Fig 2: RTCB is required for induction of XBP1s during plasma cell differentiationB220+ splenocytes of control (Rtcbfl/fl or Rtcbfl/+), Rtcbfl/+ Cd23-Cre or Rtcbfl/fl Cd23-Cre mice were stimulated with 20 μg/ml LPS for 4 days. A, B Protein levels of RTCB, tRNA ligase complex members (DDX1, FAM98B, CGI-99) and XBP1 were monitored by Western blot analysis (n > 3).C FACS-sorted CD138+ CD22− plasmablasts or CD138− CD22low pre-plasmablasts were probed for expression of the indicated proteins by Western blot analysis (n = 2).D Relative mRNA levels of Rtcb, Xbp1s, total Xbp1, Edem1, μM and μS were analyzed by RT–qPCR in fractionated LPS-stimulated cells at day 4 (n = 4, mean expression levels and SEM are displayed). Expression levels were normalized to Actb mRNA levels and to B cells from control mice. An unpaired Student's t-test was used to analyze the statistical significance of differences in mRNA levels (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig 3: Functional characterization of protein-RNA interactions in SARS-CoV-2-infected cells(A and B) Proteins with identified phenotypes in genome-wide screens using viruses. RBPs enriched in SARS-CoV-2 vRIC (A) or upregulated in the cRIC experiment (B) are displayed along the x axis. The y axis indicates the number of screens in which the protein has caused a phenotype in infection.(C) Effects of RBP inhibitors on SARS-CoV-2 infection. The red line indicates the effects in infection measured by protein ELISA at each drug dose. The black line shows cell viability at each drug dose. Error bars are SEM from three independent experiments.(D) RNA-binding profiles of the components of the tRNA ligase complex in cells infected with SARS-CoV-2 (red) and SINV (blue) (as in Figure 4D). *FDR < 20%, **FDR < 10%, and ***FDR < 1%.(E) Confocal immunofluorescence images of SARS-CoV-2 and mock-infected Calu-3 cells using antibodies against DDX1 and dsRNA. Fluorescence plot shows green and red fluorescence intensity profiles across an 8 µm section (white line).(F) Western blot analysis showing the nucleocapsid (NCAP), components of the tRNA-LC complex, and ß-actin (ACTB) levels in control cells and upon DDX1 or FAM98A knockdown.(G) SARS-CoV-2 RNA levels in control cells and upon DDX1 or FAM98A knockdown measured by qRT-PCR and normalized to ß-actin mRNA. Error bars are SEM from three independent experiments.(H) RNA-seq analysis of wild-type (WT) and DDX1 knockdown A549-ACE2 cells infected with SARS-CoV-2. Data are represented in a boxplot, showing different mRNA groups (all, SARS-CoV-2, tRNA-LC, and UPR genes). Significant changes (p < 0.05) are shown in red and blue.

Fig 4: Characterization of Ddx1 cKO ESCs. (A) Scheme of Ddx1 targeting. Arrows and arrowheads represent primers used for genomic PCR. The sizes of PCR products generated with these primer sets are shown. The probe position for Southern blotting is depicted in the figure. B, BsrGI recognition site. (B) Thirteen targeted allele-positive clones were analyzed by three-primer PCR. Primers for the 5′ junction of the targeted site were used. The upper band represents the targeted allele, while the lower band represents the non-targeted allele. C, wild-type control. (C) The 3′ junction of the targeted site was analyzed by three-primer PCR. The upper band represents the non-targeted allele, while the lower band represents the targeted allele. C, wild-type control. (D) BsrGI-digested genomic DNA of clones #1 and #2 was analyzed by Southern blotting. C, wild-type control. Clone #2 was used as Ddx1F/F ESCs hereafter. (E) DDX1 expression in the indicated cells was analyzed via western blotting. (F) Expression of Ddx1 mRNA in parental ESCs and Ddx1F/F cells was analyzed by quantitative RT-PCR (n = 3; NS, not significant). (G) EGFP expression in Ddx1F/F cells was analyzed via fluorescence microscopy. (H) Expression of EGFP was analyzed via flow cytometry. Gray, parental ESCs; Blue, Ddx1F/F ESCs. (I) Dox-inducible FLPERT2 expression system of TetFE ESC. (J) Expression of fluorescent proteins under the indicated conditions was analyzed via flow cytometry. (K) DDX1 expression in sorted EGFP+ (F/F) and EGFP− (–/–) cells was analyzed via western blotting. Cells were harvested on day 2.

Fig 5: Proliferation of Ddx1−/− ESCs. (A) The numbers of Ddx1F/F or Ddx1−/− cells were counted on the indicated days after sorting (n = 3; *P < 0.05). (B) Apoptosis of Ddx1F/F and Ddx1−/− cells was analyzed on day 3 (n = 3; ***P < 0.005). (C) The p53 expression levels in Ddx1F/F or Ddx1−/− cells were analyzed via western blotting. (D) The expression levels of p53-regulated genes were analyzed via qRT-PCR (n = 3; *P < 0.05; ***P < 0.005).