressing NLS-MCP-Halo, NLS-PCP-GFP, and ER-SNAP. Overlay of SPT (50 frames) in both mRNA channels (NLS-MCP-Halo, indicated in red; NLS-PCP-GFP, indicated in yellow) with the ER channel (gray). Scale bar, 10 μm. Inset, solid frame: overlay of SPT of untranslated mRNA (red, yellow) in cytosol with ER (white). Inset, dashed frame: overlay of SPT of translated mRNA (red only) in cytosol with ER (white). (B) Correlated diffusion and ER co-localization analysis of individual Gaussia mRNAs. y axis: instantaneous diffusion coefficients. x axis: cumulative ER localization index. px, pixels. See also Table S1D. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 mRNAs Remain on the ER for Time Frames Consistent with Several Rounds of Translation (A) Overlays show selected frames of image series acquired for Gaussia (red) and Renilla (blue) reporter transcripts that co-localize with the ER (gray). Acquisition times are shown in seconds. Asterisks indicate tracked mRNAs. Scale bar, 1 μm. (B) Sinaplot depicting all Gaussia transcripts imaged over time frames of up to 10 min (200 frames, 500 ms exposure time, 2.5 s intervals). Each dot represents a single transcript. The left y axis shows time length of track, and the right y axis illustrates the number of proteins that could be translated in this time, assuming an average ribosomal elongation rate of 7.7 amino acids (aa) per second. (C) Same as (B), but depicting Renilla tracks. See also Table S1E. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 ER-Associated Translation Sites Are Brighter Than Cytosolic Ones (A) Representative live-cell image of the SunTag-Renilla reporter construct in a HeLa cell expressing scAB-GFP and ER-SNAP. Ribosomes translating SunTag-Renilla mRNAs (green) can co-localize with the ER (gray). Scale bar, 5 μm. (B) Correlated diffusion and ER co-localization analysis of individual SunTag-Renilla translation sites (green) superposed with Renilla mRNAs (blue), as shown in Figure 4C. (C) Density plot of SunTag-Renilla translation site intensities. Solid line: cytosolic translation sites; quadrant 1 in (B). Dashed line: ER-associated translation sites; quadrant 4 in (B). The average intensity signal for translation sites regardless of their localization corresponds to four ribosomes per mRNA. Additional peaks at higher intensity values observed for ER-associated translation sites correspond to five and six ribosomes per mRNA. px, pixels. See also Tables S1G and S1H. Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 3740-3753DOI: (10.1016/j.celrep.2017.12.008) Copyright © 2017 The Author(s) Terms and Conditions