FXR1 facilitates recruitment of translation machinery. (A) Ribosomes (green) and viral RNA (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (B) Ribosomes (green) and Nsp3N (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (C) The schematic diagram shows the TRICK assay to assess translation of viral RNA. The viral RNA is engineered to contain PP7 and MS2 sequences for two RNA-binding reporter proteins: GFP-NLS-PCP (PP7 coat protein) in the translated region and RFP-NLS-MCP (MS2 coat protein) in the 3′ untranslated region. If the viral RNA is actively translated, ribosomes will displace GFP-NLS-PCP but not RFP-NLS-MCP (top). If translation is inhibited (for example, by puromycin), both reporter proteins will be present on the RNA (bottom). (D) RFP signal, but not GFP, is detected at foci positive for FXR1 and Nsp3/4. Stalled translation, induced by puromycin treatment, causes both GFP and RFP signals to localize in FXR1 condensates. GFP-NLS-PCP, PCP fused to an NLS and GFP; RFP-NLS-MCP, MCP fused to an NLS and RFP. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. (E) In siFXRs cells expressing RNAi-resistant BFP-FXR1, Nsp3/4 forms big puncta positive for FXR1 and RFP-NLS-PCP, but not GFP-NLS-PCP. Small dispersed Nsp3/4⁺ puncta are negative for FXR1, RFP-NLS-MCP, or GFP-NLS-PCP RFP in cells expressing RNAi-resistant BFP-FXR1(I355A/L358A/K359A) or BFP-FXR1(R315D/R317D). Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm.