Supplementary Figure 6: Non-translational role of EPRS in regulating antiviral immune responses.

(a,b) Purified His-tagged EPRS (aa 1–196) (a) or EPRS (aa 1–168) (b) was mixed with the GST-fused PCBP2 KH1 (aa 11-82). After His-tag precipitation, proteins were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. (c) The purified His-tagged ERS (aa 1-732) and its mutant that is inactive for tRNA glutamylation (MT). Black arrows denote protein fragments derived from ERS during purification (as in Fig. 6j). (d) Aminoacylation assay for ERS (WT) and its mutant (MT). CPM, counts per minute. Ctrl, buffer without protein. (e) IFNB promoter activity in 293T cells transfected with N-RIG-I plus empty vector (EV), Strep-EPRS (WT), or its mutants inactive for tRNA glutamylation only (E-MT), tRNA prolylation only (P-MT), or both (EP-MT). (f) Immunoblot analysis of endogenous EPRS, MAVS, and RIG-I expression in sgEPRS 293T cells or non-targeting control (sgCtrl) cells. (g–k) Non-translational function of EPRS in antiviral immune responses. Virus replication assay (examined by fluorescence microscopy) (g) and plaque assay (h) at 24 h post-infection with VSV-GFP (MOI = 0.0001). Immunoblot analysis of Strep-EPRS or endogenous EPRS expression (i). IFN-β (j) and IL-6 (k) secreted by sgEPRS cells infected with VSV-GFP. sgCtrl or sgEPRS 293T cells were reconstituted with EV, Strep-tagged EPRS (WT), or its catalytic mutant (EP-MT) (g–k). Scale bar, 100 μm (g). *P < 0.01; NS, not significant (Student’s t-test; d,e,h,j,k). Data are representative of two (a-k) independent experiments (mean and s.d. of triplicate in d,e,h,j,k).