A canonical RQC substrate reporter mRNA does not depend on VCP activity for localization to SGs. Reporter mRNAs encoding eGFP and RFP separated by a linker region that encodes 20 lysine residues in a 60-nt poly(A) tract (K20), or no lysines (K0; from Juszkiewicz and Hegde, 2017) were expressed in U-2 OS cells. Cells were stressed for 45 min with 0.5 mM arsenite (As) in the absence or presence of 0.1% DMSO (As + DMSO), 10 µM DBeQ (As + DBeQ), or 10 µM DBeQ and 10 µg/ml puromycin (As + DBeQ + Puro). Cells were fixed and G3BP detected by IMF staining (blue) to mark SGs. The reporter mRNA was detected by smFISH using probes against the eGFP ORF (white); eGFP is shown in green and RFP in red. Cells expressing eGFP were imaged at 100× using a DeltaVision microscope, and maximum intensity projections of 25 z-stacks are shown. (A) Left: Diagram of reporter mRNAs. Right: Quantification of the relative eGFP and RFP intensities in individual cells (shown as dots) was performed from cells treated with As, with the average ± SEM reported from two independent experiments (K0n = 12 cells; K20n = 9 cells). (B) Representative images are shown at left with average ± SEM of the percent eGFP mRNA that colocalized with SGs shown at right from two independent experiments. Scale bars, 10 µm (whole cell) or 5 µm (magnified panels). Individual points represent a single frame with one to five cells counted per frame. For K0, As n = 12 frames; As + DMSO n = 8 frames; As + DBeQ n = 8 frames; As + DBeQ + Puro n = 12 frames; for K20, As n = 9 frames; As + DMSO n = 10 frames; As + DBeQ n = 9 frames; As + DBeQ + Puro n = 10 frames. Student’s t test was done to assess significance, with *, P ≤ 0.05 and **, P ≤ 0.01. Exact P values and source data are provided in Table S1.
