Fig 2: RESIST counteracts repression of IFN-I by TTP family proteins, a function which requires a RESIST C-terminal region and CNOT9a. AlphaFold predictions of RESIST with CNOT1 M-HEAT and CNOT9. TTP peptide:CNOT1 M-HEAT is PDB code 4J8S41. Roquin peptide:CNOT9 is PDB code 5LSW47. N and C-terminal ends of RESIST are marked by “N” or “C” respectively.b. Ifnb1 RT-qPCR from BMMs electroporated with non-targeting control gRNA (NTC) or gRNAs targeting indicated CCR4-NOT subunits, treated for 8 hours with 100 μg/mL DMXAA.c. Strep pulldown of purified recombinant full-length human His-MBP-RESIST-Strep or His-MBP-Strep with human CNOT9 and CNOT1 (aa 1351–1588). First lane indicates purified CNOT9/CNOT1.d. Ifnb1 RT-qPCR for BMMs transduced with indicated lentiviral constructs, treated with doxycycline and DMXAA for 6 hours. Results include data also shown in Fig. 3f.e. Immunoblot for anti-HA IP of BMMs transduced with indicated constructs in d. RESIST construct is C-terminally tagged with HA. For gel source data, please see Supplementary Figure 1.f. Ifnb1 RT-qPCR from BMMs electroporated with indicated gRNAs, treated for 8 hours with 100 μg/mL DMXAA.g. Immunoblot for FLAG IP of FLAG-TTP (mouse) co-expressed with mouse HA-RESIST in HEK293Ts. For gel source data, please see Supplementary Figure 1.h. Schematic for how RESIST may interact with CCR4-NOT subunits CNOT1 and CNOT9 to mediate stabilization of Ifnb1 mRNAs. * = p < 0.05, ** = p < 0.005, ***= p < 0.0005, **** = p < 0.00005, ns = not significant, calculated with one-way ANOVA tests with Dunnett’s T3 multiple comparison correction post-hoc.

Fig 3: AlphaFold predictions suggest RESIST likely binds the CNOT1 M-HEAT domain.a. Schematic of CCR4-NOT subunit and RESIST complexes produced by Alphafold Multimer.b. Aligned AlphaFold models of RESIST with the CNOT1 M-HEAT domain.c. The top-scoring structural prediction of the RESIST and CNOT1 M-HEAT complex, colored by pLDDT.d. AlphaFold PAE plot of predicted RESIST and CNOT1 M-HEAT complex. Plot generated with PAEViewer79.e. Depiction of RESIST binding to a hydrophobic patch on the CNOT-1 M-HEAT domain (CNOT1 colored by hydrophobicity).

Fig 4: AlphaFold predictions suggest RESIST likely does not bind CNOT11.a. Aligned AlphaFold structure predictions of RESIST with the CNOT1 NMIF4G-NHEAT domains, CNOT10, and CNOT11.b. Highest-scoring AlphaFold structure prediction of RESIST and CNOT1/CNOT10/CNOT11 complex colored by pLDDT.c. AlphaFold PAE plot of predicted RESIST and CNOT1/CNOT10/CNOT11 complex. Plot generated with PAEViewer79.

Fig 5: AlphaFold predictions suggest RESIST likely binds the CNOT1 M-HEAT domain.a. Schematic of CCR4-NOT subunit and RESIST complexes produced by Alphafold Multimer. Schematic was generated in BioRender.com. b. Aligned AlphaFold models of RESIST with the CNOT1 M-HEAT domain. c. The top-scoring structural prediction of the RESIST and CNOT1 M-HEAT complex, coloured by pLDDT. d. AlphaFold PAE plot of predicted RESIST and CNOT1 M-HEAT complex. Plot generated with PAEViewer77. e. Depiction of RESIST binding to a hydrophobic patch on the CNOT-1 M-HEAT domain (CNOT1 coloured by hydrophobicity).