Fig 2: Unconjugated Ubiquitin Localizes to SGs in a UAE-Independent Manner(A–E) Immunofluorescence staining of HeLa cells treated with NaAsO2 (250 μM) for 0’, 45’, or 120’ prior to fixation (A) or pretreated with DMSO or UbE1i (1 μ M) for 90’, followed by treatment with NaAsO2 (250 mM) for 120’ prior to fixation (B–E). Cells were stained with antibodies against G3BP1 (A–E), pan-ubiquitin (A), polyubiquitin (B and D), centrosome marker pericentrin (D), proteasome subunits (D), VCP/p97 (E), SQSTM/p62 (E), HDAC6 (E), UBAP2L (E), or TRIM 25 (E). (C) Cells stably expressing either mCherry-tagged wild-type ubiquitin (mCh-Ub-WT) or mCherry-tagged ubiquitin, in which all internal lysine residues were mutated to arginine and the C-terminal diglycine residues were removed (mCh-Ub-K0DGG [K0dGG]) were stained with antibodies against G3BP1. G3BP1-positive SGs are indicated by arrows (both red and white). G3BP1-negative perinuclear foci are indicated by solid yellow arrowheads. G3BP1-negative punctate signal for VCP/p97 and SQSTM/p62 is indicated by blue open arrowheads. Images from Ub-E1i-treated cells in all panels were taken at the same exposure time and acquisition settings in the ubiquitin channel as those for images from DMSO-treated cells. Nuclei were stained using DAPI. Scale bars, 20 μ m in all panels.See also Figure S4.

Fig 3: Interaction of FXR1 to UBAP2L.(A) ITC measurements of a reported RNA binding to FXR1. Binding to FXR1 215–360 was monitored and as a control the SARS-CoV-2 N protein (n = 1). (B) Mass spectrometry analysis of the interactomes of affinity-purified YFP-tagged FXR1 WT and FXR1 I304N. Proteins specifically binding to FXR1 WT indicated in the volcano plot. Data from 4 technical repeats. (C) The indicated YFP-tagged FXR1 proteins were expressed and purified from HeLa cells and binding to UBAP2L determined by western blot. Representative of 2 biological replicates. (D) A panel of YFP-UBAP2L constructs were expressed and purified from HeLa cells and binding to FXR1 determined. Representative of 2 biological replicates. Data information: In (B) a two-sided unpaired t test was used for statistical analysis.

Fig 4: NSP3 disrupts the UBAP2L-FMRP complex.(A) FXR1 was affinity-purified and incubated with either WT NSP3 or mutant NSP3 peptide and interactomes determined by MS to determine proteins specifically displaced by WT NSP3. Data from four technical replicates. (B) As (A) but using UBAP2L as a bait. Data from four technical replicates. (C) Schematic of UBAP2L and truncation analysis to identify FXR1 binding site (n = 1). (D) Peptide array of UBAP2L 199–400 to identify FXR1 binding region and lower part single Ala scan through UBAP2L 247–266 to identify critical residues (n = 1). (E) Immunopurification of indicated UBAP2L constructs to determine binding to FXR1 and G3BP1. Representative of two biological replicates. (F) ITC measurements of indicated UBAP2L peptides to FXR1 215–360 (n = 1). (G) Competition between NSP3 peptide and UBAP2L peptide for binding to FXR1 215–360. The black trace is NSP3 binding to FXR1 while the red trace is NSP3 binding to FXR1 preincubated with UBAP2L peptide (n = 1). (H) Alphafold model of the FXR1-UBAP2L complex highlighting critical residues in yellow and phosphorylation sites. Data information: In (A, B), the statistical comparison was done using a two-sided unpaired t test. Source data are available online for this figure.

Fig 5: NSP3 prevents the incorporation of FMRPs into stress granules.(A) Analysis of FXR1 stress granule association in the presence of NSP3 WT or NSP3 A4. HeLa cells were treated 30 min with arsenite before fixation and number of FXR1 foci quantified per cell. Only cells expressing NSP3 was analyzed. (B) Similar as (A) but number of G3BP1 foci analyzed. (C) HeLa UBAP2L KO cells were complemented with UBAP2L-YFP constructs and cells treated with arsenite for 30 min before fixation. The fluorescence intensity of FXR1 to UBAP2L (GFP signal) was quantified. (D) As in (C) but staining for G3BP1. (A–D) representative stills from the immunofluorescence is shown with a scale bar of 10 μM indicated in lower left corner. Combined data from three and two biological replicates is shown in (A, B), respectively. (C, D) A pool of four biological replicates is shown in the graphs. Data information: Violin plots (A–D) with all points shown (A, B) with the median indicated with red line. The number of cells (A, B) or foci (C, D) analyzed per condition is indicated above the plot. Scale bars represent 10 μm. Source data are available online for this figure.